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REVIEWS Friedrich Miescher Insmute for Biomedical Research 4002 Basel Switzerland Department of Biochemisz and McGill Cancer Center McGill University MonUeal Ou ebec Canada H56 7Y6 Correspondence to IMF orNS emails wiloldfilimwicfomi ch nahumsonenberg mcgill ca doi l O l 058nrg2290 Published online l6 Jan uary 2008 answers in sight Mechanisms of post transcriptional regulation by microRNAs are the MicroRNAs miRNAs which are approximately 21 a nucleotideelong RNA regulators of gene expression have become a major focus ofresearch in molecular biology Although for a long time they were considered to be exclusive to multicellular organisms and possibly essential for the transition to a more complex organ ism design the recent identification of miRNAs in the unicellular algae Chlamydomonas reinhardtii indie cates that miRNAs are probably evolutionarily older than originally thought One to two hundred miRNAs are expressed in lower metazoans and plants but at least a thousand are predicted to operate in humans Functional studies indicate that miRNAs participate in the regulation of almost every cellular process investigated and that changes in their expression are observed in 7 and might underlie 7 human patholoe gies including cancer ls gi These ndings are perhaps not so surprising as bioinformatic predictions indicate that mammalian miRNAs can regulate 30 of all proteinecoding genes With just one possible exception noted so farg miRNAs control gene expression postetranscriptione ally by regulating mRNA translation or stability in the cytoplasm o Hi However further functions of miRNAs seem likely For example by virtue of base pairing to RNA miRNAs could regulate preemRNA processing in the nucleus or act as chaperones that modify mRNA structure or modulate mRNArprotein interactions Indications that mammalian miRNAs can be imported into the nucleus or even secreted from the cell16 will Witold Filipowiczquot Suvendra N Bhattacharyya and Nahum Sonenberg Abstractl MicroRNAs constitute a large family of small approximately 21 nucleotide long non coding RNAsthat have emerged as key post transcriptional regulators of gene expression in metazoans and plants In mammals microRNAs are predicted to control the activity of approximately 30 of all protein coding genes and have been shown to participate in the regulation of almost eveiy cellular process investigated so far By base pairing to mRNAs microRNAs mediate translational repression or mRNA degradation This Review summarizesthe current understanding of the mechanistic aspects of microRNA induced repression of translation and discusses some of the controversies regarding different modes of microRNA function motivate searches for currently unidentified functions for this class of molecule What is already certain is that the discovery of miRNAs has revealed an important new dimension in the complexity of postetranscriptional regulation of eukaryotic gene expression We are begin ning to understand why the 3 UTRs of mRNA with which the miRNAs and other factors interact are often so long and so important for gene function The mechanistic details of the function of miRNAs in repressing protein synthesis are still poorly understood miRNAs can affect both the translation and stability of mRNAs but the results from studies conducted in dif ferent systems and different laboratories have o en been contradictory a comprehensive and lucid picture of the mechanism of miRNAemediated repression is difficult to elaborate In this Review after brie y introducing miRNAs and their biogenesis we summarize what is currently known about the mechanistic aspects of their function in controlling mRNA stability and translation focusing primarily on animal cells We also discuss the cellular localization and reversibility of miRNAemediated repressioni For further recent Reviews covering these topics see REFS iiri 41718 and more general infore mation about the biogenesis diversity and function of miRNAs canbe found in REFS i731 9723 miRNA and microribonucleoprotein biogenesis miRNA precursors fold into imperfect dsRNAelike hairpins from which miRNAs are excised in two steps both of which are catalyzed by Drosha also known as 101i FEBRUARY 2008 i VOLUME 9 2008 Namre Publishing Group wwwnaturecomreviewsgenetics REVIEWS Box 1 Biogenesis of miroRNAs and their assembly into microribonucleoproteins specialized functions severaldifferent miRNAs PriimiRNAs fold into hairpin structures containing