RNA interference: traveling in the cell and gaining functions?

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44Review TRENDS in Genetics Vol.19 No.1 January 2003stem-loop precursors. In animals, precursor miRNAs (premiRNAs)are apparently encoded as imperfectly pairinginverted repeats of 60–70 nt [55,70]. In plants, however,some predicted pre-miRNAs are more than three timeslonger than those in animals and involve more extensive orcomplex stem-loop structures [57,59]. Most pre-miRNAsare encoded in intergenic regions and are probablytranscribed from autonomous promoters [57,59,70](Fig. 2); however, several pre-miRNAs are clustered ingenomic regions and are apparently synthesized as asingle polycistronic RNA [70,73]. In addition, a fewmiRNAs might also be processed from introns, asbyproducts of pre-mRNA splicing, or even from putativeprotein-coding RNAs [57,59,70].In mammalian cells, maturation of miRNAs involves atleast two steps [73]. Both single and clustered miRNAsseem to be expressed as longer transcripts that areprocessed into pre-miRNAs of 60–70 nt in the nucleus[73] (Fig. 2, nucleus, ‘pre-processing’). The pre-miRNAsare then exported to the cytoplasm and processed by Dicer,and possibly by other factors, into mature miRNAs ofabout 21 nt [73] (Fig. 2, cytoplasm). Recently, numeroushuman miRNAs have been found to be associated in a 15SRNP complex that includes Gemin3, Gemin4 and eIF2C2[55]. The human homolog of let-7 is a component of thisMonocistronicunitTRENDS in GeneticsPre-processingCAFPre-miRNARNP?Pre-miRNAPolycistronicunitDicerPmiRNANucleusCytoplasmPmiRNAA nPRNAdegradationIntron containedunitFunction(s)?PmiRNP?miRNP PA nPTranslationrepressionFig. 2. Model of the origin and potential functions of microRNAs. The indicatedpathways are mainly supported by experimental evidence in Arabidopsis, Caenorhabditiselegans, Drosophila and human. Green RNA, endogenously transcribedsingle-stranded RNA; red RNA, microRNAs and microRNA precursors. Protein orprotein complexes are indicated by yellow boxes: miRNP, ribonucleoprotein complexcontaining human Gemin3, Gemin4 and eIF2C2. Several steps related tomiRNA biogenesis and function are hypothetical (see text for details). Recent evidencesuggests that the miRNP complex is equivalent to the RISC complex (Fig. 1)in human cells [17].?microRNP (miRNP) and can direct the cleavage of aperfectly complementary target RNA [17]. These observationshave led to the proposal that the miRNP is thehuman RISC and can carry out both target cleavage in theRNAi pathway and translational control in the miRNApathway [17].The biogenesis of at least some plant miRNAs seems tobe different to that in animals. Although CAF, a homolog ofDicer, has been implicated in the generation of miRNAs inArabidopsis [59], pre-miRNAs are detected poorly or not atall in Arabidopsis, whereas mature miRNAs are observedreadily [57,59]. In addition, whereas in metazoans premiRNAsincrease when Dicer activity is reduced [74],mutants of CAF show significantly lower levels of miRNAswithout any concomitant accumulation of precursor [59].This suggests that because CAF is predicted to be anuclear protein [60], at least some plant miRNAs might beprocessed either co-transcriptionally or shortly aftertranscription from transient primary transcripts (Fig. 2,nucleus). Mature miRNAs might then be transported tothe cytosol as part of a RNP complex and/or they mighthave a role in the nuclear compartment (Fig. 2).Unlike animal miRNAs, whose targets are difficult toidentify by sequence complementarity, some Arabidopsisand rice miRNAs have been found to show perfect ornear-perfect complementarity to potential mRNA targets[71,72]. This suggested that they might function as siRNAs[71,72]. Indeed, an Arabidopsis miRNA (miR171/miRNA39)was found to direct specific cleavage of complementarytranscripts corresponding to a family of transcriptionalregulators [72]. Thus, despite the presence of miRNAs inboth plants and animals, current studies imply differencesin the structure of predicted precursor RNAs, in the timingand (presumed) subcellular localization of processing and,possibly, in the actual function of mature miRNAs.Biological functions of RNA-mediated silencingMutational inactivation of components of the RNAi andPTGS machinery affects at least three distinct eukaryoticprocesses: the defense response against viruses, transposonmobility and the development of multicellular organisms[1–5,54]. The RNAi and PTGS processes wereoriginally proposed to have evolved to counteract genomicparasites [1–4,54,75], but it is becoming apparent thatdsRNA-mediated mechanisms are also involved in thenormal regulation of endogenous genes. Drosophila malesuse an RNAi mechanism to degrade Stellate transcripts[75]; one miRNA has been implicated in the specificcleavage of target mRNAs corresponding to the SCARE-CROW-like transcription factors in Arabidopsis [72]; andseveral miRNAs that are complementary to protein-codingsequences have been identified in Arabidopsis and rice[57,59,71].Intriguingly, a high proportion of the predicted miRNAtargets function as developmental regulators in plants,suggesting that miRNAs might have a role in coordinatinggrowth and development [71]. Consistent with thisinterpretation, Arabidopsis CAF mutants, which aredefective in miRNA processing, show pronounced developmentalalterations [59,60].InC. elegans, developmentaldefects resulting from reduced function of Dicer and thehttp://tigs.trends.com
