Saturday, 22 December 2012


The three small RNA silencing pathways in flies are the small interfering RNA (siRNA), microRNA (miRNA) and Piwi-interacting RNA (piRNA) pathways. These pathways differ in their substrates, biogenesis, effector proteins and modes of target regulation. a | dsRNA precursors are processed by Dicer-2 (DCR-2) to generate siRNA duplexes containing guide and passenger strands. DCR-2 and the dsRNA-binding protein R2D2 (which together form the RISC-loading complex, RLC) load the duplex into Argonaute2 (AGO2). A subset of endogenous siRNAs (endo-siRNAs) exhibits dependence on dsRNA-binding protein Loquacious (LOQS), rather than on R2D2. The passenger strand is later destroyed and the guide strand directs AGO2 to the target RNA. b | miRNAs are encoded in the genome and are transcribed to yield a primary miRNA (pri-miRNA) transcript, which is cleaved by Drosha to yield a short precursor miRNA (pre-miRNA). Alternatively, miRNAs can be present in introns (termed mirtrons) that are liberated following splicing to yield authentic pre-miRNAs. pre-miRNAs are exported from the nucleus to the cytoplasm, where they are further processed by DCR-1 to generate a duplex containing two strands, miRNA and miRNA*. Once loaded into AGO1, the miRNA strand guides translational repression of target RNAs. c | piRNAs are thought to derive from ssRNA precursors and are made without a dicing step. piRNAs are mostly antisense, but a small fraction is in the sense orientation. Antisense piRNAs are preferentially loaded into Piwi or Aubergine (AUB), whereas sense piRNAs associate with AGO3. The methyltransferase HEN1 adds the 2'-O-methyl modification at the 3' end. Piwi and AUB collaborate with AGO3 to mediate an interdependent amplification cycle that generates additional piRNAs, preserving the bias towards antisense. The antisense piRNAs probably direct cleavage of transposon mRNA or chromatin modification at transposon loci. SAH, S-adenosyl homocysteine; SAM, S-adenosyl methionine.

miRNAa regulates development in vertebrates.  lin-4 the first miRNA from C elegans was discovered as an endogenous regulator of genes. It regulates developmental timing.  miRNAs regulate endogenous genes. siRNAs defend genome integrity in response to foreign or invasive nucleic acids eg viruses, transposons, transgenes.

Differences. miRNAs were seen as endogenous products of organism's own genome.  siRNAs thought to be exogenous derived from viruses etc. miRNAs processed from stem loop precursors with incomplete ds. soRNAs are excised from long fully complementary dsRNAs. Both depend on Dicer and Ago.

Dicer has a DEXD/H ATPase domain, a DUF283 domain, a PAZ domain, 2 tandem RNase III domains and a dsRNA-binding domain (dsRBD).  Some organisms use one Dicer. Some use multiple Dicer proteins. D. melanogaster Dicer-1 is required for miRNA biogenesis. Dicer-2 is used in siRNA pathway.

PAZ and RNAse III domains help excise siRNAs preferentially from ends of dsRNA. Dicer shares PAZ with Ago proteins. They are specialised to bind RNA ends esp duplex ends with 2nt 3'overhangs.  End engages Dicer PAZ domain. Substrate dsRNA extends by 2 helical turns on protein surface until it reaches processing centre. The centre is in the active site of RNas III domains. It cleaves one of 2 strands of 20-30nt miRNA/siRNA duplex from its precursor.  New ends have staggered 2nt 3' overhangs.  This leaves a 5' phosphate on product ends.

ATP promotes dsRNA processing by Drosophila Dicer 2 and D elegans Dicer 1.  Mutations that affect ATPase activity in Drosophila Dicer 2 abolishes dsRNA processing.

Ago consists of Piwi clade that binds piRNAs, Ago clade binds mi and siRNAs and a 3rd clade in nematodes.  

ds Dicer products enter RISC assembly pathway  Duplex unwinds. 1 of 2 strands associates stably with Ago effector protein. Guide strand directs target recognition by Watson-Crick bping. Passenger strand is discarded.

Ago proteins have 4 domains: PAZ domain, PIWI domain (unique to Ago superfamily) and N and Mid domains. Ago PAZ domain can bind RNA 3' terminus of guide strand.  Other end of guide strand binds 5'phosphate binding pocket in Mid domain.  Remainder of guide tracks along +vely charged surface.  Protein-DNa contacts mainly sugar-phosphate backbone interactions.  Guide strands nts2-6 are important for target recognition. Their WC bases are exposed and available for bping.