imperfectly baseipaired stems and are processed in two steps catalysed bythe RNase I type endonucleases Drosha also known as RN3 and Dicer Both Drosha and Dicer function in complexes with proteins containing dsRNAibinding domains dsRBDs The Drosha partners arethepashu protein in Drosophila melanoguster or DiGeorge syndrome critical region gene 8 DGC R8 in mammals The DroshaiDGCR8 complex processes priimiRNAs to 707nucleotide hairpins known as preimiRNAs 32 Some splicediout Q35 introns in Caenorhabditis elegans D melanoguster and mam ma ls correspond preciselyto preimiRNAs mirtrons thus circumventing the V requirement for DroshaiDGCR8 REFS l 254 27 Plant genomes do not encode Drosha homologues and all miRNA biogenesis steps inArabidopsis thalianu are carried out by one of four Dicerilike proteins In animals pref miRNAs are transported to the cytoplasm by exportinS where they are cleaved by Dicer complexed with TAR RNA binding protein M in mammals and the loguucious gene product in D melanogaster to yield 207bp miRNA duplexes One strand is then selected to function as a mature miRNA while the other strand is degraded Occasionally both arms ofthe preimiRNA hairpin give riseto mature miRNAs 3 2 Vertebrates and C elegans contain single dicergenes but some other organisms like D melanogasterand plants express two or more Dicer proteins that function as heterodimers with different dsRBD proteins and have 1 321 24 El transcriptsA single priimiRNA often contains sequences for a microRNAs miRNAs are processed from precursor molecules prii miRNAs which are eithertranscribed from independent miRNA genes orare portions of introns of proteinicoding RNA polymerase Following their processing miRNAs are assembled into ribonucleoprotein RNP complexes called microiRNPs miRNPs or miRNAiinduced silencing complexes miRISCs The assembly is a dynamic process usually coupled with preimiRNA processing by Dicer but its details are not well understood 32 The key components of miRNPs are proteins ofthe Argonaute AGO family Ofthe many paralogues encoded in plant and metazoan genomes usuallyonlysome 7 known as AGO proteins 7 function in miRNA or both miRNA and small interfering RNA siRNA pathways In mammals fourAGO proteins AGOl to AGO4 function in the miRNA repression but onlyAGOZ functions in RNAi In C elegans which expresses Z7 Argonaute proteins RDEl is involved in RNAi and ALG1 and ALGZ function in the miRNA pathway 5 Apart from AGOs miRNPs can contain further proteins that function as regulatoryfactors or effectors mediating inhibitoryfunction of miRNPs Bu Examples are the fragileX mental retardation protein FM RP and its Strand selection miRNP assembly i Transcription Pl l l TllRNA ertron 1 AAplicing LAW PrermlRNA portlnS Nucleus 6 E3 we W0 l Maturation Cytoplasm CCR47 D melanoguster orthologue of FXR which are RNAibi ndi ng proteins known to act as modulators oftranslation particularly in neurons reviewed in REF l 28 Some Pibody components such as GW18Z and RCKp54 see BOX 4 interact with miRNP Dicer Ah RNase lll famlly ehdohucleasethat processes dsRNA and prermlRNAs lhto small lhterfenhg RNAs and mlcroRNAs respectlvely Small interfering RNAs isNAs Small RNAs that are slmllar ll39l slze to mlcroRNAs butare denved from the progresslve cleavage of long dsRNA by cher Upon lhcorporatloh lhto ah RlSC isNAs guldethe ehdohucleolytlc cleavage of the target mRNA AGO proteins and are essentialfor inducing repression 7892104 M and the endoribonuclease cher 7 enzymes of the RNase 111 family BOX l The nal processing of the 707nucleotide preemiRNA hairpin byMyields 21 ebp miRNA duplexes with protruding 27nucleotide 3 ends similar to small lnterfenng RNAs siRNAs operating in RNA lnterference RNAi Generally the strand with the 5 terminus located at the thermodye namically lessestable end of the duplex is selected to function as a mature miRNA and the other strand is degraded3gt gt miRNAs function as components of ribonucleoproe tein RNP complexes or RNArlnduced sllencmg com plexes RISCs referred to as either mlcrornbonucleoprotems miRNPs or miRNAeinduced silencing complexes miRlSCs BOX l The most important and best characterized components of miRNPs are proteins Endonucleolytlc cleavage Translatlonal represslon or deadenylatlon ofthe Argonaute familyz m Mammals contain four Argonaute AGO proteins AGOl to AGO4 Their function in miRNA repression is demonstrated by their association with similar sets of miRNAs and their ability to repress protein synthesis when arti cially tethered to the mRNA 3 