RNA silencing

RNA silencing describes several mechanistically related pathways which are involved in controlling and regulating gene expression.{{cite journal | vauthors = Moazed D | title = Small RNAs in transcriptional gene silencing and genome defence | journal = Nature | volume = 457 | issue = 7228 | pages = 413–20 | date = Jan 2009 | pmid = 19158787 | doi = 10.1038/nature07756 | pmc=3246369| bibcode = 2009Natur.457..413M }}{{cite journal | vauthors = Pickford AS, Cogoni C | title = RNA-mediated gene silencing | journal = Cellular and Molecular Life Sciences | volume = 60 | issue = 5 | pages = 871–82 | date = May 2003 | pmid = 12827277 | doi = 10.1007/s00018-003-2245-2 | s2cid = 5822771 | pmc = 11138585 }}{{cite journal | vauthors = Tijsterman M, Ketting RF, Plasterk RH | title = The genetics of RNA silencing | journal = Annual Review of Genetics | volume = 36 | pages = 489–519 | year = 2002 | pmid = 12429701 | doi = 10.1146/annurev.genet.36.043002.091619 }} RNA silencing pathways are associated with the regulatory activity of small non-coding RNAs (approximately 20–30 nucleotides in length) that function as factors involved in inactivating homologous sequences, promoting endonuclease activity, translational arrest, and/or chromatic or DNA modification.{{cite journal | vauthors = Malecová B, Morris KV | title = Transcriptional gene silencing through epigenetic changes mediated by non-coding RNAs | journal = Current Opinion in Molecular Therapeutics | volume = 12 | issue = 2 | pages = 214–22 | date = Apr 2010 | pmid = 20373265 | pmc=2861437}}{{cite journal | vauthors = Meister G, Landthaler M, Dorsett Y, Tuschl T | title = Sequence-specific inhibition of microRNA- and siRNA-induced RNA silencing | journal = RNA | volume = 10 | issue = 3 | pages = 544–50 | date = Mar 2004 | pmid = 14970398 | pmc = 1370948 | doi = 10.1261/rna.5235104 }}{{cite journal | vauthors = Zhou H, Hu H, Lai M | title = Non-coding RNAs and their epigenetic regulatory mechanisms | journal = Biology of the Cell | volume = 102 | issue = 12 | pages = 645–55 | date = Dec 2010 | pmid = 21077844 | doi = 10.1042/BC20100029 | s2cid = 11325463 | doi-access = free }} In the context in which the phenomenon was first studied, small RNA was found to play an important role in defending plants against viruses. For example, these studies demonstrated that enzymes detect double-stranded RNA (dsRNA) not normally found in cells and digest it into small pieces that are not able to cause disease.{{cite journal | vauthors = Ding SW | title = RNA silencing | journal = Current Opinion in Biotechnology | volume = 11 | issue = 2 | pages = 152–6 | date = Apr 2000 | pmid = 10753772 | doi = 10.1016/s0958-1669(00)00074-4 }}{{cite journal | vauthors = Susi P, Hohkuri M, Wahlroos T, Kilby NJ | title = Characteristics of RNA silencing in plants: similarities and differences across kingdoms | journal = Plant Molecular Biology | volume = 54 | issue = 2 | pages = 157–74 | date = Jan 2004 | pmid = 15159620 | doi = 10.1023/B:PLAN.0000028797.63168.a7 | s2cid = 11018531 }}{{cite journal | vauthors = Baulcombe D | title = RNA silencing in plants | journal = Nature | volume = 431 | issue = 7006 | pages = 356–63 | date = Sep 2004 | pmid = 15372043 | doi = 10.1038/nature02874 | bibcode = 2004Natur.431..356B | s2cid = 4421274 }}{{cite journal | vauthors = Baulcombe D | title = RNA silencing | journal = Trends in Biochemical Sciences | volume = 30 | issue = 6 | pages = 290–3 | date = Jun 2005 | pmid = 15950871 | doi = 10.1016/j.tibs.2005.04.012 | doi-access = free }}{{cite journal | vauthors = Meister G, Tuschl T | title = Mechanisms of gene silencing by double-stranded RNA | journal = Nature | volume = 431 | issue = 7006 | pages = 343–9 | date = Sep 2004 | pmid = 15372041 | doi = 10.1038/nature02873 | url = http://www.blatny.com/Epigenetika2007/2007-12-04/papers/Meister%202004.pdf | bibcode = 2004Natur.431..343M | s2cid = 90438 | archive-date = 2011-07-08 | access-date = 2011-02-08 | archive-url = https://web.archive.org/web/20110708013038/http://www.blatny.com/Epigenetika2007/2007-12-04/papers/Meister%202004.pdf | url-status = dead }}