Most species have multiple Ago proteins.

Canonical RNAi inducer is long, linear and perfectly bped dsRNA.  It is introduced into cytoplasm or from environment. Dicer processes them into siRNAs. Transgene and virused induced silencing in plants. Centromeres, transposons and repetitive sequences can also cause siRNAs.

siRNAs can also originate from endogenous genomic loci. They differ from many exogenous siRNAs.

RISC assembly
siRISC assembly in Drosophila is nucleated by R2D2/Diver-2 heterodimer. It binds a siRNA duplez.  Factors form RISC-loading complex (RLC).  RLC assembles into pre-RISC. siRNA is still duplex.  This requires Ago2.  Ago2 cleaves passenger strand. Passenger strand is ejected. Assembly becomes holo-RISC.

In humans, 3 proteins associate. Diver, TRBP, Ago-2.  Even without dsRNA trigger.

LRC can bind dsRNA, dice into siRNA, load siRNA into Ago2 and discard passenger strand. This makes functional RISC.

Strand selection is dictated by relative thermodynamic stabilities of 2 duplex ends.  Strand with 5' terminus at less stably base paired end will be guide strand.

Posttranscriptional silencing by siRNAs
siRNA guide strand directs RISC to complementary RNA targets. They are degraded.  PIWI domain of Ago induces RNA degradation  Phosphodiester bonds between nts bped to siRNas residues 10 an 11 are cut.  Exonucleases attack fragments. New 3'end of RISC cleavage products is oligouridylated. This promotes exonucleolytic targeting. Target dissociates from siRNA after cleave. RISC is freed to cleave more targets.

Mismatches near or at centre of siRNA/target duplex suppress endonucleolytic cleavage. Some siRNA-programmed Ago proteins lack endonuclease activity even with perfectly paired targets. Partially mismatched targets or targets recognised by endonuclease-inactive siRISCs are silenced by translational repression or exonucleolytic degradation similar to miRNA silencing.

Silencing machinery is broadly distributed in cell.

RNAi is potent. A few dsRNA molecules can induce robust response.  

In C elegans, primary dsRNA trigger induces synthesis of secondary siRNAs through RNA-dependent RNA pol. RDRP. Thy amplify and sustain response. In plants and nematodes they cause systemic silencing that spreads throughout organism.  RdRP-genes are in genomes are RNA-competent eukaryotes except insects and mammals. This causes transitive RNAi. siRNAs corresponding to regions of mRNA not targeted by initial dsRNA trigger appear. Lack of transitive RNAi in insects and vertebrates increases specificity. Can target individual alternately spliced mRNA isoforms from common locus.

Secondary siRNas in worms correspond to antisense strand of mRNA targeted by primary dsRNA trigger.

siRNAs can induce heterochromatin formation
In S. pombe. It is transcriptional gene silencing (TGS). Ago1-containing effector in fission yeast is RITS complex. It is guided to spec chromosomal loci eg centromeric repeats by its bound siRNAs. siRNA recognises nascent transcripts. It is facilitated by direct interactions between RITS and RNAPII.  RITS association promotes H3 methylation by histone methyltransferases. This recruits Swi6 and chromatin compaction. Engaging nascent transcripts activates RNA-dependent RNAP complex (RDRC)  It uses its RdRP subunit (Rdp1) to generate 2dary siRNas.  

In plants DNA is directly methylated by DNA methyltransferases (DMTs).

miRNA are 19-25 ss RNAs in plants and animals. It an be encoded in independent transcriptional units, in polycistronic clusters or in introns of protein-coding genes. It regulates translation of over 60% protein coding genes.

Transcription of miRNA is done by RNAPII. Transcripts are capped and polyadenylated. A transcript can encode clusters of distinct miRNAs or a miRNA and protein. In the latter the miRNA sequence is in an intron.