UTRZHB Flc l AGOZ is the only AGO that functions in RNAi because its RNaseHelike Peelement induced wimpy testis PIWI domain but not those of the other AGOs can cleave mRNA at the cene tre of the siRNArmRNA duplex BOX l In Drosophila melanogaster Argonautel is dedicated to the miRNA pathway and Argonaute2 mainly functions in RNAiZ Apart from the AGO proteins miRNPs often include other proteins which probably function as miRNP assembly or regulatory factors or as effectors mediating the repressive miRNP functions NATURE REVIEWS l CENEY 1C5 2008 Nature Publishing Group VOLUME 9 lFEBRUARY 2008 103 REVIEWS RNA interference The dSRNArl nduced seq uencer homology dependent gene silencing mechanism The dsRNA is processed to SlRNAS which upon incorporation into an RlSC guide the endonucleolytic cleavage of the target mRNA RNArinduced silencing complex RlSC The ribonucleoprotein cornplex consisting ofsrnall interfering RNA and an AGO protein that harboum the shcer activity which cleaves an mRNA target in the middle ofsiRNArrnRNA complementarity microrribonucleoprotein miRNP A ribonucleoprotein complex containing a miRNA and one of the AGO proteins Depending on the identity of the associated AGO it might harboura shcer activity characteristic ofan RlSC mTG cap The Trmethylguanosine rnTC that is linked by a 575 triphosphate bridge to thefimt transcribed nucleoside at the 5 end of eukaryotic mRNAs a Capdependent reporter mRNAs n n f39 ApppN AAAAA b IREcontaining mRNA reporters ApppN or m7GpppN m7GpppN c Tethering reporters AAAAA AAAAA N AGOZ or N GWTBZ AAAAA 1 Figure 1 l Examples of reporters used in studies of microRNA function a l Capped reporters containing multiple mRNA binding sites mRNAs containing a nonifunctionalApppN cap instead ofthe 7methylguanosine m7G cap can be prepared by in vitra transcription with T7 phage RNA polymerase and either introduced into cells by transfection or used in studies in cellifree extractsl b l lVlonoicistronic and biicistronic reporters containing a Viral internal ribosomal entrysites lRESl Reporters containing ApppN or pppN at the S end can be prepared byin vitra m7GpppN BoxrB hairpins transcription and then transfected into cells c l Reporters used to studythe effects oftethering to mRNA of Argonaute AGO proteins or GWISZ on protein synthesisiThe investigated proteins are expressed as fusions with a phage lNipeptide which can bind the short BoxiB hairpins that are inserted tothe mRNA 3 UTRU The lNipeptideiBoxiB system can also be used totether initiation factors eF4E or eF4G tothe intercistronic region of the biicistronic reporter Reporters that are generated in vitra and used for either RNAtransfection experiments or studies in cellifree extracts can be prepared with orwithout the poly A tail M 1 5 Reporters can also differ in the number of miRNA binding sites that are present in the 3 UTR Reporters that are devoid of microRNA binding sites orthat contain mutated sites are used as controls Principles of miRNA mRNA interactions In plants miRNAs generally base pair to mRNAs with nearly perfect complementarity and trigger endonucleolytic mRNA cleavage by an RNAiilike mechanism In rare instances a similar mechanism is used by vertebrate and viral miRNAs see REF ET for examples However in most cases metazoan miRNAs pair imperfectly with their targets following a set of rules determined by experimental and bioinformatic analyses30 3 BOX ET The most stringent requirement is a contiguous and perfect base pairing of the miRNA nucleotides 278 representing the seed region which nucleates the interaction With few exceptions miRNA binding sites in metazoan mRNAs lie in the 3 UTR and are usually present in multiple copies 7 this is required for effective repression of translation30 3 i miRNAs also exert their repressive function when their binding sites are arti cially placed in 5 UTRs or coding regions35gt35 although the physiological effects of the codingiregion sites might be only marginal37i Modes oftranslational repression mRNA translation can be divided into three steps initii ation elongation and termination Initiation starts with the recognition of the mRNA S Aterminal cap structure m7GpppN in which N is any nucleotide by the eIF4E subunit of the eukaryotic translation initiation factor eIF eIF4F which also contains eIF4G an important scaffold for the assembly of the ribosome initiation complex BOX 3L Interaction of eIF4G with another initiation factor eIF3 facilitates the recruitment of the 405 ribosomal subunit eIF4G also interacts