Mechanism of RNAi in Genomics and Therapeutics: A Review
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paper_title: RNAi suppresses polyglutamine-induced neurodegeneration in a model of spinocerebellar ataxia

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RNAi suppresses polyglutamine-induced neurodegeneration in a model of spinocerebellar ataxia
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paper_title: Blocking oncogenes in malignant cells by RNA interference--new hope for a highly specific cancer treatment?

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A little more than one year after the first demonstration that silencing of endogenous human genes is possible in cell culture, the new tool of RNA interference (RNAi) enters the field of tumor therapy.
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paper_title: Therapeutic silencing of an endogenous gene by systemic administration of modified siRNAs

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RNA interference (RNAi) holds considerable promise as a therapeutic approach to silence disease-causing genes, particularly those that encode so-called 'non-druggable' targets that are not amenable to conventional therapeutics such as small molecules, proteins, or monoclonal antibodies. The main obstacle to achieving in vivo gene silencing by RNAi technologies is delivery. Here we show that chemically modified short interfering RNAs (siRNAs) can silence an endogenous gene encoding apolipoprotein B (apoB) after intravenous injection in mice. Administration of chemically modified siRNAs resulted in silencing of the apoB messenger RNA in liver and jejunum, decreased plasma levels of apoB protein, and reduced total cholesterol. We also show that these siRNAs can silence human apoB in a transgenic mouse model. In our in vivo study, the mechanism of action for the siRNAs was proven to occur through RNAi-mediated mRNA degradation, and we determined that cleavage of the apoB mRNA occurred specifically at the predicted site. These findings demonstrate the therapeutic potential of siRNAs for the treatment of disease.
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paper_title: Chalcone synthase cosuppression phenotypes in petunia flowers: comparison of sense vs. antisense constructs and single-copy vs. complex T-DNA sequences

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Flower pigmentation patterns were scored in 185 sense Chalcone synthase (Chs) transgenotes and 85 antisense Chs transgenotes; upon first flowering, 139 (75%) of sense transgenotes were found to be phenotypically altered, as were 70 (82%) of the antisense transgenotes. The observed patterns document the range of phenotypic variations that occur, as well as confirm and extend the finding that sense Chs constructs produce several types of morphology-based flower pigmentation patterns that antisense Chs constructs do not. Long-term monitoring for epigenetic variations in one population of 44 sense Chs transgenotes showed that 43 (98%) were capable of producing a cosuppression phenotype. The primary determinant of sense-specific patterns of cosuppression of Chs was found to be the repetitiveness and organization pattern of the transgene, not 'position effects' by, or 'readthrough' from, flanking plant DNA sequences. The degree of cosuppression observed in progeny of transgenotes carrying multiple, dispersed copies as compared to that observed with a single copy of the transgene suggests that sense cosuppression of Chs is subject to a transgene dosage effect.
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paper_title: Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells

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RNA interference (RNAi) is the process of sequence-specific, post-transcriptional gene silencing in animals and plants, initiated by double-stranded RNA (dsRNA) that is homologous in sequence to the silenced gene1,2,3,4. The mediators of sequence-specific messenger RNA degradation are 21- and 22-nucleotide small interfering RNAs (siRNAs) generated by ribonuclease III cleavage from longer dsRNAs5,6,7,8,9. Here we show that 21-nucleotide siRNA duplexes specifically suppress expression of endogenous and heterologous genes in different mammalian cell lines, including human embryonic kidney (293) and HeLa cells. Therefore, 21-nucleotide siRNA duplexes provide a new tool for studying gene function in mammalian cells and may eventually be used as gene-specific therapeutics.
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paper_title: RNA interference and small interfering RNAs.

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The term aRNA interferenceo (RNAi) was coined after the groundbreaking discovery that injection of double-stranded RNA (dsRNA) into the nematode Caenorhabditis elegans leads to specific silencing of genes highly homologous in sequence to the delivered dsRNA. The RNAi phenotype is either identical to the genetic null mutant or resembles an allelic series of mutants. The dsRNA can also be delivered by feeding bacteria that express dsRNA from recombinant plasmids to the worm or by soaking the worm in a solution containing the dsRNA. 3] In rapid sequence, RNAi was observed in other animals including mice, 5] and therefore this process possibly exists also in humans. RNAi appears to be related to the posttranscriptional gene silencing (PTGS) mechanism of cosuppression in plants and quelling in fungi. Cosuppression is the ability of some transgenes to silence both themselves and homologous chromosomal loci simultaneously. The initiator molecule for cosuppression is believed to be aberrant RNA, possibly dsRNA, and some components of the RNAi machinery are required for posttranscriptional silencing by cosuppression. 8, 13] The natural function of RNAi and cosuppression is thought to be protection of the genome against invasion by mobile genetic elements such as transposons and viruses, which produce aberrant RNA or dsRNA in the host cell when they become active. Thus, specific mRNA degradation is thought to prevent transposon and virus replication. This minireview will highlight recent advances in understanding the molecular mechanism of RNAi and its biological function. The reader is also referred to a number of excellent reviews that have appeared recently (see refs. [18 ± 24]).
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paper_title: Genetic interference in Trypanosoma brucei by heritable and inducible double-stranded RNA.

