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Cell_Biology_Alberts. DNA methylation helps to repress transcription in several ways. The methyl groups on methylated cytosines lie in the major groove of DNA and interfere directly with the binding of proteins (transcription regulators as well as the general transcription factors) required for transcription initiation. In addition, the cell contains a repertoire of proteins that bind specifically to methylated DNA.The best characterized of these associate with histone modifying enzymes, leading to a repressive chromatin state where chromatin structure and DNA methylation act synergistically (Figure 7–45). One reflection of the importance of DNA methylation to humans is the widespread involvement of “incorrect” DNA methylation patterns in cancer progression (discussed in Chapter 20). CG-rich islands Are Associated with Many Genes in Mammals


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Cell_Biology_Alberts. CG-rich islands Are Associated with Many Genes in Mammals Because of the way in which DNA repair enzymes work, methylated C nucleotides in the vertebrate genome tend to be eliminated in the course of evolution. Accidental deamination of an unmethylated C gives rise to U (see Figure 5–38), which is not normally present in DNA and thus is recognized easily by the DNA repair enzyme uracil DNA glycosylase, excised, and then replaced with a C (as discussed in Chapter 5). But accidental deamination of a 5-methyl C cannot be repaired in this way, for the deamination product is a T and so is indistinguishable from the other, nonmutant T nucleotides in the DNA. Although a special repair system exists to remove these mutant T nucleotides, many of the deaminations escape detection, so that those C nucleotides in the genome that are methylated tend to mutate to T over evolutionary time. 406 Chapter 7: Control of Gene expression


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Cell_Biology_Alberts. 406 Chapter 7: Control of Gene expression Figure 7–45 Multiple mechanisms contribute to stable gene repression.


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Cell_Biology_Alberts. in this schematic example, histone reader and writer proteins (discussed in Chapter 4), under the direction of transcription regulators, establish a repressive form of chromatin. A de novo DnA methylase is attracted by the histone reader and methylates nearby cytosines in DnA, which are, in turn, bound by DnA methyl-binding proteins. During DnA replication, some of the modified (blue dot) histones will be inherited by one daughter chromosome, some by the other, and in each daughter they can induce reconstruction of the same pattern of chromatin modifications (discussed in Chapter 4). At the same time, the mechanism shown in figure 7–44 will cause both daughter chromosomes to inherit the same methylation pattern. in these cases where DnA methylation stimulates the activity of the histone writer, the two inheritance mechanisms will be mutually reinforcing. this scheme can account for the inheritance by daughter cells of both the histone and the DnA modifications. it can also explain the


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Cell_Biology_Alberts. the two inheritance mechanisms will be mutually reinforcing. this scheme can account for the inheritance by daughter cells of both the histone and the DnA modifications. it can also explain the tendency of some chromatin modifications to spread along a chromosome (see figure 4–44).


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Cell_Biology_Alberts. During the course of evolution, more than three out of every four CGs have been lost in this way, leaving vertebrates with a remarkable deficiency of this dinucleotide. The CG sequences that remain are very unevenly distributed in the genome; they are present at 10 times their average density in selected regions, called CG islands, which average 1000 nucleotide pairs in length. The human genome contains roughly 20,000 CG islands and they usually include promoters of genes. For example, 60% of human protein-coding genes have promoters embedded in CG islands and these include virtually all the promoters of the so-called housekeeping genes—those genes that code for the many proteins that are essential for cell viability and are therefore expressed in nearly all cells (Figure 7–46). Over evolutionary timescales, the CG islands were spared the accelerated mutation rate of bulk CG sequences because they remained unmethylated in the germ line (Figure 7–47).


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Cell_Biology_Alberts. CG islands also remain unmethylated in most somatic tissues whether or not the associated gene is expressed. The unmethylated state is maintained by sequence-specific DNA-binding proteins, many of whose cis-regulatory sequences contain a CG. By binding to these sequences, which are spread across CG islands, they protect the DNA from methyl transferases. These proteins also recruit DNA demethylases, which convert 5-methyl C to hydroxy-methyl C, which 10,000 nucleotide pairs is later replaced by C either through DNA repair (see Figure 5–41A) or, passively, through multiple rounds of DNA replication. Unmethylated CG islands have several properties that make them particularly suitable for promoters. For example, some of the same proteins that bind to CG islands and protect them from methylation recruit histone modifying enzymes that make the islands particularly “promoter friendly.” As a result, RNA polymerase is often found bound to promoters within CG islands, even when the associated


Cell_Biology_Alberts

Cell_Biology_Alberts. recruit histone modifying enzymes that make the islands particularly “promoter friendly.” As a result, RNA polymerase is often found bound to promoters within CG islands, even when the associated gene is not being actively transcribed. At unmethylated CG islands, the balance between polymerase and nucleosome assembly is thus tipped toward the former. Additional steps are needed to “push” the bound polymerase into transcribing the adjacent gene, and these are directed by transcription regulators that bind to cis-regulatory sequences of DNA (often well upstream from the CG islands). These regulators serve to release the polymerase with the appropriate elongation factors (see Figure 7–21C and D).


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Cell_Biology_Alberts. Genomic imprinting is Based on DnA Methylation Mammalian cells are diploid, containing one set of genes inherited from the father and one set from the mother. The expression of a small minority of genes depends on whether they have been inherited from the mother or the father: when the paternally inherited gene copy is active, the maternally inherited gene copy is silent, or vice versa. This phenomenon is called genomic imprinting. Roughly 300 genes are imprinted in humans. Because only one copy of an imprinted gene is expressed, imprinting can “unmask” mutations that would normally be covered by the other, functional copy. For example, Angelman syndrome, a disorder of the nervous system in humans that causes reduced mental ability and severe speech impairment, results from a gene deletion on one chromosomal homolog and the silencing, by imprinting, of the intact gene on the other homolog.


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Cell_Biology_Alberts. The insulin-like growth factor-2 (Igf 2) gene in the mouse provides a well-studied example of imprinting. Mice that do not express Igf 2 at all are born half the size of normal mice. However, only the paternal copy of Igf 2 is transcribed, and only this gene copy matters for the phenotype. As a result, mice with a mutated paternally derived Igf 2 gene are stunted, while mice with a mutated maternally derived Igf 2 gene are normal.


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Cell_Biology_Alberts. Figure 7–47 A mechanism to explain both the marked overall deficiency of CG sequences and their clustering into CG islands in vertebrate genomes. White lines mark the location of CG dinucleotides in the DnA sequences, while red circles indicate the presence of a methyl group on the CG dinucleotide. CG sequences that lie in regulatory sequences of genes that are transcribed in germ cells are unmethylated and therefore tend to be retained in evolution. Methylated CG sequences, on the other hand, tend to be lost through deamination of 5-methyl C to t, unless the CG sequence is critical for survival.


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Cell_Biology_Alberts. Figure 7–46 The CG islands surrounding the promoter in three mammalian housekeeping genes. the yellow boxes show the extent of each island. As for most genes in mammals, the exons (dark red) are very short relative to the introns (light red). (Adapted from A.p. Bird, Trends Genet. 3:342–347, 1987. with permission from elsevier.) methylation of most CG sequences in germ line many millions of years of evolution mRNA mRNA BOTH PARENTS EXPRESS THE SAME ALLELE OF GENE Aimprinted allele of gene Aexpressed allele of gene Asomatic cell somatic cell REMOVAL OF IMPRINTING IN GERM CELLS, FOLLOWED BY MEIOSIS FEMALE IMPRINT ESTABLISHED MALE IMPRINT ESTABLISHED EGGS SPERM OFFSPRING DIFFER IN THE ALLELE OF GENE ATHAT IS EXPRESSED mRNA mRNA chromosome inherited from father


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Cell_Biology_Alberts. Figure 7–48 Imprinting in the mouse. the top portion of the figure shows a pair of homologous chromosomes in the somatic cells of two adult mice, one male and one female. in this example, both mice have inherited the top homolog from their father and the bottom homolog from their mother, and the paternal copy of a gene subject to imprinting (indicated in orange) is methylated, preventing its expression. the maternally derived copy of the same gene (yellow) is expressed. the remainder of the figure shows the outcome of a cross between these two mice. During germ-cell formation, but before meiosis, the imprints are erased and then, much later in germ-cell development, they are reimposed in a sex-specific pattern (middle portion of figure). in eggs produced from the female, neither allele of the A gene is methylated. in sperm from the male, both alleles of gene A are methylated. Shown at the bottom of the figure are two of the possible imprinting patterns inherited by the progeny mice;


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Cell_Biology_Alberts. the A gene is methylated. in sperm from the male, both alleles of gene A are methylated. Shown at the bottom of the figure are two of the possible imprinting patterns inherited by the progeny mice; the mouse on the left has the same imprinting pattern as each of the parents, whereas the mouse on the right has the opposite pattern. if the two alleles of gene A are distinct, these different imprinting patterns can cause phenotypic differences in the progeny mice, even though they carry exactly the same DnA sequences of the two A gene alleles. imprinting provides an important exception to classical genetic behavior, and several hundred mouse genes are thought to be affected in this way. however, the majority of mouse genes are not imprinted, and therefore the rules of Mendelian inheritance apply to most of the mouse genome.


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Cell_Biology_Alberts. In the early embryo, genes subject to imprinting are marked by methylation according to whether they were derived from a sperm or an egg chromosome. In this way, DNA methylation is used as a mark to distinguish two copies of a gene that may be otherwise identical (Figure 7–48). Because imprinted genes are somehow protected from the wave of demethylation that takes place shortly after fertilization (see pp. 404–405), this mark enables somatic cells to “remember” the parental origin of each of the two copies of the gene and to regulate their expression accordingly. In most cases, the methyl imprint silences nearby gene expression. In some cases, however, it can activate expression of a gene. In the case of Igf 2, for example, methylation of an insulator element on the paternally derived chromosome blocks its function and allows distant cis-regulatory sequences to activate transcription of the Igf 2 gene. On the maternally derived chromosome, the insulator is not methylated and the Igf 2


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Cell_Biology_Alberts. blocks its function and allows distant cis-regulatory sequences to activate transcription of the Igf 2 gene. On the maternally derived chromosome, the insulator is not methylated and the Igf 2 gene is therefore not transcribed (Figure 7–49A).