Review TRENDS in Genetics Vol.19 No.1 January 2003 45Argonaute-like proteins ALG-1 and ALG-2 have also beenattributed to the improper processing of miRNA precursorsand a reduction in mature stRNA expression [3,74].Itis therefore tempting to propose the existence of ancient,miRNA-mediated mechanisms that regulate endogenousgenes in eukaryotes. But most endogenous small RNAscloned from animals, and several from plants, do notmatch protein-coding or structural RNAs and theirmechanistic roles, with the exception of C. elegans stRNAs,remain unknown [55,70,74].Dicer processes precursor dsRNAs to make both siRNAsand miRNAs. In organisms encoding only one Dicer, asingle pathway might handle small RNAs. In other words,the miRNA and siRNA pathways might be interchangeablefrom biogenesis of the small RNA to interaction withits target. The final outcome, such as mRNA degradationor, for example, translational repression, would depend onthe degree of complementarity to the target RNA and,presumably, on associated effector proteins [3,17]. Thismodel is consistent with the findings that short hairpinRNAs, resembling miRNA precursors, can induce RNAi onperfectly homologous target mRNAs [3,17], and that thehuman RISC seems to be equivalent to the 15S miRNPthat is associated with many miRNAs [17,55]. Alternatively,two distinct pathways, intersecting at the Dicercatalyzed step, might be involved in the generation andfunction of at least some miRNAs and siRNAs [3]. This issupported by the requirement of different Argonauteproteins for the production of functional stRNAs or siRNAsin C. elegans [16,19,74]. In organisms where Dicer isencoded by a multigene family, such as Arabidopsis [57,58],cytoplasmic and nuclear processing pathways mightoperate.Recent findings have also implicated small RNAs inchromatin and/or DNA modifications and genomerearrangements, such as heterochromatin formationin S. pombe and DNA elimination in Tetrahymena[1,35,49,65–68]. This suggests that dsRNA-mediatedprocesses might have a role in genome organization andtranscriptional control. It is clear that despite muchprogress resulting from a combination of genetics andbiochemistry, we are only just beginning to understand themechanistic complexity of RNA-mediated silencing, itsbiological implications, and the differences and similaritiesamong different eukaryotes.AcknowledgementsI thank S. Jacobsen, T. Clemente and members of mylaboratory for helpful comments. My apologies to colleagueswhose work could not be cited due to spacelimitations. Supported by NIH grant GM62915.References1 Matzke, M. et al. (2001) RNA: guiding gene silencing. Science 293,1080–10832 Vance, V. and Vaucheret, H. (2001) RNA silencing in plants – defenseand counterdefense. Science 292, 2277–22803 Hannon, G.J. (2002) RNA interference. Nature 418, 244–2514 Plasterk, R.H.A. (2002) RNA silencing: the genome’s immune system.Science 296, 1263–12655 Zamore, P.D. (2002) Ancient pathways programmed by small RNAs.Science 296, 1265–12696 Zamore, P.D. et al. (2000) RNAi: double-stranded RNA directs theATP-dependent cleavage of mRNA at 21 to 23 nucleotide intervals. Cell101, 25–337 Bernstein, E. et al. (2001) Role for a bidentate ribonuclease in theinitiation step of RNA interference. Nature 409, 363–3668 Hammond, S.M. et al. (2000) An RNA-directed nuclease mediates posttranscriptionalgene silencing in Drosophila cells. Nature 404,293–2969 Nykänen, A. et al. (2001) ATP requirements and small interfering RNAstructure in the RNA interference pathway. Cell 107, 309–32110 Martinez, J. et al. 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Science 297, 2056–206018 Cerutti, L. et al. (2000) Domains in gene silencing and celldifferentiation proteins: the novel PAZ domain and redefinition ofthe Piwi domain. Trends Biochem. Sci. 25, 481–48219 Tabara, H. et al. (2002) The dsRNA binding protein RDE-4 interactswith RDE-1, DCR-1, and a DExH-box helicase to direct RNAi inC. elegans. Cell 109, 861–87120 Grishok, A. et al. (2000) Genetic requirements for inheritance of RNAiin C. elegans. Science 287, 2494–249721 Ketting, R.F. et al. (1999) Mut-7 of C. elegans, required for transposonsilencing and RNA interference, is a homolog of Werner syndromehelicase and RNaseD. Cell 99, 133–14122 Kennerdell, J.R. and Carthew, R.W. (1998) Use of dsRNA-mediatedgenetic interference to demonstrate that frizzled and frizzled 2 act inthe wingless pathway. Cell 95, 1017–102623 Svoboda, P. et al. (2000) Selective reduction of dormant maternalmRNAs in mouse oocytes by RNA interference. Development 127,4147–415624 Ratcliff, F. et al. 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(2001) Specific interference with gene expression inducedby long, double-stranded RNA in mouse embryonal teratocarcinomacell lines. Proc. Natl Acad. Sci. USA 98, 14428–1443331 Cikaluk, D.E. et al. (1999) GERp95, a membrane-associated proteinthat belongs to a family of proteins involved in stem cell differentiation.Mol. Biol. Cell 10, 3357–337232 Wesley, S.V. et al. (2001) Construct design for efficient, effective andhigh-throughput gene silencing in plants. Plant J. 27, 581–59033 Kalidas, S. and Smith, D.P. (2002) Novel genomic cDNA hybridshttp://tigs.trends.com
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