While some functions of RNA silencing and its machinery are understood, many are not. For example, RNA silencing has been shown to be important in the regulation of development and in the control of transposition events.{{cite journal | vauthors = Matzke MA, Birchler JA | title = RNAi-mediated pathways in the nucleus | journal = Nature Reviews Genetics | volume = 6 | issue = 1 | pages = 24–35 | date = Jan 2005 | pmid = 15630419 | doi = 10.1038/nrg1500 | s2cid = 9321759 | doi-access = free }} RNA silencing has been shown to play a role in antiviral protection in plants as well as insects.{{cite journal | vauthors = Voinnet O | title = Induction and suppression of RNA silencing: insights from viral infections | journal = Nature Reviews Genetics | volume = 6 | issue = 3 | pages = 206–20 | date = Mar 2005 | pmid = 15703763 | doi = 10.1038/nrg1555 | s2cid = 26351712 }} Also in yeast, RNA silencing has been shown to maintain heterochromatin structure.{{cite journal | vauthors = Grewal SI, Rice JC | title = Regulation of heterochromatin by histone methylation and small RNAs | journal = Current Opinion in Cell Biology | volume = 16 | issue = 3 | pages = 230–8 | date = Jun 2004 | pmid = 15145346 | doi = 10.1016/j.ceb.2004.04.002 | url = https://zenodo.org/record/1258832 | archive-date = 2022-01-22 | access-date = 2019-07-02 | archive-url = https://web.archive.org/web/20220122033707/https://zenodo.org/record/1258832 | url-status = live }} However, the varied and nuanced role of RNA silencing in the regulation of gene expression remains an ongoing scientific inquiry. A range of diverse functions have been proposed for a growing number of characterized small RNA sequences—e.g., regulation of developmental, neuronal cell fate, cell death, proliferation, fat storage, haematopoietic cell fate, insulin secretion.{{cite journal | vauthors = Poy MN, Eliasson L, Krutzfeldt J, Kuwajima S, Ma X, Macdonald PE, Pfeffer S, Tuschl T, Rajewsky N, Rorsman P, Stoffel M | title = A pancreatic islet-specific microRNA regulates insulin secretion | journal = Nature | volume = 432 | issue = 7014 | pages = 226–30 | date = Nov 2004 | pmid = 15538371 | doi = 10.1038/nature03076 | bibcode = 2004Natur.432..226P | s2cid = 4415988 }}

RNA silencing functions by repressing translation or by cleaving messenger RNA (mRNA), depending on the amount of complementarity of base-pairing. RNA has been largely investigated within its role as an intermediary in the translation of genes into proteins.{{cite journal|last=Eccleston|first=Alex|author2=Angela K. Eggleston |title=RNA Interference|journal=Nature|year=2004|volume=431|issue=7006|pages=338–42|doi=10.1038/431337a |pmid=15372040|bibcode=2004Natur.431..337E|doi-access=free}} More active regulatory functions, however, only began to be addressed by researchers beginning in the late-1990s.{{cite journal | vauthors = Eggleston AK | title = RNA silencing | journal = Nature | volume = 457 | issue = 7228 | pages = 395 | date = Jan 2009 | pmid = 19158784 | doi = 10.1038/457395a | bibcode = 2009Natur.457..395E | doi-access = free }} The landmark study providing an understanding of the first identified mechanism was published in 1998 by Fire et al.,{{cite journal | vauthors = Fire A, Xu S, Montgomery MK, Kostas SA, Driver SE, Mello CC | title = Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans | journal = Nature | volume = 391 | issue = 6669 | pages = 806–11 | date = Feb 1998 | pmid = 9486653 | doi = 10.1038/35888 | bibcode = 1998Natur.391..806F | s2cid = 4355692 | url = http://www.dspace.cam.ac.uk/handle/1810/238264 }} demonstrating that double-stranded RNA could act as a trigger for gene silencing. Since then, various other classes of RNA silencing have been identified and characterized. Presently, the therapeutic potential of these discoveries is being explored, for example, in the context of targeted gene therapy.{{cite journal | vauthors = Takeshita F, Ochiya T | title = Therapeutic potential of RNA interference against cancer | journal = Cancer Science | volume = 97 | issue = 8 | pages = 689–96 | date = Aug 2006 | pmid = 16863503 | doi = 10.1111/j.1349-7006.2006.00234.x | s2cid = 19447542 | doi-access = free | pmc = 11158528 }}{{cite journal | vauthors = Dykxhoorn DM, Novina CD, Sharp PA | title = Killing the messenger: short RNAs that silence gene expression | journal = Nature Reviews Molecular Cell Biology | volume = 4 | issue = 6 | pages = 457–67 | date = Jun 2003 | pmid = 12778125 | doi = 10.1038/nrm1129 | s2cid = 7445808 }}