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Cell_Biology_Alberts. Other cases of imprinting involve long noncoding RNAs, which are defined as RNA molecules over 200 nucleotides in length that do not code for proteins. We discuss lncRNAs broadly at the end of this chapter; here, we focus on the role of a specific lncRNA in imprinting. In the case of the Kcnq1 gene, which codes for a voltage-gated calcium channel needed for proper heart function, the lncRNA is made from the paternal allele (which is unmethylated) but it is not released by the RNA polymerase, remaining instead at its site of synthesis on the DNA template. This RNA in turn recruits histone-modifying and DNA-methylating enzymes that direct the formation of repressive chromatin, which silences the protein-coding gene associated on the paternally derived chromosome (Figure 7–49B). The maternally derived gene, on the other hand, is immune to these effects because the specific methylation present from imprinting blocks the synthesis of the lncRNA but allows transcription of the


Cell_Biology_Alberts

Cell_Biology_Alberts. The maternally derived gene, on the other hand, is immune to these effects because the specific methylation present from imprinting blocks the synthesis of the lncRNA but allows transcription of the protein-coding gene. Like Igf 2, the specificity of Kcnq1 imprinting arises from the inherited methylation patterns; the difference lies in the way these patterns bring about differential expression of the imprinted gene.


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Cell_Biology_Alberts. Why imprinting should exist at all is a mystery. In vertebrates, it is restricted to placental mammals, and many of the imprinted genes are involved in fetal development. One idea is that imprinting reflects a middle ground in the evolutionary struggle between males to produce larger offspring and females to limit offspring size. Whatever its purpose might be, imprinting provides startling evidence that features of DNA other than its sequence of nucleotides can be inherited. We have seen that DNA methylation and certain types of chromatin structure can be heritable, preserving patterns of gene expression across cell generations. Perhaps the most striking example of this effect occurs in mammals, in which an alteration in the chromatin structure of an entire chromosome can modulate the levels of expression of most genes on that chromosome. Figure 7–49 Mechanisms of imprinting.


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Cell_Biology_Alberts. Figure 7–49 Mechanisms of imprinting. On chromosomes inherited from the female, a protein called CtCf binds to an insulator (see figure 7–24), blocking communication between cis-regulatory sequences (green) and the Igf2 gene (orange). Igf2 is therefore not expressed from the maternally inherited chromosome. Because of imprinting, the insulator on the male-derived chromosome is methylated (red circles); this inactivates the insulator by blocking the binding of the CtCf protein, and allows the cis-regulatory sequences to activate transcription of the Igf2 gene. in other examples of imprinting, methylation simply blocks gene expression by interfering with the binding of proteins required for a gene’s transcription.


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Cell_Biology_Alberts. imprinting of the mouse Kcnq1 gene. On the maternally derived chromosome, synthesis of the lncrnA is blocked by methylation of the DnA (red circles), and the Kcnq1 gene is expressed. On the paternally derived chromosome, the lncrnA is synthesized, remains in place, and by directing alterations in chromatin structure blocks expression of the Kcnq1 gene. Although shown as directly binding to lncrnA, the histone-modifying enzymes are likely to be recruited indirectly, through additional proteins.


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Cell_Biology_Alberts. Males and females differ in their sex chromosomes. Females have two X chromosomes, whereas males have one X and one Y chromosome. As a result, female cells contain twice as many copies of X-chromosome genes as do male cells. In mammals, the X and Y sex chromosomes differ radically in gene content: the X chromosome is large and contains more than a thousand genes, whereas the Y chromosome is small and contains less than 100 genes. Mammals have evolved a dosage compensation mechanism to equalize the dosage of X-chromosome gene products between males and females. The correct ratio of X chromosome to auto-some (non-sex chromosome) gene products is carefully controlled, and mutations that interfere with this dosage compensation are generally lethal.


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Cell_Biology_Alberts. Mammals achieve dosage compensation by the transcriptional inactivation of one of the two X chromosomes in female somatic cells, a process known as X-inactivation. As a result of X-inactivation, two X chromosomes can coexist within the same nucleus, exposed to the same diffusible transcription regulators, yet differ entirely in their expression.


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Cell_Biology_Alberts. Early in the development of a female embryo, when it consists of a few hundred cells, one of the two X chromosomes in each cell becomes highly condensed into a type of heterochromatin. The initial choice of which X chromosome to inactivate, the maternally inherited one (Xm) or the paternally inherited one (Xp), is random. Once either Xp or Xm has been inactivated, it remains silent throughout all subsequent cell divisions of that cell and its progeny, indicating that the inactive state is faithfully maintained through many cycles of DNA replication and mitosis. Because X-inactivation is random and takes place after several hundred cells have already formed in the embryo, every female is a mosaic of clonal groups of cells in which either Xp or Xm is silenced (Figure 7–50). These clonal groups are


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Cell_Biology_Alberts. Xp Xm Xp Xm Xp Xm CONDENSATION OF A RANDOMLY SELECTED X CHROMOSOME DIRECT INHERITANCE OF THE PATTERN OF CHROMOSOME CONDENSATION DIRECT INHERITANCE OF THE PATTERN OF CHROMOSOME CONDENSATION Figure 7–50 X-inactivation. the clonal inheritance in female mammals of a only Xm active in this clone only Xp active in this clone condensed, inactive X chromosome.


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Cell_Biology_Alberts. Figure 7–51 Photoreceptor cells in the retina of a female mouse showing patterns of X-chromosome inactivation. Using genetic engineering techniques (described in Chapter 8), the germ line of a mouse was modified so that one copy of the X chromosome (if active) makes a green fluorescent protein and the other a red fluorescent protein. Both proteins concentrate in the nucleus and, in the field of cells shown here, it is clear that only one of the two X chromosomes is active in each cell. (from h. wu et al., Neuron 81:103–119, 2014. with permission from elsevier.) distributed in small clusters in the adult animal because sister cells tend to remain close together during later stages of development (Figure 7–51). For example, X-chromosome inactivation causes the orange and black “tortoiseshell” coat coloration of some female cats. In these cats, one X chromosome carries a gene that produces orange hair color, and the other X chromosome carries an allele of the same gene that results in


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Cell_Biology_Alberts. coat coloration of some female cats. In these cats, one X chromosome carries a gene that produces orange hair color, and the other X chromosome carries an allele of the same gene that results in black hair color; it is the random X-inactivation that produces patches of cells of two distinctive colors. In contrast, male cats of this genetic stock are either solid orange or solid black, depending on which X chromosome they inherit from their mothers. Although X-chromosome inactivation is maintained over thousands of cell divisions, it is reversed during germ-cell formation, so that all haploid oocytes contain an active X chromosome and can express X-linked gene products.


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Cell_Biology_Alberts. How is an entire chromosome transcriptionally inactivated? X-chromosome inactivation is initiated and spreads from a single site near the middle of the X chromosome, the X-inactivation center (XIC). Within the XIC is a transcribed 20,000-nucleotide lncRNA (called Xist), which is expressed solely from the inactive X chromosome. Xist RNA spreads from the XIC over the entire chromosome and directs gene silencing. Although we do not know exactly how this is accomplished, it likely involves recruitment of histone-modifying enzymes and other proteins to form a repressive form of chromatin analogous to that of Figure 7–45. Curiously, about 10% of the genes on the X chromosome (including Xist itself) escape this silencing and remain active.


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Cell_Biology_Alberts. The spread of Xist RNA along the X chromosome does not proceed linearly along the DNA. Rather, starting at its site of synthesis, it is first handed off across the base of the DNA loops that make up the chromosome; these shortcuts explain how Xist can spread rapidly, by a “hand-over-hand” mechanism, along the X chromosome once the inactivation process begins (Figure 7–52). It also helps to explain why the inactivation does not spread to the other, active X chromosome.


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Cell_Biology_Alberts. Imprinting and X-chromosome inactivation are examples of monoallelic gene expression, where in a diploid genome, only one of the two copies of a gene is expressed. In addition to the approximately 1000 genes on the X chromosome and the 300 or so genes that are imprinted, there are another 1000–2000 human genes that exhibit monoallelic expression. Like X-chromosome inactivation (but unlike imprinting), the choice of which copy of the gene is to be expressed and which is to be silenced often appears random. Yet once the choice is made, it can persist for many cell divisions. Because the choice is often made relatively late in development, cells of the same tissue in the same individual can express different copies of a given gene. In other words, somatic tissues are often mosaics, where different clones of cells have subtly different patterns of gene expression. The mechanisms responsible for this type of monoallelic expression are not known in detail, and its general purpose—if any—is


Cell_Biology_Alberts

Cell_Biology_Alberts. clones of cells have subtly different patterns of gene expression. The mechanisms responsible for this type of monoallelic expression are not known in detail, and its general purpose—if any—is poorly understood. Several different mechanisms may contribute to such epigenetic inheritance, as we explain next.


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Cell_Biology_Alberts. epigenetic Mechanisms ensure that Stable patterns of Gene expression Can Be transmitted to Daughter Cells


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Cell_Biology_Alberts. As we have seen, once a cell in an organism differentiates into a particular cell type, it generally remains specialized in that way; if it divides, its daughters inherit the same specialized character. Perhaps the simplest way for a cell to remember transcription of Xist RNA Xist RNA spreads Xist RNA binds histone-modifying from one X chromosome “hand-over-hand” and continues to spread its identity is through a positive feedback loop in which a key transcription regulator activates, either directly or indirectly, the transcription of its own gene (see Figure 7–39). Interlocking positive feedback loops of the type shown in Figure 7–37 provide greater stability by buffering the circuit against fluctuations in the level of any one transcription regulator. Because transcription regulators are synthesized in the cytosol and diffuse throughout the nucleus, feedback loops based on this mechanism will affect both copies of a gene in a diploid cell. However, as discussed in this section, the


Cell_Biology_Alberts

Cell_Biology_Alberts. synthesized in the cytosol and diffuse throughout the nucleus, feedback loops based on this mechanism will affect both copies of a gene in a diploid cell. However, as discussed in this section, the expression pattern of a gene on one chromosome can differ from the copy of the same gene on the other chromosome (as in X-chromosome inactivation or in imprinting), and such differences can also be inherited through many cell divisions.


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Cell_Biology_Alberts. The ability of a daughter cell to retain a memory of the gene expression patterns that were present in the parent cell is an example of epigenetic inheritance: a heritable alteration in a cell or organism’s phenotype that does not result from changes in the nucleotide sequence of DNA (discussed in Chapter 4). (Unfortunately, the term epigenetic is sometimes also used to refer to all covalent modifications to histones and DNA, whether or not they are self-propagating; many of these modifications are erased each time a cell divides and do not generate cell memory.) In Figure 7–53, we contrast two self-propagating epigenetic mechanisms that work in cis, affecting only one chromosomal copy with two self-propagating mechanisms that work in trans, affecting both chromosomal copies of a gene. Cells can combine these mechanisms to ensure that patterns of gene expression are maintained and inherited accurately and reliably—over a period of up to a hundred years or more, in our own case.