with the polyadenylateibinding protein 1 PABPI I The ability of eIF4G to interact simultaneously with eIF4E and PABPI brings the two ends of the mRNA in close proximity mli This circularization stimulates transla7 tion initiation by increasing the affinity of eIF4E for m7GpppN and might facilitate ribosome recycling h Some cellular and viral mRNAs initiate translation independently ofthe mic cap and eIF4E in this case 405 ribosomes are recruited to the mRNA through 104 l FEBRUARY 2008 l VOLUME 9 2008 Namre Publishing Group wwwnaturecomreviewsgenetics Box 2 Principles of microRNA m RNA interactions Bulge I gtl5 nucleotides 1 N NN 039l l quot 8 i AAAAAA miRNA l l6 l3 Bulge Seed 3 complementarity reg on MicroRNAs miRNAs interactwith their mRNA targets by base pairing In plants most miRNAs base pairto mRNAs with nearly perfect complementarity and induce mRNA degradation byan RNAielike mechanism ithe mRNA is cleaved endonucleolytically in the middle ofthe miRNAimRNA duplex By contrast with few exceptions metazoan miRNAs base pairwith theirtargets imperfectly following a set of rules that have been identified by experimentaland bioinformatics analysesw One rule for miRNAitarget base paring is perfect and contiguous base pairing of miRNA nucleotides Zto 8 representing the seed region shown in dark red and green which nucleates the miRNAimRNA association GU pairs or mismatches and bulges in the seed region greatly affect repression However an A residue across position 1 ofthe miRNA and an Aor U across position 9 shown inyellow improve the site efficiency although theydo not need to base pairwith miRNA nucleotides Another rule isthat bulges or mismatches must be present inthe central region ofthe miRNAim RNA duplex precluding theArgonaute AGO7mediated endonucleolytic cleavage omeNA Thethird rule isthat there must be reasonable complementarityto the miRNA 3 halfto stabilizethe interaction Mismatches and bulges are generallytolerated in this region although good base pairing particularlyto residues 13716 ofthe miRNA shown in orange becomes important when matching inthe seed region is suboptimal31 33 Other factors that can improve site efficacy include an AUerich neighbourhood and for long 3 UTRs a positionthat is not too far away from the polyAtailorthe termination codon these factors can makethe 3 UTR regions less structured and hence more accessible to miRNP recognition3334 19 Indeed accessibility of binding sites might have an important effect on miRNAemediated repressionm Some experimentally characterized sites deviate significantly from these rules and can for example even require a bulged nucleotide in the seed region pairing131 2 In addition combinations of sites can require a specific configuration for example separation by a stretch of nucleotides ofspecific sequence and length forefficient repression Usually miRNAebinding sites in metazoan mRNAs lie inthe3 UTR and are present in multiple copies Importantly multiple sites forthe sameordifferent miRNAs are generally required for effective repressionm39 When they are present close to each other 10740 nucleotides apart they tend to act cooperatively that is their effect exceeds that expected from the independent contributions oftwo single sitesa39m interaction with an internal ribosome entry site IRES Joining ofthe 60 subunit at the AUG codon precedes the elongation phase of translation Although it is now clear that the effects of miRNAs on protein synthesis can result from mRNA destabilie zation or translational repression whether the latter occurs at the initiation or posteinitiation step or both remains a matter of debate Several recently published papers provide important mechanistic insights into the repressioneatetheeinitiation step giving extra credence to this model Internal ribosomal entry site lRES An RNAelement usually present in the 5 UTR that allows micrca pr independent association of riboso me With mRNA Repression ut the initiation step Investigations were carried out using HeLa cells and reporter mRNAs that had multiple binding sites for either natural or synthetic miRNAs in their 3 UTR The investigations revealed that the translation of m7G7capped mRNAs but not of mRNAs containing an IRES or a nonefunctional ApppN ADPPN cap An u n methylated cap a na logue that is not bound by elFliE The mRNAs With an artificially introduced ApppN cap instead ofa physiological mlcpppN cap are translated inefficiently REVIEWS cap is repressed by miRNAs gt As in numerous subse7 quent studies the specificity