While RNA silencing is an evolving class of mechanisms, a common theme is the fundamental relationship between small RNAs and gene expression. It has also been observed that the major RNA silencing pathways currently identified have mechanisms of action which may involve both post-transcriptional gene silencing (PTGS){{cite journal | vauthors = Hammond SM, Caudy AA, Hannon GJ | title = Post-transcriptional gene silencing by double-stranded RNA | journal = Nature Reviews Genetics | volume = 2 | issue = 2 | pages = 110–9 | date = Feb 2001 | pmid = 11253050 | doi = 10.1038/35052556 | s2cid = 2864720 }} as well as chromatin-dependent gene silencing (CDGS) pathways. CDGS involves the assembly of small RNA complexes on nascent transcripts and is regarded as encompassing mechanisms of action which implicate transcriptional gene silencing (TGS) and co-transcriptional gene silencing (CTGS) events.{{cite journal | vauthors = Bühler M | title = RNA turnover and chromatin-dependent gene silencing | journal = Chromosoma | volume = 118 | issue = 2 | pages = 141–51 | date = Apr 2009 | pmid = 19023586 | doi = 10.1007/s00412-008-0195-z | s2cid = 2790637 }} This is significant at least because the evidence suggests that small RNAs play a role in the modulation of chromatin structure and TGS.{{cite journal | vauthors = Gonzalez S, Pisano DG, Serrano M | title = Mechanistic principles of chromatin remodeling guided by siRNAs and miRNAs | journal = Cell Cycle | volume = 7 | issue = 16 | pages = 2601–8 | date = Aug 2008 | pmid = 18719372 | doi = 10.4161/cc.7.16.6541 | doi-access = free }}{{cite journal | vauthors = Kim JK, Gabel HW, Kamath RS, Tewari M, Pasquinelli A, Rual JF, Kennedy S, Dybbs M, Bertin N, Kaplan JM, Vidal M, Ruvkun G | title = Functional genomic analysis of RNA interference in C. elegans | journal = Science | volume = 308 | issue = 5725 | pages = 1164–7 | date = May 2005 | pmid = 15790806 | doi = 10.1126/science.1109267 | bibcode = 2005Sci...308.1164K | s2cid = 15510615 | doi-access = free }}

Despite early focus in the literature on RNA interference (RNAi) as a core mechanism which occurs at the level of messenger RNA translation, others have since been identified in the broader family of conserved RNA silencing pathways acting at the DNA and chromatin level.{{cite journal | vauthors = Bühler M, Moazed D | title = Transcription and RNAi in heterochromatic gene silencing | journal = Nature Structural & Molecular Biology | volume = 14 | issue = 11 | pages = 1041–8 | date = Nov 2007 | pmid = 17984966 | doi = 10.1038/nsmb1315 | s2cid = 39098216 }} RNA silencing refers to the silencing activity of a range of small RNAs and is generally regarded as a broader category than RNAi. While the terms have sometimes been used interchangeably in the literature, RNAi is generally regarded as a branch of RNA silencing. To the extent it is useful to craft a distinction between these related concepts, RNA silencing may be thought of as referring to the broader scheme of small RNA related controls involved in gene expression and the protection of the genome against mobile repetitive DNA sequences, retroelements, and transposons to the extent that these can induce mutations.{{cite journal | vauthors = Dombroski BA, Feng Q, Mathias SL, Sassaman DM, Scott AF, Kazazian HH, Boeke JD | title = An in vivo assay for the reverse transcriptase of human retrotransposon L1 in Saccharomyces cerevisiae | journal = Molecular and Cellular Biology | volume = 14 | issue = 7 | pages = 4485–92 | date = Jul 1994 | pmid = 7516468 | pmc = 358820 | doi=10.1128/mcb.14.7.4485}} The molecular mechanisms for RNA silencing were initially studied in plants but have since broadened to cover a variety of subjects, from fungi to mammals, providing strong evidence that these pathways are highly conserved.{{cite book | vauthors = Svoboda P | chapter = RNA Silencing in Mammalian Oocytes and Early Embryos | series = Current Topics in Microbiology and Immunology | title = RNA Interference | volume = 320 | pages = 225–56 | year = 2008 | pmid = 18268847 | doi = 10.1007/978-3-540-75157-1_11 | isbn = 978-3-540-75156-4 }}

At least three primary classes of small RNA have currently been identified, namely: small interfering RNA (siRNA), microRNA (miRNA), and piwi-interacting RNA (piRNA).

{{Main|Small interfering RNA}}

siRNAs act in the nucleus and the cytoplasm and are involved in RNAi as well as CDGS. siRNAs come from long dsRNA precursors derived from a variety of single-stranded RNA (ssRNA) precursors, such as sense and antisense RNAs. siRNAs also come from hairpin RNAs derived from transcription of inverted repeat regions. siRNAs may also arise enzymatically from non-coding RNA precursors.{{cite journal | vauthors = Ghildiyal M, Seitz H, Horwich MD, Li C, Du T, Lee S, Xu J, Kittler EL, Zapp ML, Weng Z, Zamore PD | title = Endogenous siRNAs derived from transposons and mRNAs in Drosophila somatic cells | journal = Science | volume = 320 | issue = 5879 | pages = 1077–81 | date = May 2008 | pmid = 18403677 | doi = 10.1126/science.1157396 | pmc=2953241| bibcode = 2008Sci...320.1077G }} The volume of literature on siRNA within the framework of RNAi is extensive. One of the potent applications of siRNAs is the ability to distinguish the target versus non-target sequence with a single-nucleotide difference. This approach has been considered as therapeutically crucial for the silencing dominant gain-of-function (GOF) disorders, where mutant allele causing disease is differed from wt-allele by a single nucleotide (nt). This type of siRNAs with capability to distinguish a single-nt difference are termed as allele-specific siRNAs.{{cite journal|vauthors=Monga I, Qureshi A, Thakur N, Gupta AK, Kumar M|year=2017|title=ASPsiRNA: A Resource of ASP-siRNAs Having Therapeutic Potential for Human Genetic Disorders and Algorithm for Prediction of Their Inhibitory Efficacy|url=http://crdd.osdd.net/servers/aspsirna/asptar.php|journal=G3: Genes, Genomes, Genetics |volume=7|issue=9|pages=2931–2943|doi=10.1534/g3.117.044024|pmc=5592921|pmid=28696921}}