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Cell_Biology_Alberts. We can get some idea of the prevalence of epigenetic changes by comparing identical twins. Their genomes have the same sequence of nucleotides, and, obviously, many features of identical twins—such as their appearance—are strongly determined by the genome sequences they inherit. When their gene expression, histone modification, and DNA methylation patterns are compared, however, many differences are observed. Because these differences are roughly correlated not only with age but also with the time that the twins have spent apart from each other, it has been proposed that some of these differences are heritable from cell to cell and are the result of environmental factors. Although these studies are in early stages, the idea that environmental events can be permanently registered as epigenetic changes in our cells is a fascinating one that presents an important challenge to the next generation of biological scientists.


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Cell_Biology_Alberts. Figure 7–52 Mammalian X-chromosome inactivation. X-chromosome inactivation begins with the synthesis of Xist (X-inactivation specific transcript) rnA from the XiC (X-inactivation center) locus and moves outward to the chromosome ends. According to the model depicted here, the long (≈20,000 nucleotides) Xist rnA has many low-affinity binding sites for the structural components of chromosomes and spreads by releasing its hold on one portion of the chromosome while grasping another. the continued synthesis of Xist from the center of the chromosome drives it to the ends. As shown, Xist rnA does not move linearly along the chromosomal DnA, but, instead, moves first across the base of chromosome loops. it has been proposed that the portions of chromosomal DnA at the tips of long loops contain the 10% of genes that escape X-chromosome inactivation.


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Cell_Biology_Alberts. Eukaryotic cells can use inherited forms of DNA methylation and inherited states of chromatin condensation as additional mechanisms for generating cell memory of gene expression patterns. An especially dramatic case that involves chromatin condensation is the inactivation of an entire X chromosome in female mammals. DNA methylation underlies the phenomenon in mammals of genomic imprinting, in which the expression of a gene depends on whether it was inherited from the mother or the father.


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Cell_Biology_Alberts. In principle, every step required for the process of gene expression can be controlled. Indeed, one can find examples of each type of regulation, and many genes are regulated by multiple mechanisms. As we have seen, controls on the initiation of gene transcription are a critical form of regulation for all genes. But other controls can act later in the pathway from DNA to protein to modulate the amount of gene product that is made—and in some cases, to determine the exact amino acid sequence of the protein product. These post-transcriptional controls, which operate after RNA polymerase has bound to the gene’s promoter and has begun RNA synthesis, are crucial for the regulation of many genes. In the following sections, we consider the varieties of post-transcriptional regulation in temporal order, according to the sequence of events that an RNA molecule might experience after its transcription has begun (Figure 7–54).


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Cell_Biology_Alberts. Figure 7–53 Four distinct mechanisms that can produce an epigenetic form of inheritance in an organism. (A) epigenetic mechanisms that act in cis. As discussed in this chapter, a maintenance methylase can propagate specific patterns of cytosine methylation (see figure 7–44). As discussed in Chapter 4, a histone modifying enzyme that replicates the same modification that attracts it to chromatin can result in the modification being self-propagating (see figure 4–44). (B) epigenetic mechanisms that act in trans. positive feedback loops, formed by transcriptional regulators are found in all species and are probably the most common form of cell memory. As discussed in Chapter 3, some proteins can form self-propagating prions (figure 3–33). if these proteins are involved in gene expression, they can transmit patterns of gene expression to daughter cells.


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Cell_Biology_Alberts. Figure 7–54 Post-transcriptional controls of gene expression. the final synthesis rate of a protein can, in principle, be controlled at any of the steps listed in capital letters. in addition, rnA splicing, rnA editing, and translation recoding can also alter the sequence of amino acids in a protein, making it possible for the cell to produce more than one protein variant from the same gene. Only a few of the steps depicted here are likely to be critical for the regulation of any one particular protein. transcription Attenuation Causes the premature termination of Some rnA Molecules


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Cell_Biology_Alberts. transcription Attenuation Causes the premature termination of Some rnA Molecules It has long been known that the expression of some genes is inhibited by premature termination of transcription, a phenomenon called transcription attenuation. In some of these cases, the nascent RNA chain adopts a structure that causes it to interact with the RNA polymerase in such a way as to abort its transcription. When the gene product is required, regulatory proteins bind to the nascent RNA chain and remove the attenuation, allowing the transcription of a complete RNA molecule.


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Cell_Biology_Alberts. A well-studied example of transcription attenuation occurs during the life cycle of HIV, the human immunodeficiency virus that is the causative agent of acquired immune deficiency syndrome, or AIDS. Once the HIV genome has been integrated into the host genome, the viral DNA is transcribed by the cell’s RNA polymerase II (see Figure 5–62). However, this polymerase usually terminates transcription after synthesizing transcripts of several hundred nucleotides and therefore fails to efficiently transcribe the entire viral genome. When conditions for viral growth are optimal, a virus-encoded protein called Tat, which binds to a specific stem-loop structure in the nascent RNA that contains a “bulged base,” prevents this premature termination (see Figure 6–89). Once bound to this specific RNA structure (called TAR), Tat assembles several host-cell proteins that allow the RNA polymerase to continue transcribing. The normal role of at least some of these proteins is to prevent pausing and


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Cell_Biology_Alberts. structure (called TAR), Tat assembles several host-cell proteins that allow the RNA polymerase to continue transcribing. The normal role of at least some of these proteins is to prevent pausing and premature termination by RNA polymerase when it transcribes normal cell genes. Thus, a normal cell mechanism has apparently been highjacked by HIV to permit transcription of its genome to be controlled by a single viral protein.


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Cell_Biology_Alberts. riboswitches probably represent Ancient forms of Gene Control In Chapter 6, we discussed the idea that, before modern cells arose on Earth, RNA played the role of both DNA and proteins, both storing hereditary information and catalyzing chemical reactions (see pp. 362–366). The discovery of riboswitches shows that RNA can also form control devices. Riboswitches are short sequences of RNA that change their conformation on binding small molecules, such as metabolites. Each riboswitch recognizes a specific small molecule and the resulting conformational change is used to regulate gene expression. Riboswitches are often located near the 5ʹ end of mRNAs, and they fold while the mRNA is being synthesized, blocking or permitting progress of the RNA polymerase according to whether the regulatory small molecule is bound (Figure 7–55).


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Cell_Biology_Alberts. Riboswitches are particularly common in bacteria, in which they sense key small metabolites in the cell and adjust gene expression accordingly. Perhaps their most remarkable feature is the high specificity and affinity with which each recognizes only the appropriate small molecule; in many cases, every chemical feature of the small molecule is read by the RNA (Figure 7–55C). Moreover, the binding affinities observed are as tight as those typically observed between small molecules and proteins.


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Cell_Biology_Alberts. Figure 7–55 A riboswitch that responds to guanine. (A) in this example from bacteria, the riboswitch controls expression of the purine biosynthetic genes. when guanine levels in cells are low, an elongating rnA polymerase transcribes the purine biosynthetic genes, and the enzymes needed for guanine synthesis are therefore expressed. (B) when guanine is abundant, it binds the riboswitch, causing it to undergo a conformational change that forces the rnA polymerase to terminate transcription (see figure 6–11). (C) Guanine (red) bound to the riboswitch. Only those nucleotides that form the guanine-binding pocket are shown. Many other riboswitches exist, including those that recognize S-adenosylmethionine, coenzyme B12, flavin mononucleotide, adenine, lysine, and glycine. (Adapted from M. Mandal and r.r. Breaker, Nat. Rev. Mol. Cell Biol. 5:451–463, 2004. with permission from Macmillan publishers ltd; and C.K. vanderpool and S. Gottesman, Mol. Microbiol. 54:1076–1089, 2004. with permission


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Cell_Biology_Alberts. and r.r. Breaker, Nat. Rev. Mol. Cell Biol. 5:451–463, 2004. with permission from Macmillan publishers ltd; and C.K. vanderpool and S. Gottesman, Mol. Microbiol. 54:1076–1089, 2004. with permission from Blackwell publishing.)


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Cell_Biology_Alberts. Figure 7–56 Five patterns of alternative RNA splicing. in each case, a single type of rnA transcript is spliced in two alternative ways to produce two distinct mrnAs (1 and 2). the dark blue boxes mark exon sequences 1 that are retained in both mrnAs. the light blue boxes mark possible exon 2 sequences that are included in only one of the mrnAs. the boxes are joined by red lines to indicate where intron sequences (yellow) are removed. (Adapted from h. Keren et al. Nat. Rev. Genet. 11:345–355, 2010. with permission from Macmillan publishers ltd.)


Cell_Biology_Alberts

Cell_Biology_Alberts. Riboswitches are perhaps the most economical examples of gene control devices, inasmuch as they bypass the need for regulatory proteins altogether. In the example shown in Figure 7–55, the riboswitch controls transcription elongation, but they can also regulate other steps in gene expression, as we shall see later 1 in this chapter. Clearly, highly sophisticated gene control devices can be made from short sequences of RNA, a fact that supports the hypothesis of an early “RNA world.” 1 Alternative rnA Splicing Can produce Different forms of a protein 2 from the Same Gene


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Cell_Biology_Alberts. As discussed in Chapter 6 (see Figure 6–26), RNA splicing shortens the transcripts of many eukaryotic genes by removing the intron sequences from the mRNA pre-1 cursor. We also saw that a cell can splice an RNA transcript differently and thereby 2 make different polypeptide chains from the same gene—a process called alternative RNA splicing (Figure 7–56). A substantial proportion of animal genes (estimated at 90% in humans) produce multiple proteins in this way.