Resulting primary or pri-miRNA transcript extends 5' and 3' from miRNA sequence. Processings trim transcript into mature miRNA.  mirNA folds into a stem loop.  First processing step occurs in nucleus. pri-miRNA stem loop is cleaved in nucleus by RNase III enzyme Dosha. This releases shorter (65nt) precursor miRNA (pre-miRNA) hairpin.

pre-miRNA is exported to cytoplasm by exportin 5. Dicer processes pre-miRNA. This excises 19-25nt dsRNA. Temrinal loop is excised from pre-miRNA stem. Duplex is incorported into miRISC. Mature miRNA strand is preferentially retained. miRISC contains miRNA, Ago protein and other protein effectors.
In plants Dcl1 carries it out in nucleus. In animals pre-miRNA is exported from nucleus. Dicer cleaves in cytoplasm.

miRNA differs from siRNA in end precision.   miRNA has exact ends.  siRNA are more heterogenous.  miRNAs interact specifically with substrate mRNAs.  

Mature miRNA duplex is short-lived. When it associates with Ago it is rapidly unwound. One strand is retained. The other is lost.  5' terminus of retained strand is at less stably base paired end of duplex.

miRISC is directed to mRNAs complementary to its miRNA component.

miRNA specifically recognises and regulate particular mRNA.  miRNA binding sites in animal mRNAs lie in 3' UTR. They are in multiple copies. Most animal miRNAs bind with mismatches and bulges.  miRNA nt2-8 WC bps helping recognition.  Most plant miRNAs bind perfect complementarity.

Degree of miRNA-RNA may determine regulatory mechanism. Perfect complementarity allows Ago-catalysed cleavage of mRNA. Central mismatches exclude cleavage and promote repression of mRNA translation. It is shown that tramslational repression is default mechanism of miRNA. Perfect complementarity may also engage in mRNA cleavage.

Animal miRNAs bind with imperfect complementarity to targets. Causes mismatches  Complementarity at 3' end of cognate miRNA compensates for imperfect matching.

Overexpressing single miRNAs can decrease levels of more than 100 mRNAs.

Deleting Dicer 1 and Dgcr8 causes early developmental arrest in mice. Defects in proliferation of pluripotent stem cells.  

miRNAs in early embryonic development (see Ason)
In Xenopus laevis, miR-15 and 16 have roles in translating early β-catenin dorsal ventral gradient into late blastula gradient of Nodal activity 

miRNAs in neuronal development
miRNA can specify and maintain neuronal cell type identity. miRNa has roles in neuronal cell differentiation. miR-124 which is specific to neuronal tissue acquires and maintaihs neuronal cell identity by silencing target mrNAs.

It is expressed spec in mouse brain and P19 pluripotent cells on differentiation to neuronlike cells.

One target is polypyrimide tract binding protein (PTB). PTB regulates alternative splicing that inhibits inclusion of alternative cassette exons. During neuronal differentiation switch between expression of PTB and nPTB( neuronal PTB) a homologous neuron spec protein coded by a separate gene cause changes in splicing pattern of genes involved in neuronal functions.  PTB alters splicing of nTPB by repressing inclusion of alternative exon. mir-124 activates expression of nPTB by inhibiting PTB.

In mouse embryos, PtV is expressed in areas where nondifferentiated progenitor cells are present. nPTB and mir-124 are expressed in differentiated neurons. Distribution of exon-including isoforms of genes regulated by PTB and/or nPTB overlapped with miR-124.  Hence mir 1245 antagonises PTB. In mice with Dicer-null mutation, pattern of expression of splicing isoforms of PTB target genes was perturbed. This shows miRNAs regulate splicing.

During muscle development muscle spec mir133 inhibits nPTB expression in nucleus. This changes splicing pattern of genes regulated by nPTB.

miR-124 affects REST, a reglator of neuron spec gene expression. REST is a TF. It repressed extra-neuronal transcription of genes including mir124.  mir124 inhibits REST activity by targeting SCP1, required for REST-mediated repression of neuronal genes.  Negative feedback loop. In nonneuronal cells an neuronal progenitors, expression of neuronal spec genes inc mir124 is repressed by REST and SCP1.  As cells differentiate and REST is transcriptionally inhibited, mir124 post-transcriptionally inhibits expression of required cofactor SCP1. This stops biological effects of REST.