{{Main|MicroRNA}}

The majority of miRNAs act in the cytoplasm and mediate mRNA degradation or translational arrest.{{cite journal | vauthors = Filipowicz W, Jaskiewicz L, Kolb FA, Pillai RS | title = Post-transcriptional gene silencing by siRNAs and miRNAs | journal = Current Opinion in Structural Biology | volume = 15 | issue = 3 | pages = 331–41 | date = Jun 2005 | pmid = 15925505 | doi = 10.1016/j.sbi.2005.05.006 }} However, some plant miRNAs have been shown to act directly to promote DNA methylation.{{cite journal | vauthors = Bao N, Lye KW, Barton MK | title = MicroRNA binding sites in Arabidopsis class III HD-ZIP mRNAs are required for methylation of the template chromosome | journal = Developmental Cell | volume = 7 | issue = 5 | pages = 653–62 | date = Nov 2004 | pmid = 15525527 | doi = 10.1016/j.devcel.2004.10.003 | doi-access = free }} miRNAs come from hairpin precursors generated by the RNaseIII enzymes Drosha and Dicer.{{cite journal | vauthors = Zeng Y, Yi R, Cullen BR | title = Recognition and cleavage of primary microRNA precursors by the nuclear processing enzyme Drosha | journal = The EMBO Journal | volume = 24 | issue = 1 | pages = 138–48 | date = Jan 2005 | pmid = 15565168 | pmc = 544904 | doi = 10.1038/sj.emboj.7600491 }} Both miRNA and siRNA form either the RNA-induced silencing complex (RISC) or the nuclear form of RISC known as RNA-induced transcriptional silencing complex (RITS).{{cite journal | vauthors = Irvine DV, Zaratiegui M, Tolia NH, Goto DB, Chitwood DH, Vaughn MW, Joshua-Tor L, Martienssen RA | title = Argonaute slicing is required for heterochromatic silencing and spreading | journal = Science | volume = 313 | issue = 5790 | pages = 1134–7 | date = Aug 2006 | pmid = 16931764 | doi = 10.1126/science.1128813 | bibcode = 2006Sci...313.1134I | s2cid = 42997104 }} The volume of literature on miRNA within the framework of RNAi is extensive.

{{Main|Three prime untranslated region}}

Three prime untranslated regions (3'UTRs) of messenger RNAs (mRNAs) often contain regulatory sequences that post-transcriptionally cause RNA interference. Such 3'-UTRs often contain both binding sites for microRNAs (miRNAs) as well as for regulatory proteins. By binding to specific sites within the 3'-UTR, miRNAs can decrease gene expression of various mRNAs by either inhibiting translation or directly causing degradation of the transcript. The 3'-UTR also may have silencer regions that bind repressor proteins that inhibit the expression of a mRNA.

The 3'-UTR often contains microRNA response elements (MREs). MREs are sequences to which miRNAs bind. These are prevalent motifs within 3'-UTRs. Among all regulatory motifs within the 3'-UTRs (e.g. including silencer regions), MREs make up about half of the motifs.

As of 2014, the miRBase web site,miRBase.org an archive of miRNA sequences and annotations, listed 28,645 entries in 233 biologic species. Of these, 1,881 miRNAs were in annotated human miRNA loci. miRNAs were predicted to have an average of about four hundred target mRNAs (affecting expression of several hundred genes).{{cite journal | vauthors = Friedman RC, Farh KK, Burge CB, Bartel DP | title = Most mammalian mRNAs are conserved targets of microRNAs | journal = Genome Research | volume = 19 | issue = 1 | pages = 92–105 | date = Jan 2009 | pmid = 18955434 | pmc = 2612969 | doi = 10.1101/gr.082701.108 }} Freidman et al. estimate that >45,000 miRNA target sites within human mRNA 3'UTRs are conserved above background levels, and >60% of human protein-coding genes have been under selective pressure to maintain pairing to miRNAs.

Direct experiments show that a single miRNA can reduce the stability of hundreds of unique mRNAs.{{cite journal | vauthors = Lim LP, Lau NC, Garrett-Engele P, Grimson A, Schelter JM, Castle J, Bartel DP, Linsley PS, Johnson JM | title = Microarray analysis shows that some microRNAs downregulate large numbers of target mRNAs | journal = Nature | volume = 433 | issue = 7027 | pages = 769–73 | date = Feb 2005 | pmid = 15685193 | doi = 10.1038/nature03315 | bibcode = 2005Natur.433..769L | s2cid = 4430576 }} Other experiments show that a single miRNA may repress the production of hundreds of proteins, but that this repression often is relatively mild (less than 2-fold).{{cite journal | vauthors = Selbach M, Schwanhäusser B, Thierfelder N, Fang Z, Khanin R, Rajewsky N | title = Widespread changes in protein synthesis induced by microRNAs | journal = Nature | volume = 455 | issue = 7209 | pages = 58–63 | date = Sep 2008 | pmid = 18668040 | doi = 10.1038/nature07228 | bibcode = 2008Natur.455...58S | s2cid = 4429008 }}{{cite journal | vauthors = Baek D, Villén J, Shin C, Camargo FD, Gygi SP, Bartel DP | title = The impact of microRNAs on protein output | journal = Nature | volume = 455 | issue = 7209 | pages = 64–71 | date = Sep 2008 | pmid = 18668037 | pmc = 2745094 | doi = 10.1038/nature07242 | bibcode = 2008Natur.455...64B }}