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Cell_Biology_Alberts. When different splicing possibilities exist at several positions in the transcript, a single gene can produce dozens of different proteins. In one extreme case, a Drosophila gene may produce as many as 38,000 different proteins from a single gene through alternative splicing (Figure 7–57), although only a fraction of these forms have thus far been experimentally observed. Considering that the Drosophila genome has approximately 14,000 identified genes, it is clear that the protein complexity of an organism can greatly exceed the number of its genes. This example also illustrates the perils in equating gene number with an organism’s complexity. For example, alternative splicing is rare in single-celled budding yeasts one out of 38,016 possible splicing patterns


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Cell_Biology_Alberts. Figure 7–57 Alternative splicing of RNA transcripts of the Drosophila Dscam gene. DSCAM proteins have several different functions. in cells of the fly immune system, they mediate the phagocytosis of bacterial pathogens. in cells of the nervous system, DSCAM proteins are needed for proper wiring of neurons. the final mrnA contains 24 exons, four of which (denoted A, B, C, and D) are present in the Dscam gene as arrays of alternative exons. each rnA contains 1 of 12 alternatives for exon A (red), 1 of 48 alternatives for exon B (green), 1 of 33 alternatives for exon C (blue), and 1 of 2 alternatives for exon D (yellow). this figure shows only one of the many possible splicing patterns (indicated by the red line and by the mature mrnA below it). each variant DSCAM protein would fold into roughly the same structure (predominantly a series of extracellular immunoglobulin-like domains linked to a membrane-spanning region; see figure 24–48), but the amino acid sequence of the domains vary


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Cell_Biology_Alberts. the same structure (predominantly a series of extracellular immunoglobulin-like domains linked to a membrane-spanning region; see figure 24–48), but the amino acid sequence of the domains vary according to the splicing pattern. the diversity of DSCAM variants contributes to the plasticity of the immune system as well as the formation of complex neural circuits; we take up the specific role of the DSCAM variants in more detail when we describe the development of the nervous system in Chapter 21. (Adapted from D.l. Black, Cell 103:367–370, 2000. with permission from elsevier.) but very common in flies. Budding yeast has ≈6200 genes, only about 300 of which are subject to splicing, and nearly all of these have only a single intron. To say that flies have only 2–3 times as many genes as yeasts greatly underestimates the difference in complexity of these two genomes.


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Cell_Biology_Alberts. In some cases, alternative RNA splicing occurs because there is an intron sequence ambiguity: the standard spliceosome mechanism for removing intron sequences (discussed in Chapter 6) is unable to distinguish clearly between two or more alternative pairings of 5ʹ and 3ʹ splice sites, so that different choices are made by chance on different individual transcripts. Where such constitutive alternative splicing occurs, several versions of the protein encoded by the gene are made in all cells in which the gene is expressed.


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Cell_Biology_Alberts. In many cases, however, alternative RNA splicing is regulated. In the simplest examples, regulated splicing is used to switch from the production of a nonfunctional protein to the production of a functional one (or the other way around). The transposase that catalyzes the transposition of the Drosophila P element, for example, is produced in a functional form in germ cells and a nonfunctional form in somatic cells of the fly, allowing the P element to spread throughout the genome of the fly without causing damage in somatic cells (see Figure 5–61). The difference in transposon activity has been traced to the presence of an intron sequence in the transposase RNA that is removed only in germ cells.


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Cell_Biology_Alberts. In addition to enabling switching from the production of a functional protein to the production of a nonfunctional one (or vice versa), the regulation of RNA splicing can generate different versions of a protein in different cell types, according to the needs of the cell. Tropomyosin, for example, is produced in specialized forms in different types of cells (see Figure 6–26). Cell-type-specific forms of many other proteins are produced in the same way. RNA splicing can be regulated either negatively, by a regulatory molecule that prevents the splicing machinery from gaining access to a particular splice site on the RNA, or positively, by a regulatory molecule that helps direct the splicing machinery to an otherwise overlooked splice site (Figure 7–58).


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Cell_Biology_Alberts. Because of the plasticity of RNA splicing, the blocking of a “strong” splicing site will often expose a “weak” site and result in a different pattern of splicing. Thus, the splicing of a pre-mRNA molecule can be thought of as a delicate balance between competing splice sites—a balance that can easily be tipped by effects on splicing of regulatory proteins. the Definition of a Gene has Been Modified Since the Discovery of Alternative rnA Splicing The discovery that eukaryotic genes usually contain introns and that their coding sequences can be assembled in more than one way raised new questions about the definition of a gene. A gene was first clearly defined in molecular terms in the early 1940s from work on the biochemical genetics of the fungus Neurospora. Figure 7–58 Negative and positive control of alternative RNA splicing.


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Cell_Biology_Alberts. Figure 7–58 Negative and positive control of alternative RNA splicing. in negative control, a repressor protein binds to a specific sequence in the premrnA transcript and blocks access of the splicing machinery to a splice junction. this often results in the use of a secondary splice site, thereby producing an altered pattern of splicing (see figure 7–56). in positive control, the splicing machinery is unable to remove a particular intron sequence efficiently without assistance from an activator protein. Because rnA is flexible, the nucleotide sequences that bind these activators can be located many nucleotide pairs from the splice junctions they control, and they are often called splicing enhancers, by analogy with the transcriptional enhancers mentioned earlier in this chapter.


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Cell_Biology_Alberts. Until then, a gene had been defined operationally as a region of the genome that segregates as a single unit during meiosis and gives rise to a definable phenotypic trait, such as a red or a white eye in Drosophila or a round or wrinkled seed in peas. The work on Neurospora showed that most genes correspond to a region of the genome that directs the synthesis of a single enzyme. This led to the hypothesis that one gene encodes one polypeptide chain. The hypothesis proved fruitful for subsequent research; as more was learned about the mechanism of gene expression in the 1960s, a gene became identified as that stretch of DNA that was transcribed into the RNA coding for a single polypeptide chain (or a single structural RNA such as a tRNA or an rRNA molecule). The discovery of split genes and introns in the late 1970s could be readily accommodated by the original definition of a gene, provided that a single polypeptide chain was specified by the RNA transcribed from any one DNA sequence.


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Cell_Biology_Alberts. introns in the late 1970s could be readily accommodated by the original definition of a gene, provided that a single polypeptide chain was specified by the RNA transcribed from any one DNA sequence. But it is now clear that many DNA sequences in higher eukaryotic cells can produce a set of distinct (but related) proteins by means of alternative RNA splicing. How, then, is a gene to be defined?


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Cell_Biology_Alberts. In those relatively rare cases in which a single transcription unit produces two very different eukaryotic proteins, the two proteins are considered to be produced by distinct genes that overlap on the chromosome. It seems unnecessarily complex, however, to consider most of the protein variants produced by alternative RNA splicing as being derived from overlapping genes. A more sensible alternative is to modify the original definition to count a DNA sequence that is transcribed as a single unit and encodes one set of closely related polypeptide chains (protein isoforms) as a single protein-coding gene. This definition also accommodates those DNA sequences that encode protein variants produced by post-transcriptional processes other than RNA splicing, such as transcript cleavage and RNA editing (discussed below). A Change in the Site of rnA transcript Cleavage and poly-A Addition Can Change the C-terminus of a protein


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Cell_Biology_Alberts. We saw in Chapter 6 that the 3ʹ end of a eukaryotic mRNA molecule is not formed by the termination of RNA synthesis by the RNA polymerase, as it is in bacteria. Instead, it results from an RNA cleavage reaction that is catalyzed by additional proteins while the transcript is elongating (see Figure 6–34). A cell can control the site of this cleavage so as to change the C-terminus of the resultant protein. In the simplest cases, one protein variant is simply a truncated version of the other; in many other cases, however, the alternative cleavage and polyadenylation sites lie within intron sequences and the pattern of splicing is thereby altered. This process can produce two closely related proteins differing only in the amino acid sequences at their C-terminal ends. Close analysis of RNAs produced from the human genome in a variety of cell types (see Figure 7–3) indicate that as many as 50% of human protein-coding genes produce mRNA species that differ at their site of polyadenylation.


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Cell_Biology_Alberts. A well-studied example of regulated polyadenylation is the switch from the synthesis of membrane-bound to secreted antibody molecules that occurs during the development of B lymphocytes (see Figure 24–22). Early in the life history of a B lymphocyte, the antibody it produces is anchored in the plasma membrane, where it serves as a receptor for antigen. Antigen stimulation causes B lymphocytes to multiply and to begin secreting their antibody. The secreted form of the antibody is identical to the membrane-bound form except at the extreme C-terminus. In this part of the protein, the membrane-bound form has a long string of hydrophobic amino acids that traverses the lipid bilayer of the membrane, whereas the secreted form has a much shorter string of hydrophilic amino acids. The switch from membrane-bound to secreted antibody is generated through a change in the site of RNA cleavage and polyadenylation, as shown in Figure 7–59.


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Cell_Biology_Alberts. The change is caused by an increase in the concentration of a subunit of a protein (CstF) that promotes RNA cleavage (see Figure 6–34). The first cleavage/ poly-A addition site that a transcribing RNA polymerase encounters is suboptimal and is usually skipped in unstimulated B lymphocytes, leading to production of the longer RNA transcript. When activated to produce antibodies, the B lymphocyte increases its CstF concentration; as a result, cleavage now occurs at the suboptimal site, and the shorter transcript is produced. In this way, a change in concentration of a general RNA-processing factor has a dramatic effect on the expression of a particular gene. rnA editing Can Change the Meaning of the rnA Message


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Cell_Biology_Alberts. rnA editing Can Change the Meaning of the rnA Message The molecular mechanisms used by cells are a continual source of surprises. An example is the process of RNA editing, which alters the nucleotide sequences of RNA transcripts once they are synthesized and thereby changes the coded message they carry. We saw in Chapter 6 that tRNA and rRNA molecules are chemically modified after they are synthesized: here we focus on changes to mRNAs.


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Cell_Biology_Alberts. In animals, two principal types of mRNA editing occur: the deamination of adenine to produce inosine (A-to-I editing) and, less frequently, the deamination of cytosine to produce uracil (C-to-U editing), as shown in Figure 5–43. Because these chemical modifications alter the pairing properties of the bases (I pairs with C, and U pairs with A), they can have profound effects on the meaning of the RNA. If the edit occurs in a coding region, it can change the amino acid sequence of the protein or produce a truncated protein by creating a premature stop codon. Edits that occur outside coding sequences can affect the pattern of pre-mRNA splicing, the transport of mRNA from the nucleus to the cytosol, the efficiency with which the RNA is translated, or the base-pairing between microRNAs (miRNAs) and their mRNA targets, a form of regulation that will be discussed later in the chapter.


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Cell_Biology_Alberts. The process of A-to-I editing is particularly prevalent in humans, where it occurs in approximately 1000 genes. Enzymes called ADARs (adenosine deaminases acting on RNA) perform this type of editing; these enzymes recognize a double-stranded RNA structure that is formed through base-pairing between the site to be edited and a complementary sequence located elsewhere on the same RNA molecule, typically in an intron (Figure 7–60). The structure of the double-stranded RNA specifies whether the mRNA is to be edited, and if so, where the edit should be made. An especially important example of A-to-I editing takes place in the mRNA that codes for a transmitter-gated ion channel in the brain. A single edit changes a glutamine to an arginine; the affected amino acid lies on the inner wall of the channel, and the editing change alters the Ca2+ permeability of the channel. Mutant mice that cannot make this edit are prone to epileptic seizures and die during or shortly after weaning, showing


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Cell_Biology_Alberts. channel, and the editing change alters the Ca2+ permeability of the channel. Mutant mice that cannot make this edit are prone to epileptic seizures and die during or shortly after weaning, showing that editing of the ion channel RNA is normally crucial for proper brain development.