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The use of double-stranded RNA (dsRNA) to disrupt gene expression has become a powerful method of achieving RNA interference (RNAi) in a wide variety of organisms. However, in Trypanosoma brucei this tool is restricted to transient interference, because the dsRNA is not stably maintained and its effects are diminished and eventually lost during cellular division. Here, we show that genetic interference by dsRNA can be achieved in a heritable and inducible fashion. To show this, we established stable cell lines expressing dsRNA in the form of stem-loop structures under the control of a tetracycline-inducible promoter. Targeting a-tubulin and actin mRNA resulted in potent and specific mRNA degradation as previously observed in transient interference. Surprisingly, 10-fold down regulation of actin mRNA was not fatal to trypanosomes. This type of approach could be applied to study RNAi in other organisms that are difficult to microinject or electroporate. Furthermore, to quickly probe the consequences of RNAi for a given gene we established a highly efficient in vivo T7 RNA polymerase system for expression of dsRNA. Using the alpha-tubulin test system we obtained greater than 98% transfection efficiency and the RNAi response lasted at least two to three cell generations. These new developments make it possible to initiate the molecular dissection of RNAi both biochemically and genetically.
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paper_title: Stable suppression of gene expression by RNAi in mammalian cells

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In a diverse group of organisms including plants, Caenorhabditis elegans, Drosophila, and trypanosomes, double-stranded RNA (dsRNA) is a potent trigger of gene silencing. In several model systems, this natural response has been developed into a powerful tool for the investigation of gene function. Use of RNA interference (RNAi) as a genetic tool has recently been extended to mammalian cells, being inducible by treatment with small, ≈22-nt RNAs that mimic those produced in the first step of the silencing process. Here, we show that some cultured murine cells specifically silence gene expression upon treatment with long dsRNAs (≈500 nt). This response shows hallmarks of conventional RNAi including silencing at the posttranscriptional level and the endogenous production of ≈22-nt small RNAs. Furthermore, enforced expression of long, hairpin dsRNAs induced stable gene silencing. The ability to create stable “knock-down” cell lines expands the utility of RNAi in mammalian cells by enabling examination of phenotypes that develop over long time periods and lays the groundwork for by using RNAi in phenotype-based, forward genetic selections.
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paper_title: Prospects of RNA interference therapy for cancer

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RNA interference (RNAi) is a powerful gene-silencing process that holds great promise in the field of cancer therapy. The discovery of RNAi has generated enthusiasm within the scientific community, not only because it has been used to rapidly identify key molecules involved in many disease processes including cancer, but also because RNAi has the potential to be translated into a technology with major therapeutic applications. Our evolving understanding of the molecular pathways important for carcinogenesis has created opportunities for cancer therapy employing RNAi technology to target the key molecules within these pathways. Many gene products involved in carcinogenesis have already been explored as targets for RNAi intervention, and RNAi targeting of molecules crucial for tumor-host interactions and tumor resistance to chemo- or radiotherapy has also been investigated. In most of these studies, the silencing of critical gene products by RNAi technology has generated significant antiproliferative and/or proapoptotic effects in cell-culture systems or in preclinical animal models. Nevertheless, significant obstacles, such as in vivo delivery, incomplete suppression of target genes, nonspecific immune responses and the so-called off-target effects, need to be overcome before this technology can be successfully translated into the clinical arena. Significant progress has already been made in addressing some of these issues, and it is foreseen that early phase clinical trials will be initiated in the very near future.
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paper_title: MicroRNAs as Oncogenes and Tumor Suppressors

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The struggle to combat cancer — including the discovery of oncogenes, tumor suppressors, telomerase, and cancer stem cells — has revealed the complex nature of cancer in humans. Chang-Zheng Chen as...
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Title: Mechanism of RNAi in Genomics and Therapeutics: A Review

Section 1: INTRODUCTION
Description 1: Provide an overview of the role of RNAi in gene regulation, its significance in genomics and therapeutics, and outline the structure of the paper.

Section 2: CENTRAL DOGMA OF MOLECULAR BIOLOGY
Description 2: Explain the fundamental principles of molecular biology, including DNA replication, transcription, and translation.

Section 3: DNA Replication
Description 3: Describe the process of DNA replication and its importance in biological inheritance.

Section 4: Transcription
Description 4: Discuss how genetic information is copied from DNA to RNA, highlighting the roles of introns, exons, and mRNA.

Section 5: Translation
Description 5: Explain the process by which mRNA is translated into proteins, involving ribosomes and tRNA.

Section 6: Biogenesis of siRNA
Description 6: Review the discovery of RNAi, its pathway, and its application in gene silencing across various organisms.

Section 7: dsRNA cleavage by Dicer generating siRNAs
Description 7: Detail the role of the enzyme Dicer in cleaving dsRNA to generate siRNAs.

Section 8: Formation of RISC
Description 8: Describe the formation of the RNA Induced Silencing Complex (RISC) and its initial inactivity.

Section 9: SiRNA unwinding and RISC activation
Description 9: Discuss the unwinding of siRNA duplexes by RISC and the activation process involving sense and anti-sense strands.

Section 10: mRNA targeting and degradation
Description 10: Explain how the activated RISC complex targets and degrades mRNA to inhibit its translation.

Section 11: RNAI IN GENOMICS AND THERAPEUTICS
Description 11: Explore the applications of RNAi in gene regulation and its therapeutic potential for various diseases.

Section 12: Role of siRNA in Genomics
Description 12: Examine how siRNA contributes to gene regulation, normal cellular processes, and the implications of its malfunction.

Section 13: Role of siRNA in Therapeutics
Description 13: Discuss the therapeutic applications of siRNA in targeting cancer and other diseases by controlling cell growth and apoptosis.

Section 14: CONCLUSION
Description 14: Summarize the significance of RNAi in genomics and therapeutics, and the future research directions.