The effects of miRNA dysregulation of gene expression seem to be important in cancer.{{cite journal | vauthors = Palmero EI, de Campos SG, Campos M, de Souza NC, Guerreiro ID, Carvalho AL, Marques MM | title = Mechanisms and role of microRNA deregulation in cancer onset and progression | journal = Genetics and Molecular Biology | volume = 34 | issue = 3 | pages = 363–70 | date = Jul 2011 | pmid = 21931505 | pmc = 3168173 | doi = 10.1590/S1415-47572011000300001 }} For instance, in gastrointestinal cancers, nine miRNAs have been identified as epigenetically altered and effective in down regulating DNA repair enzymes.{{cite journal | vauthors = Bernstein C, Bernstein H | title = Epigenetic reduction of DNA repair in progression to gastrointestinal cancer | journal = World Journal of Gastrointestinal Oncology | volume = 7 | issue = 5 | pages = 30–46 | date = May 2015 | pmid = 25987950 | pmc = 4434036 | doi = 10.4251/wjgo.v7.i5.30 | doi-access = free }}

The effects of miRNA dysregulation of gene expression also seem to be important in neuropsychiatric disorders, such as schizophrenia, bipolar disorder, major depression, Parkinson's disease, Alzheimer's disease and autism spectrum disorders.{{cite journal | vauthors = Maffioletti E, Tardito D, Gennarelli M, Bocchio-Chiavetto L | title = Micro spies from the brain to the periphery: new clues from studies on microRNAs in neuropsychiatric disorders | journal = Frontiers in Cellular Neuroscience | volume = 8 | pages = 75 | year = 2014 | pmid = 24653674 | pmc = 3949217 | doi = 10.3389/fncel.2014.00075 | doi-access = free }}{{cite journal | vauthors = Mellios N, Sur M | title = The Emerging Role of microRNAs in Schizophrenia and Autism Spectrum Disorders | journal = Frontiers in Psychiatry | volume = 3 | pages = 39 | year = 2012 | pmid = 22539927 | pmc = 3336189 | doi = 10.3389/fpsyt.2012.00039 | doi-access = free }}{{cite journal | vauthors = Geaghan M, Cairns MJ | title = MicroRNA and Posttranscriptional Dysregulation in Psychiatry | journal = Biological Psychiatry | volume = 78 | issue = 4 | pages = 231–9 | date = Aug 2015 | pmid = 25636176 | doi = 10.1016/j.biopsych.2014.12.009 | doi-access = free | hdl = 1959.13/1335073 | hdl-access = free }}

{{Main|Piwi-interacting RNA}}

piRNAs represent the largest class of small non-coding RNA molecules expressed in animal cells, deriving from a large variety of sources, including repetitive DNA and transposons.{{cite journal | vauthors = Klattenhoff C, Theurkauf W | title = Biogenesis and germline functions of piRNAs | journal = Development | volume = 135 | issue = 1 | pages = 3–9 | date = Jan 2008 | pmid = 18032451 | doi = 10.1242/dev.006486 | url = https://escholarship.umassmed.edu/oapubs/1252 | doi-access = free | archive-date = 2020-06-07 | access-date = 2019-07-02 | archive-url = https://web.archive.org/web/20200607124512/https://escholarship.umassmed.edu/oapubs/1252/ | url-status = live }} However, the biogenesis of piRNAs is also the least well understood.{{cite journal | vauthors = Ishizu H, Siomi H, Siomi MC | title = Biology of PIWI-interacting RNAs: new insights into biogenesis and function inside and outside of germlines | journal = Genes & Development | volume = 26 | issue = 21 | pages = 2361–73 | date = Nov 2012 | pmid = 23124062 | doi = 10.1101/gad.203786.112 | pmc=3489994}} piRNAs appear to act both at the post-transcriptional and chromatin levels. They are distinct from miRNA due to at least an increase in terms of size and complexity. Repeat associated small interfering RNA (rasiRNAs) are considered to be a subspecies of piRNA.{{cite journal | vauthors = Gunawardane LS, Saito K, Nishida KM, Miyoshi K, Kawamura Y, Nagami T, Siomi H, Siomi MC | title = A slicer-mediated mechanism for repeat-associated siRNA 5' end formation in Drosophila | journal = Science | volume = 315 | issue = 5818 | pages = 1587–90 | date = Mar 2007 | pmid = 17322028 | doi = 10.1126/science.1140494 | bibcode = 2007Sci...315.1587G | s2cid = 11513777 | doi-access = free }}

File:MiRNA processing.svg

The most basic mechanistic flow for RNA Silencing is as follows:

(For a more detailed explanation of the mechanism, refer to the RNAi:Cellular mechanism article.)