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Cell_Biology_Alberts. C-to-U editing, which is carried out by a different set of enzymes, is also crucial in mammals. For example, in certain cells of the gut, the mRNA for apolipoprotein B undergoes a C-to-U edit that creates a premature stop codon and therefore


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Cell_Biology_Alberts. Figure 7–59 Regulation of the site of RNA cleavage and poly-A addition determines whether an antibody molecule is secreted or remains membrane-bound. in unstimulated B lymphocytes (left), a long rnA transcript is produced, and the intron sequence (yellow) near its 3ʹ end is removed by rnA splicing to provide an mrnA molecule that codes for a membrane-bound antibody molecule. Only a portion of the antibody gene is shown in the figure; the actual gene and its mrnA would extend further to the left of the diagram. After antigen stimulation (right), the rnA transcript is cleaved and polyadenylated upstream from the intron’s 3′ splice site. As a result, some of the intron sequence remains as a coding sequence in the short transcript and specifies the hydrophilic C-terminal portion of the secreted antibody molecule (brown). (Adapted from D. Di Giammartino et al., Mol. Cell 43:853–866, 2011. with permission from elsevier.)


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Cell_Biology_Alberts. Figure 7–60 Mechanism of A-to-I RNA editing in mammals. typically, a sequence complementary to the position of the edit is present in an intron, and the resulting double-stranded rnA structure attracts an A-to-i editing enzyme (ADAr). in the case illustrated, the edit is made in an exon; in most cases, however, this occurs in noncoding portions of the mrnA. editing by ADAr takes place in the nucleus, before the pre-mrnA has been fully processed. Mice and humans have two ADAr genes: ADR1 is expressed in many tissues and is required in the liver for proper red blood cell development; ADR2 is expressed only in the brain, where it is required for proper brain development.


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Cell_Biology_Alberts. no editing editing, CAA UAA produces a shorter form of the protein. In cells of the liver, the editing enzyme is not expressed, and the full-length apolipoprotein B is produced. The two protein isoforms have different properties, and each plays a role in lipid metabolism that is specific to the organ that produces it (Figure 7–61).


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Cell_Biology_Alberts. Why RNA editing exists at all is a mystery. One idea is that it arose in evolution to correct “mistakes” in the genome. Another is that it arose as a somewhat slapdash way for the cell to produce subtly different proteins from the same gene. A third possibility is that RNA editing originally evolved as a defense mechanism against retroviruses and retrotransposons and was later adapted by the cell to change the meanings of certain mRNAs. Indeed, RNA editing still plays important roles in cell defense. Some retroviruses, including HIV, are extensively edited after they infect cells. This hyperediting creates many harmful mutations in the viral RNA genome and also causes viral mRNAs to be retained in the nucleus, where they are eventually degraded. Although some modern retroviruses protect themselves against this defense mechanism, RNA editing presumably helps to hold many viruses in check. rnA transport from the nucleus Can Be regulated


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Cell_Biology_Alberts. rnA transport from the nucleus Can Be regulated It has been estimated that in mammals only about one-twentieth of the total mass of RNA synthesized ever leaves the nucleus. We saw in Chapter 6 that most mammalian RNA molecules undergo extensive processing and that the “leftover” RNA fragments (excised introns and RNA sequences 3ʹ to the cleavage/poly-A site) are degraded in the nucleus. Incompletely processed and otherwise damaged RNAs are also eventually degraded as part of the quality control system for RNA production. As described in Chapter 6, the export of RNA molecules from the nucleus is delayed until processing has been completed. However, mechanisms that deliberately override this control point can be used to regulate gene expression. This strategy forms the basis for one of the best-understood examples of regulated nuclear transport of mRNA, which occurs in the human AIDS virus, HIV.


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Cell_Biology_Alberts. As we saw in Chapter 5, HIV, once inside the cell, directs the formation of a double-stranded DNA copy of its genome, which is then inserted into the genome of the host (see Figure 5–62). Once inserted, the viral DNA can be transcribed as one long RNA molecule by the host cell’s RNA polymerase II. This transcript is then spliced in many different ways to produce over 30 different species of mRNA, which in turn are translated into a variety of different proteins (Figure 7–62). In order to make progeny virus, entire, unspliced viral transcripts must be exported from the nucleus to the cytosol, where they are packaged into viral capsids and serve as the viral genome. This large transcript, as well as alternatively spliced HIV mRNAs that the virus needs to move to the cytoplasm for protein synthesis, still carries complete introns. The host cell’s normal block to the nuclear export of unspliced RNAs therefore presents a special problem for HIV.


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Cell_Biology_Alberts. Figure 7–61 C-to-U RNA editing produces a truncated form of apolipoprotein B. integrated into host genome1,000 nucleotide pairs The block is overcome in an ingenious way. The virus encodes a protein (called Rev) that binds to a specific RNA sequence (called the Rev responsive element, RRE) located within a viral intron. The Rev protein interacts with a nuclear export receptor (Crm1), which directs the movement of viral RNAs through nuclear pores into the cytosol despite the presence of intron sequences. We discuss in detail the way in which export receptors function in Chapter 12.


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Cell_Biology_Alberts. The regulation of nuclear export by Rev has several important consequences for HIV growth and pathogenesis. In addition to ensuring the nuclear export of specific unspliced RNAs, it divides the viral infection into an early phase (in which Rev is translated from a fully spliced RNA and all of the intron-containing viral RNAs are retained in the nucleus and degraded) and a late phase (in which unspliced RNAs are exported due to Rev function). This timing helps the virus replicate by providing the gene products in roughly the order in which they are needed (Figure 7–63). Regulation by Rev and by Tat, the HIV protein that counteracts premature transcription termination (see p. 414), allows the virus to achieve latency, a condition in which the HIV genome has become integrated into the host-cell genome but the production of viral proteins has temporarily ceased.


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Cell_Biology_Alberts. Figure 7–62 The compact genome of HIV, the human AIDS virus. the positions of the nine hiv genes are shown in green. the red double line indicates a DnA copy of the viral genome that has become integrated into the host DnA (gray). note that the coding regions of many genes overlap, and that those of Tat and Rev are split by introns. the blue line at the bottom of the figure represents the pre-mrnA transcript of the viral DnA and shows the locations of all the possible splice sites (arrows). there are many alternative ways of splicing the viral transcript; for example, the Env mrnAs retain the intron that has been spliced out of the Tat and Rev mrnAs. the rev response element (rre) is indicated by a blue ball and stick. it is a 234-nucleotide-long stretch of rnA that folds into a defined structure; rev recognizes a particular hairpin within this larger structure.


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Cell_Biology_Alberts. the Gag gene codes for a protein that is cleaved into several smaller proteins that form the viral capsid. the Pol gene codes for a protein that is cleaved to produce reverse transcriptase (which transcribes rnA into DnA), as well as the integrase involved in integrating the viral genome (as double-stranded DnA) into the host genome. the Env gene codes for the envelope proteins (see figure 5–62). tat, rev, vif, vpr, vpu, and nef are small proteins with a variety of functions. for example, rev regulates nuclear export (see figure 7–63) and tat regulates the elongation of transcription across the integrated viral genome (see p. 414).


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Cell_Biology_Alberts. Figure 7–63 Regulation of nuclear export by the HIV Rev protein. (A) early in hiv infection, only the fully spliced rnAs (which contain the coding sequences for rev, tat, and nef) are exported from the nucleus and translated. (B) Once sufficient rev protein has accumulated and been transported into the nucleus, unspliced viral rnAs can be exported from the nucleus. Many of these rnAs are translated into protein, and the full-length transcripts are packaged into new viral particles. If, after its initial entry into a host cell, conditions become unfavorable for viral transcription and replication, Rev and Tat are made at levels too low to promote transcription and export of unspliced RNA. This situation stalls the viral growth cycle until conditions improve, whereupon Rev and Tat levels increase, and the virus enters the replication cycle. Some mrnAs Are localized to Specific regions of the Cytosol


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Cell_Biology_Alberts. Once a newly made eukaryotic mRNA molecule has passed through a nuclear pore and entered the cytosol, it is typically met by ribosomes, which translate it into a polypeptide chain (see Figure 6–8). Once the first round of translation “passes” the nonsense-mediated decay test (see Figure 6–76), the mRNA is usually translated in earnest. If the mRNA encodes a protein that is destined to be secreted or expressed on the cell surface, a signal sequence at the protein’s N-terminus will direct it to the endoplasmic reticulum (ER). In this case, as discussed in Chapter 12, components of the cell’s protein-sorting apparatus recognize the signal sequence as soon as it emerges from the ribosome and direct the entire complex of ribosome, mRNA, and nascent protein to the membrane of the ER, where the remainder of the polypeptide chain is synthesized. In other cases, free ribosomes in the cytosol synthesize the entire protein, and signals in the completed polypeptide chain may then direct the


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Cell_Biology_Alberts. the remainder of the polypeptide chain is synthesized. In other cases, free ribosomes in the cytosol synthesize the entire protein, and signals in the completed polypeptide chain may then direct the protein to other sites in the cell.


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Cell_Biology_Alberts. Many mRNAs are themselves directed to specific intracellular locations before their efficient translation begins, allowing the cell to position its mRNAs close to the sites where the encoded protein is needed. RNA localization has been observed in many organisms, including unicellular fungi, plants, and animals, and it is likely to be a common mechanism that cells use to concentrate high-level production of proteins at specific sites. This strategy also provides the cell with other advantages. For example, it allows the establishment of asymmetries in the cytosol of the cell, a key step in many stages of development. Localized mRNA, coupled with translational control, also allows the cell to regulate gene expression independently in different regions. This feature is particularly important in large, highly polarized cells such as neurons, where it plays a central role in synaptic function.