of repression was assessed using reporters containing mutated miRNA sites or by antisense oligonucleotides that specifically block the targeting miRNA The conclusion that the m7G cap is essential for translational repression was corroborated by experiments with biecistronic mRNAs In these experi7 ments the activity of the rst capedependent cistron but not the second cistron placed under the control of eIF4E or eIF4G artificially tethered to the mRNA was repressed by the endogenous le miRNA Pic i Polysome gradient analysis independently supports an effect on the initiation step reporter mRNAs that either contained functional let777binding sites or that were repressed by AGOZ arti cially tethered to the 3 UTR showed a marked shift in sedimentation toward the top of the gradient indicating reduced ribosome loading on the repressed mRNA Likewise the aminoeacide starvationeinduced release of endogenous cationic amino acid transporter 1 CATl mRNA from repression that was mediated by the miRNA miR7122 was accompa7 nied by a more effective recruitment of CATl mRNA to polysomes in human hepatoma cells There is substantial evidence that factors bound at the 3 UTR exert their inhibitory effect on translational initiation by recruiting proteins that either interfere with the eIF4EreIF4G interaction or bind directly to the cap but unlike eIF4E are unable to associate with eIF4G and promote assembly of the 405 initiation complex Hg Could miRNPs or tethered AGO proteins function in a similar manner Kiriakidou et a1 recently reported that the central domain of AGO proteins contains limited sequence homology to the capebmding region of eIF4E Importantly the similarity includes two aromatic resi7 dues Pic 2 which are crucial for cap binding in eIF4E and other capebmding proteinsm Mutations of one or both aromatic amino acids in AGOZ to valine but signif icantly not to other aromatic amino acids prevented the interaction with m7GTPeSepharose and abolished the ability of AGOZ to repress translation when tethered to the mRNA 3 UTR These data indicate that AGOZ and related proteins can compete with eIF4E for m7G binding and thus prevent translation of capped but not IREsecontaining mRNAs The data also provide a plausible explanation for the requirement of multiple miRNPs or tethered AGO molecules for robust represe sion27gt3 gt43gt5 Multiple copies of AGO with an apparently lower affinity for m7G than eIF4E would increase the likelihood ofAGO association with the cap It will be important to determine whether the AGO aromatic residues are essential for miRNAemediated repression in a physiological assay Additional evidence for example from crosselinking experiments should be obtained in support of direct interaction of AGO with the mRNA m7G cap structure Lessons from in vitro studies Four different cellefree extracts that recapitulate many features of the miRNA mediated in vivo effects have recently been described In all of them the presence of the m7G cap was required for translational repressionsz ss the mRNAs containing NATURE REVIEWS l C EN E 39 ICS 2008 Nature Publishing Group VOLUME 9 lFEBRUARY 2008 i105 REVIEWS Box 3 Steps in eukaryotic translation VWWWVWW N K WWWWWW N Translation of mRNA consists ofthree steps initiation elongation and termination Initiation is the most complex step and is subject to a large number of interventions with the phosphorylation of initiation factors being the key regulator Translation requires the participation of at least 10 initiation factors many ofthem multisubunit complexesamg Initiation oftranslation of most cellular mRNAs starts with the recognition ofthe mRNA 5 7terminal 7methylguanosine mIG cap represented bythe red circle in the figure bythe eukaryotic translation initiation factor eI F 4E subunit ofthe initiation factor eIF4F which also contains eIF4A an RNA helicase and eIF4G a large multidomain protein that functions as a scaffold for the assembly ofthe translation initiation complex Interaction of eIF4G with another multiisubunit initiation factor eIF3 facilitates the recruitment of the 405 subunit which then begins scanning the mRNA 5 UTR in search ofthe AUG or in rare cases its cognate initiation codon Following thejoining ofthe 60 ribosomalsubunit the elongation phase ensues The elongation step can also be regulated by phosphorylation ofthe elongation factor eEFZ REF l34 When the ribosome encounters a termination codon translation release factors mediate thetermination process in which the ribosomal subunits dissociate