1: RNA with inverted repeats hairpin/panhandle constructs --> 2: dsRNA --> 3: miRNAs/siRNAs --> 4: RISC --> 5: Destruction of target mRNA

1. It has been discovered that the best precursor to good RNA silencing is to have single stranded antisense RNA with inverted repeats which, in turn, build small hairpin RNA and panhandle constructs. The hairpin or panhandle constructs exist so that the RNA can remain independent and not anneal with other RNA strands.
2. These small hairpin RNAs and/or panhandles then get transported from the nucleus to the cytosol through the nuclear export receptor called exportin-5, and then get transformed into a dsRNA, a double stranded RNA, which, like DNA, is a double stranded series of nucleotides. If the mechanism didn't use dsRNAs, but only single strands, there would be a higher chance for it to hybridize to other "good" mRNAs. As a double strand, it can be kept on call for when it is needed.
3. The dsRNA then gets cut up by a Dicer into small (21-28 nt = nucleotides long) strands of miRNAs (microRNAs) or siRNAs (short interfering RNAs.) A Dicer is an endoribonuclease RNase, which is a complex of a protein mixed with strand(s) of RNA.
4. Lastly, the double stranded miRNAs/siRNAs separate into single strands; the antisense RNA strand of the two will combine with another endoribonuclease enzyme complex called RISC (RNA-induced silencing complex), which includes the catalytic component Argonaute, and will guide the RISC to break up the "perfectly complementary" target mRNA or viral genomic RNA so that it can be destroyed.
5. It means that based on a short sequence specific area, a corresponding mRNA will be cut. To make sure, it will be cut in many other places as well. (If the mechanism only worked with a long stretch, then there would be higher chance that it would not have time to match to its complementary long mRNA.) It has also been shown that the repeated-associated short interference RNAs (rasiRNA) have a role in guiding chromatin modification.

RNA silencing is the mechanism that our cells (and cells from all kingdoms) use to fight RNA viruses and transposons (which originate from our own cells as well as from other vehicles). In the case of RNA viruses, these get destroyed immediately by the mechanism cited above. In the case of transposons, it's a little more indirect. Since transposons are located in different parts of the genome, the different transcriptions from the different promoters produce complementary mRNAs that can hybridize with each other. When this happens, the RNAi machinery goes into action, debilitating the mRNAs of the proteins that would be required to move the transposons themselves.{{cite book |author1=Matthew P Scott |author2=Lodish, Harvey F. |author3=Arnold Berk |author4=Kaiser, Chris |author5=Monty Krieger |author6=Anthony Bretscher |author7=Hidde Ploegh |author8=Angelika Amon |title=Molecular Cell Biology |publisher=W. H. Freeman |location=San Francisco |year=2012 |isbn=978-1-4292-3413-9 }}

For a detailed explanation of the down-regulation of genes, see RNAi:downregulation of genes

For a detailed explanation of the up-regulation of genes, see RNAi:upregulation of genes

The same way that RNA silencing regulates downstream target mRNAs, RNA silencing itself is regulated. For example, silencing signals get spread between cells by a group of enzymes called RdRPs (RNA-dependent RNA polymerases) or RDRs.

Growing understanding of small RNA gene-silencing mechanisms involving dsRNA-mediated sequence-specific mRNA degradation has directly impacted the fields of functional genomics, biomedicine, and experimental biology. The following section describes various applications involving the effects of RNA silencing. These include uses in biotechnology, therapeutics, and laboratory research. Bioinformatics techniques are also being applied to identify and characterize large numbers of small RNAs and their targets.

Artificial introduction of long dsRNAs or siRNAs has been adopted as a tool to inactivate gene expression, both in cultured cells and in living organisms. Structural and functional resolution of small RNAs as the effectors of RNA silencing has had a direct impact on experimental biology. For example, dsRNA may be synthesized to have a specific sequence complementary to a gene of interest. Once introduced into a cell or biological system, it is recognized as exogenous genetic material and activates the corresponding RNA silencing pathway. This mechanism can be used to effect decreases in gene expression with respect to the target, useful for investigating loss of function for genes relative to a phenotype. That is, studying the phenotypic and/or physiologic effects of expression decreases can reveal the role of a gene product. The observable effects can be nuanced, such that some methods can distinguish between “knockdown” (decrease expression) and “knockout” (eliminate expression) of a gene.{{cite journal | vauthors = Voorhoeve PM, Agami R | title = Knockdown stands up | journal = Trends in Biotechnology | volume = 21 | issue = 1 | pages = 2–4 | date = Jan 2003 | pmid = 12480342 | doi = 10.1016/S0167-7799(02)00002-1 }} RNA interference technologies have been noted recently as one of the most widely utilized techniques in functional genomics.{{cite journal | vauthors = Karagiannis TC, El-Osta A | title = RNA interference and potential therapeutic applications of short interfering RNAs | journal = Cancer Gene Therapy | volume = 12 | issue = 10 | pages = 787–95 | date = Oct 2005 | pmid = 15891770 | doi = 10.1038/sj.cgt.7700857 | doi-access = free }} Screens developed using small RNAs have been used to identify genes involved in fundamental processes such as cell division, apoptosis and fat regulation.