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Cell_Biology_Alberts. Several mechanisms for mRNA localization have been discovered (Figure 7–64), all of which require specific signals in the mRNA itself. These signals are usually concentrated in the 3ʹ untranslated region (UTR), the region of RNA that


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Cell_Biology_Alberts. Figure 7–64 Mechanisms for the localization of mRNAs. the mrnA to be localized leaves the nucleus through nuclear pores (top). Some localized mrnAs (left diagram) travel to their destination by associating with cytoskeletal motors, which use the energy of Atp hydrolysis to move the mrnAs unidirectionally along filaments in the cytoskeleton (red) (see Chapter 16). At their destination, the mrnAs are held in place by anchor proteins (black). Other mrnAs randomly diffuse through the cytosol and are simply trapped by anchor proteins and at their sites of localization (center diagram). Some mrnAs (right diagram) are degraded in the cytosol unless they have bound, through random diffusion, a localized protein complex that anchors and protects the mrnA from degradation (black). each mechanism requires specific signals on the mrnA, which are typically located in the 3ʹ Utr. Additional components can block the translation of the mrnA until it is properly localized. (Adapted from h.D. lipshitz


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Cell_Biology_Alberts. specific signals on the mrnA, which are typically located in the 3ʹ Utr. Additional components can block the translation of the mrnA until it is properly localized. (Adapted from h.D. lipshitz and C.A. Smibert, Curr. Opin. Genet. Dev. 10:476–488, 2000. with permission from elsevier.)


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Cell_Biology_Alberts. Figure 7–65 An experiment demonstrating the importance of the 3ʹ UTR in localizing mRNAs to specific regions of the cytoplasm. for this experiment, two different fluorescently labeled rnAs were prepared by transcribing DnA in vitro in the presence of fluorescently labeled derivatives of Utp. One rnA (labeled with a red fluorochrome) contains the coding region for the Drosophila hairy protein and includes the adjacent 3ʹ Utr (see figure 6–21). the other rnA (labeled green) contains the hairy coding region with the 3ʹ Utr deleted. the two rnAs were mixed and injected into a Drosophila embryo at a stage of development when multiple nuclei reside in a common cytoplasm (see figure 7–26). when the fluorescent rnAs were visualized 10 minutes later, the full-length hairy rnA (red) was localized to the apical side of nuclei (blue) but the transcript missing the 3ʹ Utr (green) failed to localize. hairy is one of many transcriptional regulators that specify positional information in the


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Cell_Biology_Alberts. to the apical side of nuclei (blue) but the transcript missing the 3ʹ Utr (green) failed to localize. hairy is one of many transcriptional regulators that specify positional information in the developing Drosophila embryo (discussed in Chapter 21), and the localization of its mrnA (shown in this experiment to depend on its 3ʹ Utr) is critical for proper fly development. (Courtesy of Simon Bullock and David ish-horowicz.) extends from the stop codon that terminates protein synthesis to the start of the poly-A tail (Figure 7–65). The mRNA localization is usually coupled with translational controls to ensure that the mRNA remains quiescent until it has been moved into place.


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Cell_Biology_Alberts. The Drosophila egg exhibits an especially striking example of mRNA localization. The mRNA encoding the Bicoid transcription regulator is localized by attachment to the cytoskeleton at the anterior tip of the developing egg. When fertilization triggers the translation of this mRNA, it generates a gradient of the Bicoid protein that plays a crucial part in directing the development of the anterior part of the embryo (see Figure 7–26). Many mRNAs in somatic cells are also localized in a similar way. The mRNA that encodes actin, for example, is localized to the actin-filament-rich cell cortex in mammalian fibroblasts by means of a 3ʹ UTR signal.


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Cell_Biology_Alberts. We saw in Chapter 6 that mRNA molecules exit from the nucleus bearing numerous markings in the form of RNA modifications (the 5ʹ cap and the 3ʹ poly-A tail) and bound proteins (exon-junction complexes, for example) that signify the successful completion of the different pre-mRNA processing steps. As just described, the 3ʹ UTR of an mRNA can be thought of as a “zip code” that directs mRNAs to different places in the cell. Below, we will also see that mRNAs carry information specifying their average lifetime in the cytosol and the efficiency with which they are translated into protein. In a broad sense, the untranslated regions of eukaryotic mRNAs resemble the transcriptional control regions of genes: their nucleotide sequences contain information specifying the way the RNA is to be used, and proteins interpret this information by binding specifically to these sequences. Thus, over and above the specification of the amino acid sequences of proteins, mRNA molecules are rich with


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Cell_Biology_Alberts. used, and proteins interpret this information by binding specifically to these sequences. Thus, over and above the specification of the amino acid sequences of proteins, mRNA molecules are rich with information.


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Cell_Biology_Alberts. the 5ʹ and 3ʹ Untranslated regions of mrnAs Control their translation Once an mRNA has been synthesized, one of the most common ways of regulating the levels of its protein product is to control the step that initiates translation. Even though the details of translation initiation differ between eukaryotes and bacteria (as we saw in Chapter 6), they each use some of the same basic regulatory strategies. In bacterial mRNAs, a conserved stretch of nucleotides, the Shine–Dalgarno sequence, is always found a few nucleotides upstream of the initiating AUG codon. In bacteria, translational control mechanisms are carried out by proteins or by RNA molecules, and they generally involve either exposing or blocking the Shine– Dalgarno sequence (Figure 7–66).


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Cell_Biology_Alberts. Eukaryotic mRNAs do not contain such a sequence. Instead, as discussed in Chapter 6, the selection of an AUG codon as a translation start site is largely determined by its proximity to the cap at the 5ʹ end of the mRNA molecule, which is the site at which the small ribosomal subunit binds to the mRNA and begins scanning for an initiating AUG codon. In eukaryotes, translational repressors can bind to the 5ʹ end of the mRNA and thereby inhibit translation initiation. Other repressors recognize nucleotide sequences in the 3ʹ UTR of specific mRNAs and decrease translation initiation by interfering with the communication between the 5ʹ cap and 3ʹ poly-A tail, a step required for efficient translation (see Figure 6–70). A particularly important type of translational control in eukaryotes relies on small RNAs (termed microRNAs or miRNAs) that bind to mRNAs and reduce protein output, as described later in this chapter.


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Cell_Biology_Alberts. the phosphorylation of an initiation factor regulates protein Synthesis Globally Eukaryotic cells decrease their overall rate of protein synthesis in response to a variety of situations, including deprivation of growth factors or nutrients, infection by viruses, and sudden increases in temperature. Much of this decrease is caused by the phosphorylation of the translation initiation factor eIF2 by specific protein kinases that respond to the changes in conditions.


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Cell_Biology_Alberts. The normal function of eIF2 was outlined in Chapter 6. It forms a complex with GTP and mediates the binding of the methionyl initiator tRNA to the small ribosomal subunit, which then binds to the 5ʹ end of the mRNA and begins scanning along the mRNA. When an AUG codon is recognized, the eIF2 protein hydrolyzes the bound GTP to GDP, causing a conformational change in the protein and releasing it from the small ribosomal subunit. The large ribosomal subunit then joins the small one to form a complete ribosome that begins protein synthesis. Figure 7–66 Mechanisms of translational control. Although these examples are from bacteria, many of the same principles operate in eukaryotes.


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Cell_Biology_Alberts. (A) Sequence-specific rnA-binding proteins repress translation of specific mrnAs by blocking access of the ribosome to the Shine–Dalgarno sequence (orange). for example, some ribosomal proteins repress translation of their own rnA. this mechanism allows the cell to maintain correctly balanced quantities of the various components needed to form ribosomes. (B) An rnA “thermosensor” permits efficient translation initiation only at elevated temperatures at which the stem-loop structure has been melted. An example occurs in the human pathogen Listeria monocytogenes, in which the translation of its virulence genes increases at 37°C, the temperature of the host. (C) Binding of a small molecule to a riboswitch causes a major rearrangement of the rnA forming a different set of stem-loop structures. in the bound structure, the Shine–Dalgarno sequence (orange) is sequestered and translation initiation is thereby blocked. in many bacteria, S-adenosylmethionine acts in this manner to block


Cell_Biology_Alberts

Cell_Biology_Alberts. in the bound structure, the Shine–Dalgarno sequence (orange) is sequestered and translation initiation is thereby blocked. in many bacteria, S-adenosylmethionine acts in this manner to block production of the enzymes that synthesize it. (D) An “antisense” rnA produced elsewhere from the genome base-pairs with a specific mrnA and blocks its translation. Many bacteria regulate expression of iron-storage proteins in this way.


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Cell_Biology_Alberts. Because eIF2 binds very tightly to GDP, a guanine nucleotide exchange factor (see p. 157), designated eIF2B, is required to cause GDP release so that a new GTP molecule can bind and eIF2 can be reused (Figure 7–67A). The reuse of eIF2 is inhibited when it is phosphorylated—the phosphorylated eIF2 binds to eIF2B unusually tightly, inactivating eIF2B. There is more eIF2 than eIF2B in cells, and even a fraction of phosphorylated eIF2 can trap nearly all of the eIF2B. This prevents the reuse of the nonphosphorylated eIF2 and greatly slows protein synthesis (Figure 7–67B). Regulation of the level of active eIF2 is especially important in mammalian cells; eIF2 is part of the mechanism that allows cells to enter a nonproliferating, resting state (called G0) in which the rate of total protein synthesis is reduced to about one-fifth the rate in proliferating cells. initiation at AUG Codons Upstream of the translation Start Can regulate eukaryotic translation initiation


Cell_Biology_Alberts

Cell_Biology_Alberts. We saw in Chapter 6 that eukaryotic translation typically begins at the first AUG downstream of the 5ʹend of the mRNA, which is the first AUG encountered by a scanning small ribosomal subunit. But the nucleotides immediately surrounding the AUG also influence the efficiency of translation initiation. If the recognition site is poor enough, scanning ribosomal subunits will sometimes ignore the first AUG codon in the mRNA and skip to the second or third AUG codon instead. This phenomenon, known as “leaky scanning,” is a strategy frequently used to produce two or more closely related proteins, differing only in their N-termini, from the same mRNA. A particularly important use of this mechanism is the production of the same protein with and without a signal sequence attached at its N-terminus. This allows the protein to be directed to two different locations in the cell (for example, to both mitochondria and the cytosol). Cells can regulate the relative abundance of the protein isoforms


Cell_Biology_Alberts

Cell_Biology_Alberts. This allows the protein to be directed to two different locations in the cell (for example, to both mitochondria and the cytosol). Cells can regulate the relative abundance of the protein isoforms produced by leaky scanning; for example, a cell-type-specific increase in the abundance of the initiation factor eIF4F favors the use of the AUG closest to the 5ʹ end of the mRNA.