Polypyrimidine tract-binding protein (PTB) and its neuron-specific homologue nPTB are regulators of gene expression at the interface between RNA silencing and splicing. a | In undifferentiated cells, the ubiquitous splicing regulator PTB represses the expression of nPTB by affecting the pattern of pre-mRNA splicing. As differentiation proceeds, miR-124 activates the expression of nPTB by inhibiting PTB. nPTB, in turn, shifts the alternative splicing of an array of genes to a neuron-specific pattern. Furthermore, miR-124 silences REST, a transcriptional inhibitor of neuron-specific genes that is expressed outside the neural system. b| As myogenesis progresses from the myoblast stage to the myotube stage, the level of the muscle-specific miR-133 increases. miR-133 inhibits the expression of nPTB, indirectly affecting the pattern of alternative splicing of several target genes.

miR-1 and development of heart and muscle.
 | miR-1 regulates cardiac morphogenesis by optimizing the level of the HAND2 transcription factor. Electric conduction is abnormal in mice that lack miR-1 as a consequence of de-inhibition of IRX5, a homeodomain-containing transcription factor that represses the expression of the KCND2 potassium channel. b | In normal conditions in wild-type animals, miR-208 maintains an optimal level of the thyroid hormone receptor (TR) cascade activity by acting on THRAP1 (thyroid hormone receptor-associated protein complex 240 kDa component) in a negative feedback loop. In transgenic mice that overexpress miR-208, inhibition of the TR pathways allows aberrant expression of beta-myosin heavy chain (betaMHC) in the adult. Similarly, in conditions of stress or hypothyroidism, decreased activity of the TR cascade leads to expression of betaMHC and hypertrophy. In the absence of miR-208 in null mice, THRAP1 is de-repressed and baseline levels of TR activity are abnormally high and resistant to inhibition by stress signals. Therefore, Mir-208-null mice do not express elevated levels of betaMHC or undergo cardiac hypertrophy in conditions of stress and hypothyroidism. T3, tri-iodothyronine; TRE, T3 response element.

miR-1 is highly expressed and skeletal and heart muscles.
Expressing mir-1 was induced on growth in a differentiating medium. Muscle spec molecular markers appeared. Overexpressing mir-1 in myoblasts promotes differentiation and reduces cell proliferation. In developing hjeart, mir1 overexpression reduced cell proliferation causing thinner ventricular wall.

mir-1 controls balance between proliferation and differentation of myocardiocytes by inhibiting translation of HAND2, which encodes aTF.

mir-1's electrophysiological effects are mdiated by its control of TF IRX5 which inhibits expression of KCND2 a gene which encodes a potassium channel. It is impotant in cardiac repolarisation.

miRNAs involved in cardiac hypertrophy.
In cardiac hypertrophy, aberrant postnatal activation of foetal genes. β-myosin heavy chain (βMHC) is aberrantly expressed during hypertrophy at expense of adult form, αMHC. β has lower ATPase activity than adult form. Causes contractile dysfunction in adult heart.

Heart spec miRNA mir208 is encoded in α MHC intron.  mir208 was deleted by homologous recomniation without affecting levels of αMHC expression. Untreated mutant animals showed decreased ontratility and expression of fast skeletal muscle spec genes in heart. In hypertrophy models Mir208 null animals failed to show heart hypertrophy and induction of βMHC. Transgenic overexpression ofmif208 induced βMHC.

thyroid hormone receptor (TR)with THRAP1 represed expression of βMHC. It promotes αMHC at transcriptional level. mir208 maintains optimal level of TR activity by negatively controlling expression of THRAP1 on negative feedback loop/ In wt animals mir208-mediated inhibition is not sufficient to allow βMHC expression. 3fold increase of mif 208 in transgenic mice induces βMHC. Without mir108 threshold of inhibition of TR cascade is elevated beyond hyperthyroidism.

miRNAs in lymphocyte development

a | Higher levels of miR-181 in double positive lymphocytes (DP cells) compared with mature T cells is accompanied by the higher sensitivity of the T-cell receptor to stimulation by MHC–peptide complexes. As miR-181 levels decrease during maturation, the activation threshold of T-cell receptors increases as a result of increased levels of several phosphatases modulated by miR-181 (see graph). b | Mir-155-null mice are characterized by complex defects in homeostasis of the immune system and globally impaired immune responses. Among the defects that were characterized in detail, the loss of miR-155-mediated inhibition of the transcription factor c-MAF led to increased production of interleukin-4 (IL-4) and T helper-2 (Th2) cells. The germinal centre reaction was disrupted, resulting in impaired T cell-dependent antibody responses (see main text).

miRNA content of haemotopoietic system was studied with DNA microarray and deep sequencing techniques.

miRNA181 is elevated at double positive DP stage during T lymphocyte maturation. Thymocytes expressing both CD4 and CD8 undergo positive and negative selection.

mir181 increases sensitivity of DP cells to stimulation of Tcell receptor TCR.  Blocking mir181 suppressed positive and negative selection.