from both the mRNA and from each other An important function of eIF4G is its interaction with the polyAbinding protein 1 PABPI which is associated with the polyA tail This interaction brings about the circularization ofthe mRNA which stimulates translation initiation and possibly recycling of ribosomes 4 eI F6 is required for 605 subunit biogenesis and might also act as an initiation factorthat regulates subunitjoiningSH Some cellular and manyviral mRNAs initiatetranslation independently ofthe mlG cap and eIF4E and sometimes also independentlyof other initiation factors During this mode oftranslation ribosomes are recruited to the mRNA through interaction with internal ribosome entry sites IRES which are usually highly structured regions in the SCUTR The bestistudied IRES are those of the encephalomyocarditis and polio viruses hepatitis C virus and the insect cricket paralysisvirus Biicistronic constructs inwhichtranslation of the upstream cistron requires the presence ofthe cap and eIF4E and that ofthe downstream cistron requires internal initiation are widely used as one ofthe means to identify a putative IRES 42 FIG I an IRES or an ApppN cap were not inhibitedsls Extracts derived from D melanogaster embryos and mouse Krebs2 ascites cells were used to de ne the repression step more precisely In both systems miRNAs inhibited the association of mRNA with either the 40S or the 80S ribosome consistent with miRNAs targeting translation initiation probably at the 40S7mRNA complex assembly steps In agreement with the model that AGO proteins compete with eIF4E for cap binding the addition of puri ed initiation factor eIF4F to the ascites extract resi cued mRNA from the miRNAimediated inhibition Extracts that were prepared from rabbit reticulocytes and from human HEK293 cells were also tested for the polyA7taiI requirement 7 translational repression occurred only when target mRNAs contained both an m7G cap and a polyA tail In the reticulocyte lysate m7G cap dependence could be partially relieved by the addition of polyA tails of noniphysiological length 7 08 kb orlonger 7 implying that polyadenylation might have a role in miRNAimediated repression Studies in HEK293 cell extracts showed that mRNAs containing miRNAibinding sites underwent deadenylation irrei spective of whether they contained an m7G cap transi lationally repressed mRNAs or an ApppN cap or IRES nonirepressed mRNAs Thus although the miRNA7 mediated deadenylation had no apparent effect on the translation of IRESicontaining or ApppNicontaining mRNAs it might have contributed to the repression of m7G7capp ed mRNAs by disrupting the eIF4G7mediated mRNA circularizationss Taken together the data support the notion that by targeting one of the two terminal mRNA structures miRNAs prevent the synergy between the 5 cap and 3 polyA tail Notably HEK293 cell lysate was supple7 mented with an extract of cells overexpressing GW182 REF 55 a protein that recruits the CCR47NOT deadi enylation complex to the miRNAibound mRNA dis7 cussed below Hence in this system miRNAimediated repression might be biased towards deadenylation Of the remaining three systems the D melanogaster and mouse ascites extracts originated from nonimodified cells and responded to endogenous miRNPs53gt5 By con7 trast the repression in the reticulocyte lysate required preiannealing of the synthetic miRNA to the template mRNA It is not known how effectively such a pre7 formed miRNA7mRNA duplex associates with AGO proteins and thus to what extent this system recapitulates the physiological miRNA response Interestingly bind7 ing of miRNPs to the 3 UTR in D melanoguster extracts resulted in the formation of heavy aggregates termed pseudoipolysomes even in the absence of translation Whether they are related to Pibodies discussed below remains unknown Data on the requirement of a polyA tail for represi sion in vitro differ from some findings obtained in intact cells Reporter RNA transcripts that were directly trans7 fected to HeLa cells were repressed even in the absence of a polyA tail In one study its presence resulted in stronger repression but this effect was not seen in a different study Although it is unlikely the possibility that the polyA7free RNA becomes polyadenylated in 105 I FEBRUARY 2008 I VOLUME 9 2008 Nature Publishing Group wwwnaturecomreviewsgenetics Polysome gradient analysis Atechhiquethat involves the sedimentation of cell extracts through a gradiehtofsucrose orglycerol thereby allowrhg the determination ofthe number of