Cell_Biology_Alberts

Cell_Biology_Alberts. Another type of control found in eukaryotes uses one or more short open reading frames—short stretches of DNA that begin with a start codon (ATG) and end with a stop codon, with no stop codons in between—that lie between the 5ʹend of the mRNA and the beginning of the gene. Often, the amino acid sequences coded by these upstream open reading frames (uORFs) are not important; rather, the uORFs serve a purely regulatory function. An uORF present on an mRNA molecule will generally decrease translation of the downstream gene by trapping a guanine nucleotide exchange factor, eIF2B IN ABSENCE OF ACTIVE eIF2B, THE REMAINING UNBOUND eIF2 REMAINS IN ITS Figure 7–67 The eIF2 cycle. (A) the INACTIVE, GDP-recycling of used eif2 by a guanine BOUND FORM nucleotide exchange factor (eif2B). (B) eif2 (B) INACTIVE COMPLEX rates by tying up eif2B.


Cell_Biology_Alberts

Cell_Biology_Alberts. scanning ribosome initiation complex and causing the ribosome to translate the uORF and dissociate from the mRNA before it reaches the bona fide protein-coding sequence. When the activity of a general translation factor (such as the eIF2 discussed above) is reduced, one might expect that the translation of all mRNAs would be reduced equally. Contrary to this expectation, however, the phosphorylation of eIF2 can have selective effects, even enhancing the translation of specific mRNAs that contain uORFs. This can enable cells, for example, to adapt to starvation for specific nutrients by shutting down the synthesis of all proteins except those that are required for synthesis of the missing nutrients. The details of this mechanism have been worked out for a specific yeast mRNA that encodes a protein called Gcn4, a transcription regulator that activates many genes that encode proteins that are important for amino acid synthesis.


Cell_Biology_Alberts

Cell_Biology_Alberts. The Gcn4 mRNA contains several short uORFs, and when amino acids are abundant, ribosomes translate the uORFs and generally dissociate before they reach the Gcn4 coding region. A global decrease in eIF2 activity brought about by amino acid starvation makes it more likely that a scanning small ribosomal subunit will move across the uORFs (without translating them) before it acquires a molecule of eIF2 (see Figure 6–70). Such a ribosomal subunit is then free to initiate translation on the actual Gcn4 sequences. The increased level of this transcription regulator increases production of the amino acid biosynthetic enzymes.


Cell_Biology_Alberts

Cell_Biology_Alberts. Although approximately 90% of eukaryotic mRNAs are translated beginning with the first AUG downstream from the 5ʹ cap, certain AUGs, as we saw in the previous section, can be skipped over during the scanning process. In this section, we discuss yet another way that cells can initiate translation at positions distant from the 5ʹ end of the mRNA, using a specialized type of RNA sequence called an internal ribosome entry site (IRES). In some cases, two distinct protein-coding sequences are carried in tandem on the same eukaryotic mRNA; translation of the first occurs by the usual scanning mechanism, and translation of the second occurs through an IRES. IRESs are typically several hundred nucleotides in length and fold into specific structures that bind many, but not all, of the same proteins that are used to initiate normal 5ʹ cap-dependent translation (Figure 7–68). In fact, different IRESs require different subsets of initiation factors. However, all of them bypass the need for a 5ʹ


Cell_Biology_Alberts

Cell_Biology_Alberts. that are used to initiate normal 5ʹ cap-dependent translation (Figure 7–68). In fact, different IRESs require different subsets of initiation factors. However, all of them bypass the need for a 5ʹ cap structure and the translation initiation factor that recognizes it, eIF4E.


Cell_Biology_Alberts

Cell_Biology_Alberts. Figure 7–68 Two mechanisms of translation initiation. (A) the normal, cap-dependent mechanism requires a set of initiation factors whose assembly on the mrnA is stimulated by the presence of a 5ʹ cap and a poly-A tail (see also figure 6–70). (B) the ireS-dependent mechanism, seen mainly in viruses, requires only a subset of the normal translation initiating factors, and these assemble directly on the folded ireS. (Adapted from A. Sachs, Cell 101:243–245, 2000. with permission from elsevier.)


Cell_Biology_Alberts

Cell_Biology_Alberts. Some viruses use IRESs as part of a strategy to get their own mRNA molecules translated while blocking normal 5ʹ cap-dependent translation of host mRNAs. On infection, these viruses produce a protease (encoded in the viral genome) that cleaves the host-cell translation factor eIF4G, rendering it unable to bind to eIF4E, the cap-binding complex. This shuts down most of the host cell’s translation and effectively diverts the translation machinery to the IRES sequences present on the viral mRNAs. (The truncated eIF4G remains competent to initiate translation at these internal sites.) The many ways in which viruses manipulate their host’s protein-synthesis machinery for their own advantage continue to surprise cell biologists. Studying this “arms race” between humans and pathogens has led to many fundamental insights into the workings of the cell, and we revisit this topic in more detail in Chapter 23.


Cell_Biology_Alberts

Cell_Biology_Alberts. Most mRNAs in a bacterial cell are very unstable, having half-lives of less than a couple of minutes. Exonucleases, which degrade in the 3ʹ-to-5ʹ direction, are usually responsible for the rapid destruction of these mRNAs. Because its mRNAs are both rapidly synthesized and rapidly degraded, a bacterium can adapt quickly to environmental changes. As a general rule, the mRNAs in eukaryotic cells are more stable. Some, such as that encoding β globin, have half-lives of more than 10 hours, but most have considerably shorter half-lives, typically less than 30 minutes. The mRNAs that code for proteins such as growth factors and transcription regulators, whose production rates need to change rapidly in cells, have especially short half-lives.


Cell_Biology_Alberts

Cell_Biology_Alberts. We saw in Chapter 6 that the cell has several mechanisms that rapidly destroy incorrectly processed RNAs; here, we consider the fate of the typical “normal” eukaryotic mRNA. Two general mechanisms exist for eventually destroying each mRNA that is made by the cell. Both begin with the gradual shortening of the poly-A tail by an exonuclease, a process that starts as soon as the mRNA reaches the cytosol. In a broad sense, this poly-A shortening acts as a timer that counts down the lifetime of each mRNA. Once the poly-A tail is reduced to a critical length (about 25 nucleotides in humans), the two pathways diverge. In one, the 5ʹ cap is removed (a process called decapping) and the “exposed” mRNA is rapidly degraded from its 5ʹ end. In the other, the mRNA continues to be degraded from the 3ʹ end, through the poly-A tail, into the coding sequences (Figure 7–69).


Cell_Biology_Alberts

Cell_Biology_Alberts. Nearly all mRNAs are subject to both types of decay, and the specific sequences of each mRNA determine how fast each step occurs and therefore how long each mRNA will persist in the cell and be able to produce protein. The 3ʹ UTR sequences are especially important in controlling mRNA lifetimes, and they often carry binding sites for specific proteins that increase or decrease the rate of poly-A shortening, decapping, or 3ʹ-to-5ʹ degradation. The half-life of an mRNA is also affected by how efficiently it is translated. Poly-A shortening and decapping compete directly with the machinery that translates the mRNA; therefore, any factors that affect the translation efficiency of an mRNA will tend to have the opposite effect on its degradation (Figure 7–70). Although poly-A shortening controls the half-life of most eukaryotic mRNAs, some mRNAs can be degraded by a specialized mechanism that bypasses this step altogether. In these cases, specific nucleases cleave the mRNA internally,


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Cell_Biology_Alberts. Figure 7–69 Two mechanisms of eukaryotic mRNA decay. A critical threshold of poly-A tail length induces rapid 3ʹ-to-5ʹ degradation, which may be triggered by the loss of the poly-Abinding proteins. As shown in figure 7–70, a deadenylase associates with both the 3ʹ poly-A tail and the 5ʹ cap, and this connection may be involved in signaling decapping after poly-A shortening. Although 5ʹ-to-3ʹ and 3ʹ-to-5ʹ degradation are shown here on separate rnA molecules, these two processes can occur together on the same molecule. (Adapted from C.A. Beelman and r. parker, Cell 81:179–183, 1995. with permission from elsevier.)


Cell_Biology_Alberts

Cell_Biology_Alberts. Figure 7–70 The competition between effectively decapping one end and removing the poly-A tail from the other so that both halves are rapidly degraded. The mRNAs that are destroyed in this way carry specific nucleotide sequences, often in the 3ʹ UTRs, that serve as recognition sequences for these endonucleases. This strategy makes it especially simple to tightly regulate the stability of these mRNAs by blocking or exposing the endonuclease site in response to extracellular signals. For example, the addition of iron to cells decreases the stability of the mRNA that encodes the receptor protein that binds the iron-transporting protein transferrin, causing less of this receptor to be made. This effect is mediated by the iron-sensitive RNA-binding protein aconitase. Aconitase can bind to the 3ʹ UTR of the transferrin receptor mRNA and increase receptor production by blocking endonucleolytic cleavage of the mRNA. On the addition of iron, aconitase is released from the mRNA, exposing the


Cell_Biology_Alberts

Cell_Biology_Alberts. 3ʹ UTR of the transferrin receptor mRNA and increase receptor production by blocking endonucleolytic cleavage of the mRNA. On the addition of iron, aconitase is released from the mRNA, exposing the cleavage site and thereby decreasing the stability of the mRNA (Figure 7–71).


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Cell_Biology_Alberts. regulation of mrnA Stability involves p-bodies and Stress Granules We saw in Chapters 3 and 6 that large aggregates of proteins and nucleic acids that work together are often held in proximity by loose, low-affinity connections (see Figure 3–36). In this way, they function as “organelles” even though they are not surrounded by membranes. Many of the events discussed in the previous mRNA translation and mRNA decay. the same two features of an mrnA molecule—its 5ʹ cap and the 3ʹ poly-A decay (see figure 7–69). the deadenylase that shortens the poly-A tail in the 3ʹ-to5ʹ direction associates with the 5ʹ cap. As described in Chapter 6 (see figure 6–70), the translation initiation machinery also associates with both the 5ʹ cap and the poly-A tail. (Adapted from M. Gao et al., Mol. Cell 5:479–488, 2000. with permission from elsevier.)


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Cell_Biology_Alberts. Figure 7–71 Two post-translational controls mediated by iron. (A) During iron starvation, the binding of aconitase to the 5ʹ Utr of the ferritin mrnA blocks translation initiation; its binding to the 3ʹ Utr of the transferrin receptor mrnA blocks an endonuclease cleavage site and thereby stabilizes the mrnA.