Since at least the mid-2000s, there has been intensifying interest in developing short interfering RNAs for biomedical and therapeutic applications.{{cite journal | vauthors = Kim DH, Rossi JJ | title = Strategies for silencing human disease using RNA interference | journal = Nature Reviews Genetics | volume = 8 | issue = 3 | pages = 173–84 | date = Mar 2007 | pmid = 17304245 | doi = 10.1038/nrg2006 | s2cid = 5781886 }} Bolstering this interest is a growing number of experiments which have successfully demonstrated the clinical potential and safety of small RNAs for combatting diseases ranging from viral infections to cancer as well as neurodegenerative disorders.{{cite journal | vauthors = Stevenson M | title = Therapeutic potential of RNA interference | journal = The New England Journal of Medicine | volume = 351 | issue = 17 | pages = 1772–7 | date = Oct 2004 | pmid = 15496626 | doi = 10.1056/NEJMra045004 | s2cid = 1531568 }} In 2004, the first Investigational New Drug applications for siRNA were filed in the United States with the Food and Drug Administration; it was intended as a therapy for age-related macular degeneration. RNA silencing in vitro and in vivo has been accomplished by creating triggers (nucleic acids that induce RNAi) either via expression in viruses or synthesis of oligonucleotides.{{cite journal | vauthors = Davidson BL, McCray PB | title = Current prospects for RNA interference-based therapies | journal = Nature Reviews Genetics | volume = 12 | issue = 5 | pages = 329–40 | date = May 2011 | pmid = 21499294 | doi = 10.1038/nrg2968 | pmc = 7097665 }} Optimistically many studies indicate that small RNA-based therapies may offer novel and potent weapons against pathogens and diseases where small molecule/pharmacologic and vaccine/biologic treatments have failed or proved less effective in the past. However, it is also warned that the design and delivery of small RNA effector molecules should be carefully considered in order to ensure safety and efficacy.

The role of RNA silencing in therapeutics, clinical medicine, and diagnostics is a fast developing area and it is expected that in the next few years some of the compounds using this technology will reach market approval. A report has been summarized below to highlight the many clinical domains in which RNA silencing is playing an increasingly important role, chief among them are ocular and retinal disorders, cancer, kidney disorders, LDL lowering, and antiviral. The following table displays a listing of RNAi based therapy currently in various phases of clinical trials. The status of these trials can be monitored on the ClinicalTrials.gov website, a service of the National Institutes of Health (NIH).{{Cite web|url=https://clinicaltrials.gov/|title=ClinicalTrials.gov|website=clinicaltrials.gov|access-date=2024-03-04|archive-date=2020-12-18|archive-url=https://web.archive.org/web/20201218140935/https://www.clinicaltrials.gov/|url-status=live}} Of note are treatments in development for ocular and retinal disorders, that were among the first compounds to reach clinical development. AGN211745 (sirna027) (Allergan) and bevasiranib (Cand5) (Opko) underwent clinical development for the treatment of age-related macular degeneration, but trials were terminated before the compounds reached the market. Other compounds in development for ocular conditions include SYL040012 (Sylentis) and QPI-007 (Quark). SYL040012 (bamosinan) is a drug candidate under clinical development for glaucoma, a progressive optic neurdegeneration frequently associated to increased intraocular pressure; QPI-007 is a candidate for the treatment of angle-closure glaucoma and Non-arteritic anterior ischaemic optic neuropathy; both compounds are currently undergoing phase II clinical trials. Several compounds are also under development for conditions such as cancer and rare diseases.

As with conventional manufactured drugs, the main challenge in developing successful offshoots of the RNAi-based drugs is the precise delivery of the RNAi triggers to where they are needed in the body. The reason that the ocular macular degeneration antidote was successful sooner than the antidote with other diseases is that the eyeball is almost a closed system, and the serum can be injected with a needle exactly where it needs to be. The future successful drugs will be the ones who are able to land where needed, probably with the help of nanobots. Below is a rendition of a table that shows the existing means of delivery of the RNAi triggers.

The scientific community has been quick to harness RNA silencing as a research tool. The strategic targeting of mRNA can provide a large amount of information about gene function and its ability to be turned on and off. Induced RNA silencing can serve as a controlled method for suppressing gene expression. Since the machinery is conserved across most eukaryotes, these experiments scale well to a range of model organisms.{{cite journal | vauthors = Zeng Y, Wagner EJ, Cullen BR | title = Both natural and designed micro RNAs can inhibit the expression of cognate mRNAs when expressed in human cells | journal = Molecular Cell | volume = 9 | issue = 6 | pages = 1327–33 | date = Jun 2002 | pmid = 12086629 | doi = 10.1016/s1097-2765(02)00541-5 | doi-access = free }} In practice, expressing synthetic short hairpin RNAs can be used to reach stable knock-down.{{cite journal | vauthors = Paddison PJ, Caudy AA, Bernstein E, Hannon GJ, Conklin DS | title = Short hairpin RNAs (shRNAs) induce sequence-specific silencing in mammalian cells | journal = Genes & Development | volume = 16 | issue = 8 | pages = 948–58 | date = Apr 2002 | pmid = 11959843 | doi = 10.1101/gad.981002 | pmc=152352}} If promoters can be made to express these designer short hairpin RNAs, the result is often potent, stable, and controlled gene knock-down in both in vitro and in vivo contexts.{{cite journal | vauthors = Dickins RA, Hemann MT, Zilfou JT, Simpson DR, Ibarra I, Hannon GJ, Lowe SW | title = Probing tumor phenotypes using stable and regulated synthetic microRNA precursors | journal = Nature Genetics | volume = 37 | issue = 11 | pages = 1289–95 | date = Nov 2005 | pmid = 16200064 | doi = 10.1038/ng1651 | s2cid = 15586239 }} Short hairpin RNA vector systems can be seen as roughly analogous in scope to using cDNA overexpression systems.{{cite book | vauthors = Rigó G, Papdi C, Szabados L | chapter = Transformation Using Controlled cDNA Overexpression System | title = Plant Salt Tolerance | series = Methods in Molecular Biology | volume = 913 | pages = 277–90 | year = 2012 | pmid = 22895767 | doi = 10.1007/978-1-61779-986-0_19 | isbn = 978-1-61779-985-3 }} Overall, synthetic and natural small RNAs have proven to be an important tool for studying gene function in cells as well as animals.{{cite journal | vauthors = Silva JM, Li MZ, Chang K, Ge W, Golding MC, Rickles RJ, Siolas D, Hu G, Paddison PJ, Schlabach MR, Sheth N, Bradshaw J, Burchard J, Kulkarni A, Cavet G, Sachidanandam R, McCombie WR, Cleary MA, Elledge SJ, Hannon GJ | title = Second-generation shRNA libraries covering the mouse and human genomes | journal = Nature Genetics | volume = 37 | issue = 11 | pages = 1281–8 | date = Nov 2005 | pmid = 16200065 | doi = 10.1038/ng1650 | s2cid = 17346898 }}