MIC encodes a 1700 polyadenylated and splcied transcript that lacks a recognisable protein coding sequence. Conserved region encodes miR155.  BIC/mir155 expression increases in activated B adn T cells, macrophages and dendritic cells. High levels correlated with poor prognosis in lung caner.

Genetic deletions of mir155. Alters immune response. Lung histopathology.  Altered equilibrium between Th1 and Th2. In favour of Th 2 in mir155 KO mice. Loss of mir155-mediated inhibition of c-MAF. a TF that promoted expression of IL-4, an output of T2 cells. B lymphocytes decreased in germinal centres.

miRNA in cancer
a | miR-200 is an example of a microRNA (miRNA) whose role in cancer is well characterized. Alterations in the epigenetic regulation of the miR-200 family are involved in epithelial-to-mesenchymal transition (EMT) in cancer. Specifically, CpG island hypermethylation-associated silencing of these miRNAs in human tumours causes an upregulation of the zinc finger E-box-binding homeobox (HOX) 1 (ZEB1) and ZEB2 transcriptional repressors, which, in turn, leads to a downregulation of E-cadherin (CDH1) — these are changes that promote EMT.
miRNAs can act as oncogenes or tumour suppressors. mir200 regulates epithelial tomesenchymal transition.

Dysregulation of miRNAs in cancer can occur through epigenetic changes eg promoter CpG hypermethylation in miR200 family.  Genetic alterations which affect production of primary miNRA transcript, their processing to mature miRNAs and/or interactions with mRNA targets.  miRNAs are often in fragile regions of hcromosomes involved in ovarian and breast carcinomas and melanomas.

Tumour spec defects in miRNA machinery include genes that encode TARBP2, DICER1, XPO5 (exportin 5).

a | MicroRNAs (miRNAs) are transcribed as individual units (primary miRNA (pri-miRNA)) or together with host genes (mirtrons). Following processing by the Drosha complex or the lariat-debranching enzyme, respectively, precursor miRNAs (pre-miRNAs) are exported from the nucleus by exportin 5 (XPO5). Further processing by Dicer and TAR RNA-binding protein 2 (TARBP2) generates mature miRNAs, which are loaded into the RNA-induced silencing complex (RISC). miRNAs function through degradation of protein-coding transcripts or translational repression. b | PIWI-interacting RNAs (piRNAs) are mainly expressed as ssRNAs from mono- or bidirectional clusters. Subsequently, additional piRNAs are produced through a PIWI-protein-catalysed amplification loop (called the 'ping-pong cycle') via sense and antisense intermediates. The PIWI ribonucleoprotein (piRNP) complex functions in transposon repression through target degradation and epigenetic silencing. 
ncRNas of 24-30nt. They are Dicer-independent. They arise from sequences antisense to transposons. Recognise and silence transposon nRNA.

They bind PIWI subfamily of Ago family proteins involved in maintaining genome stability in germline cells.   Transcribed from regions in genome that contain transcribed transposable elements and other repetitive elements.

PIWI protein family in Drosophila consists of 3 members : PIWI, Aubergine and Ago3.

Complex formed by piRNA and PIWI proteins suppress TE expression and mobilisation. They mediate cleavage of TE transcripts by PIWI proteins. This is mediated through bping recognition by piRNA. They also mediate heterochromatin mediated gene silencing. PIWIL1, PIWIL2, and PIWIL4 are involved in ping pong amplification cycle. This creates antisense piRNAs that can repress transcript of origin.

Transposon antisense piRNAs bound to Piwi and Aubbergine associate with sense transposon transcripts and cleave 10 nt from 5' end A 2nd clevage event creates 3'end of piNRA, release a sense 2dary piRNA which associates with Ago3.  Ago3 bound sense piRNAs target antisense transposon transcripts and ago-3mediated cleavage  A 2nd cleavage event generates antisense piRNAs which silence target and reinforce the cycle by creating new sense piRNAs.

Fig. 3. Ping-pong cycle for the generation of piRNAs and transposon repression. Maternally deposited antisense piRNAs or primary piRNAs derived from initial processing of piRNA clusters bound to Aubergine and Piwi proteins identify and cleave expressed transposon transcripts, resulting in new sense secondary piRNAs. Bound to Ago3, these newly generated piRNAs are targeting antisense transposon sequences and Ago3-mediated cleavage generates additional antisense piRNAs, leading to a feed forward amplification mechanism.

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