Cell_Biology_Alberts

Cell_Biology_Alberts. (B) in response to an increase in iron concentration in the cytosol, a cell increases its synthesis of ferritin in order to bind the extra iron and decreases its synthesis of transferrin receptors in order to import less iron across the plasma membrane. Both responses are mediated by the same iron-responsive regulatory protein, aconitase, which recognizes common features in a stem-loop structure in the mrnAs encoding ferritin and the transferrin receptor. Aconitase dissociates from the mrnA when it binds iron. But because the transferrin receptor and ferritin are regulated by different types of mechanisms, their levels respond oppositely to iron concentrations even though they are regulated by the same iron-responsive regulatory protein. section—including decapping and RNA degradation—take place in aggregates known as Processingor P-bodies, which are present in the cytosol (Figure 7–72).


Cell_Biology_Alberts

Cell_Biology_Alberts. Although many mRNAs are eventually degraded in P-bodies, some remain intact and are later returned to the pool of translating mRNAs. To be “rescued” in this way, mRNAs move from P-bodies to another type of aggregate known as a stress granule, which contains translation initiation factors, poly-A-binding protein, and small ribosomal subunits. Translation itself does not occur in stress granules, but mRNAs can become “translation-ready” as the proteins bound to them in P-bodies are replaced with those in stress granules. The movement of mRNAs between active translation, P-bodies, and stress granules can be seen as an mRNA cycle (Figure 7–73) where the competition between translation and mRNA degradation is carefully controlled. Thus, when translation initiation is blocked (by starvation, drugs, or genetic manipulation), stress granules enlarge as more and more nontranslated mRNAs are moved directly into them for storage. Clearly, once a cell has made the large investment in producing a


Cell_Biology_Alberts

Cell_Biology_Alberts. or genetic manipulation), stress granules enlarge as more and more nontranslated mRNAs are moved directly into them for storage. Clearly, once a cell has made the large investment in producing a properly processed mRNA molecule, it carefully controls its subsequent fate.


Cell_Biology_Alberts

Cell_Biology_Alberts. Many steps in the pathway from RNA to protein are regulated by cells in order to control gene expression. Most genes are regulated at multiple levels, in addition to being controlled at the initiation stage of transcription. The regulatory mechanisms include (1) attenuation of the RNA transcript by its premature termination, (2) alternative RNA splice-site selection, (3) control of 3ʹ-end formation by cleavage and poly-A addition, (4) RNA editing, (5) control of transport from the nucleus to the cytosol, (6) localization of mRNAs to particular parts of the cell, (7) control of translation initiation, and (8) regulated mRNA degradation. Most of these control processes require the recognition of specific sequences or structures in the RNA molecule being regulated, a task performed by either regulatory proteins or regulatory RNA molecules. Figure 7–72 Visualization of P-bodies.


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Cell_Biology_Alberts. Figure 7–72 Visualization of P-bodies. human cells were stained with antibodies to a component of the mrnA decapping enzyme Dcp1a (left panels) and to the Argonaute protein (middle panels). As described later in this chapter, Argonaute is a key component of rnA interference pathways. the merged image (right panels) shows that the two proteins co-localize to p-bodies in the cytoplasm. (Adapted from J. liu et al., Nat. Cell Biol. 7:719–723, 2005. with permission from Macmillan publishers ltd.) Figure 7–73 Possible fates of an mRNA molecule. An mrnA molecule released from the nucleus can be actively translated (center), stored in stress granules (right), or degraded in p-bodies (left). As the needs of the cell change, mrnAs can be shuffled from one pool to the next, as indicated by the arrows.


Cell_Biology_Alberts

Cell_Biology_Alberts. In the previous chapter, we introduced the central dogma, according to which the flow of genetic information proceeds from DNA through RNA to protein (Figure 6–1). But we have seen throughout this book that RNA molecules perform many critical tasks in the cell besides serving as intermediate carriers of genetic information. Among these noncoding RNAs are the rRNA and tRNA molecules, which are responsible for reading the genetic code and synthesizing proteins. The RNA molecule in telomerase serves as a template for the replication of chromosome ends, snoRNAs modify ribosomal RNA, and snRNAs carry out the major events of RNA splicing. And we saw in the previous section that Xist RNA has an important role in inactivating one copy of the X chromosome in females.


Cell_Biology_Alberts

Cell_Biology_Alberts. A series of recent discoveries has revealed that noncoding RNAs are even more prevalent than previously imagined. We now know that such RNAs play widespread roles in regulating gene expression and in protecting the genome from viruses and transposable elements. These newly discovered RNAs are the subject of this section.


Cell_Biology_Alberts

Cell_Biology_Alberts. We begin our discussion with a group of short RNAs that carry out RNA interference or RNAi. Here, short single-stranded RNAs (20–30 nucleotides) serve as guide RNAs that selectively reorganize and bind—through base-pairing—other RNAs in the cell. When the target is a mature mRNA, the small noncoding RNAs can inhibit its translation or even catalyze its destruction. If the target RNA molecule is in the process of being transcribed, the small noncoding RNA can bind to it and direct the formation of certain types of repressive chromatin on its attached DNA template (Figure 7–74). Three classes of small noncoding RNAs work in this way—microRNAs (miRNAs), small interfering RNAs (siRNAs), and piwi-interacting RNAs (piRNAs)—and we discuss them in turn in the next sections. Although they differ in the way the short pieces of single-stranded RNA are generated, all three types of short RNAs locate their targets through RNA–RNA base-pairing, and they generally cause reductions in gene


Cell_Biology_Alberts

Cell_Biology_Alberts. differ in the way the short pieces of single-stranded RNA are generated, all three types of short RNAs locate their targets through RNA–RNA base-pairing, and they generally cause reductions in gene expression.


Cell_Biology_Alberts

Cell_Biology_Alberts. Over 1000 different microRNAs (miRNAs) are produced from the human genome, and these appear to regulate at least one-third of all human protein-coding genes. Once made, miRNAs base-pair with specific mRNAs and fine-tune their translation and stability. The miRNA precursors are synthesized by RNA polymerase II and are capped and polyadenylated. They then undergo a special type of processing, after which the miRNA (typically 23 nucleotides in length) is assembled with a set of proteins to form an RNA-induced silencing complex or RISC. Once formed, the RISC seeks out its target mRNAs by searching for complementary nucleotide translational repression formation of heterochromatin cleavage of of target RNA is being transcribed


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Cell_Biology_Alberts. Figure 7–74 RNA interference in eukaryotes. Single-stranded interfering rnAs are generated from double-stranded rnA. they locate target rnAs through base-pairing and, at this point, several fates are possible, as shown. As described in the text, there are several types of rnA interference; the way the double-stranded rnA is produced and processed and the ultimate fate of the target rnA depends on the particular system. sequences (Figure 7–75). This search is greatly facilitated by the Argonaute protein, a component of RISC, which holds the 5ʹ region of the miRNA so that it is optimally positioned for base-pairing to another RNA molecule (Figure 7–76). In animals, the extent of base-pairing is typically at least seven nucleotide pairs, and this pairing most often occurs in the 3ʹ UTR of the target mRNA.


Cell_Biology_Alberts

Cell_Biology_Alberts. Once an mRNA has been bound by an miRNA, several outcomes are possible. If the base-pairing is extensive (which is unusual in humans but common in many plants), the mRNA is cleaved (sliced) by the Argonaute protein, effectively removing the mRNA’s poly-A tail and exposing it to exonucleases (see Figure 7–69). Following cleavage of the mRNA, the RISC with its associated miRNA is released, and it can seek out additional mRNAs (see Figure 7–75). Thus, a single miRNA can act catalytically to destroy many complementary mRNAs. These miRNAs can be thus thought of as guide sequences that bring destructive nucleases into contact with specific mRNAs.


Cell_Biology_Alberts

Cell_Biology_Alberts. If the base-pairing between the miRNA and the mRNA is less extensive (as observed for most human miRNAs), Argonaute does not slice the mRNA; rather, translation of the mRNA is repressed and the mRNA is shuttled to P-bodies (see Figure 7–73) where, sequestered from ribosomes, it eventually undergoes poly-A tail shortening, decapping, and degradation.


Cell_Biology_Alberts

Cell_Biology_Alberts. Several features make miRNAs especially useful regulators of gene expression. First, a single miRNA can regulate a whole set of different mRNAs, so long as the mRNAs carry a common short sequence in their UTRs. This situation is common in humans, where a single miRNA can control hundreds of different mRNAs. Second, regulation by miRNAs can be combinatorial. When the base-pairing between the miRNA and mRNA fails to trigger cleavage, additional miRNAs binding to the same mRNA lead to further reductions in its translation. As discussed earlier for transcription regulators, combinatorial control greatly expands the possibilities available to the cell by linking gene expression to a combination of different regulators rather than a single regulator. Third, an miRNA occupies relatively little


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Cell_Biology_Alberts. Figure 7–75 miRNA processing and mechanism of action. the precursor mirnA, through complementarity between one part of its sequence and another, forms a double-stranded structure. this rnA is cropped while still in the nucleus and then exported to the cytosol, where it is further cleaved by the Dicer enzyme to form the mirnA proper. Argonaute, in conjunction with other components of riSC, initially associates with both strands of the mirnA and then cleaves and discards one of them. the other strand guides riSC to specific mrnAs through base-pairing. if the rnA–rnA match is extensive, as is commonly seen in plants, Argonaute cleaves the target mrnA, causing its rapid degradation. in mammals, the mirnA–mrnA match often does not extend beyond a short seven-nucleotide “seed” region near the 5ʹ end of the mirnA. this less extensive base-pairing leads to inhibition of translation, mrnA destabilization, and transfer of the mrnA to p-bodies, where it is eventually degraded.


Cell_Biology_Alberts

Cell_Biology_Alberts. 3˜ OH (end of RNA) active site, showing 2 Mg2+ atoms needed for “slicing” space in the genome when compared with a protein. Indeed, their small size is one reason that miRNAs were discovered only recently. Although we are only beginning to appreciate the full impact of miRNAs, it is clear that they represent an important part of the cell’s equipment for regulating the expression of genes. We discuss specific examples of miRNAs that have key roles in development in Chapter 21. rnA interference is Also Used as a Cell Defense Mechanism


Cell_Biology_Alberts

Cell_Biology_Alberts. rnA interference is Also Used as a Cell Defense Mechanism Many of the proteins that participate in the miRNA regulatory mechanisms just described also serve a second function as a defense mechanism: they orchestrate the degradation of foreign RNA molecules, specifically those that occur in double-stranded form. Many transposable elements and viruses produce double-stranded RNA, at least transiently, in their life cycles, and RNA interference helps to keep these potentially dangerous invaders in check. As we shall see, this form of RNAi also provides scientists with a powerful experimental technique to turn off the expression of individual genes.