Bioinformatics approaches to identify small RNAs and their targets have returned several hundred, if not thousands of, small RNA candidates predicted to affect gene expression in plants, C. elegans, D. melanogaster, zebrafish, mouse, rat, and human.{{cite journal | vauthors = Berezikov E, Guryev V, van de Belt J, Wienholds E, Plasterk RH, Cuppen E | title = Phylogenetic shadowing and computational identification of human microRNA genes | journal = Cell | volume = 120 | issue = 1 | pages = 21–4 | date = Jan 2005 | pmid = 15652478 | doi = 10.1016/j.cell.2004.12.031 | s2cid = 16403721 | doi-access = free }} These methods are largely directed to identifying small RNA candidates for knock-out experiments but may have broader applications. One bioinformatics approach evaluated sequence conservation criteria by filtering seed complementary target-binding sites. The cited study predicted that approximately one third of mammalian genes were to be regulated by, in this case, miRNAs.{{cite journal | vauthors = Lewis BP, Burge CB, Bartel DP | title = Conserved seed pairing, often flanked by adenosines, indicates that thousands of human genes are microRNA targets | journal = Cell | volume = 120 | issue = 1 | pages = 15–20 | date = Jan 2005 | pmid = 15652477 | doi = 10.1016/j.cell.2004.12.035 | s2cid = 17316349 | doi-access = free }}

One aspect of RNA silencing to consider is its possible off-target affects, toxicity, and delivery methods. If RNA silencing is to become a conventional drug, it must first pass the typical ethical issues of biomedicine.{{Cite book|title=Principles of Biomedical Ethics, 6th ed|last1=Beauchamp|first1=TL|last2=Childress|first2=JF|publisher=Oxford University Press|year=2009|location=Oxford}} Using risk-benefit analysis, researchers can determine whether RNA silencing conforms to ethical ideologies such as nonmaleficence, beneficence, and autonomy.{{Cite journal|last1=Ebbesen|first1=Mette|last2=Jensen|first2=Thomas G.|last3=Andersen|first3=Svend|last4=Pedersen|first4=Finn Skou|date=2008-06-25|title=Ethical Perspectives on RNA Interference Therapeutics|journal=International Journal of Medical Sciences|volume=5|issue=3|pages=159–168|issn=1449-1907|pmc=2443345|pmid=18612370|doi=10.7150/ijms.5.159}}

There is a risk of creating infection-competent viruses that could infect non-consenting people.{{Cite journal|last=Cullen|first=RC|date=2006|title=Enhancing and Confirming the Specificity of RNAi Experiments|journal=Nature Methods|volume=3|issue=9|pages=677–681|doi=10.1038/nmeth913|pmid=16929311|s2cid=13320443}} There is also a risk of affecting future generations based on these treatments. These two scenarios, in respect to autonomy, is possible unethical. At this moment, unsafe delivery methods and unintended aspects of vector viruses add to the argument against RNA silencing.

In terms of off-target effects, siRNA can induce innate interferon responses, inhibit endogenous miRNAs through saturation, and may have complementary sequences to other non-target mRNAs. These off-targets could also have target up-regulations such as oncogenes and antiapoptotic genes. The toxicity of RNA silencing is still under review as there are conflicting reports.{{Cite journal|last1=Elbashir|first1=SM|last2=Martinez|first2=J|last3=Patkaniowska|first3=A|last4=Lendeckel|first4=W|last5=Tuschl|first5=T|date=2001|title=Functional Anatomy of siRNA for Mediating Efficient RNAi in Drosophilia melanogaster Embryo Lysate|journal=EMBO Journal |volume=20|issue=23|pages=6877–88|doi=10.1093/emboj/20.23.6877|pmid=11726523|pmc=125328}}

File:RNAi publication statistics.png

RNA silencing is quickly developing, because of that, the ethical issues need to be discussed further. With the knowledge of general ethical principles, we must continuously perform risk-benefit analysis.

• RNAi
• siRNA
• miRNA
• piwiRNA
• rasiRNA

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{{DEFAULTSORT:Rna Silencing}}

Category:RNA

Category:Gene expression