ribosomes that are associated With a specific mRNA Repression of translational ll39lltlatlol39lWlilCli results iii the less efficient loadihgof ribosomes ohto mRNA is usually associated With a shift of mRNA towards thetop ofthegradieht transfected cells was not excluded by these studies VVrth this caveat in mind the data suggest that a polyA tail per se is not absolutely required for the repression a conclusion supported by the observation that mRNA containing a 3 histone stemiloop in place of a polyA tail also undergoes translational repression in HEK293 cells55r Repression by preventing 60S subunit joining An alt ernative mechanism of miRNA action was recently proposed by Chendrimada et al57 The authors reported that elF6 and 605 ribosomal subunit proteins co immunoprecipitate with the AGOZrDiceriTRBP com plex elF6 was first described as a protein that binds the 605 subunit to prevent its precocious interaction with the 405 subunit and was thought to act as an initiai tion factor However it was shown later that elF6 is not involved in translation in yeast but rather has a crucial function both in yeast and mammals in the biogenesis of the 605 subunit in the nucleolus and accompanies the 605 subunit to the cytoplasmsg s r Chendrimada et al57 showed that partial depletion of elF6 in either human cells or Caenorhabditis elegans rescues mRNA tare gets from miRNA inhibition possibly by reducing elF67mediated impediment of 605 joining The involvement of elF6 in ribosome biogenesis complicates the interpretation of the data that support its role in miRNA repression and invites another possible scenario Sachs and Davissm demonstrated that muta7 tions in a ribosomal protein and a helicase involved in yeast 60 biogenesis could act as bypass suppressors of complete deletion of the gene encoding polyA bind ing protein Pab1r As in metazoans the yeast Pabl is an essential protein contributing to translation initiai tion through its function in mRNA circularization The Cap binding Human AGOZ DUF PAZ PlWl l875 RNase l Hike fold Drosophla melanogaster GWlBZ W AGO binding UBA REVIEWS bypass suppressor mutations which all resulted in a 605 ribosomal subunit deficit allowed growth albeit reduced in the absence of Pablr This rescue can be explained by an increase in the free 405 subunit pool resulting from a partial depletion of 605 ribosomes leading to an enhanced rate of their recruitment to mRNA This would partially compensate for the lack of Pabl which stimulates 40 recruitment and would switch the rateilimiting step from the 405 subunitiloading step to the 605 joining step A similar switch negating the advantages of the circularization of bulk mRNAs could be caused by the knockdown of elF6r The resulting limited 60 defii cit57 would bring some relief of the miRNAimediated repression because the target mRNAs could now come pete with the bulk of mRNAs on a more equal footing If this explanation is correct the work of Chendrimada et al57 would be consistent with the idea that miRNA mediated repression affects the initiation of translation by targeting the 5 cap and polyA tail although perhaps not because of a direct involvement of elF6 in represi sionr Admittedly this model does not explain why elF6 coipuri es with the R150 Repression utposteinitiution steps Despite compelling in vitro and in viva evidence targeting of translation initiation is unlikely to be the only mechanism by which miRNAs bring about mRNA repressionr Early studies in C elegans showed that M and M mRNAs which are targets ofm miRNA remain associated with polysomes despite a strong reduction in their protein products at a specific stage of larval development Similar results which are incompatible with the initiai tion model were seen in mammalian cells In two studies that used reporter mRNAs targeted by either synthetic C GW M GW RBD Pr body targeting Figure 2 l Domain organization of Argonaute and GW182 proteins The schemes represent humanArgonauteZ A602 and Drosophila melanogaster GW182two proteins extensively characterized in mediating the microRNA miRNAhmediated repression AGOZ isthe only mammalian AGO protein that in addition to miRNA repression also functions in RNAir Its RNAseHalike Paelement induced wimpytestis PIWI domain is competent in endonucleolytically cleaving the mRNA The region separating the PIWI Argona ute Zwille PAZ and PIWI domains of A602 contains two aromatic aminoacids phenylalanines Fmand F505 mutation of which was reported to prevent both the repression of translation in the A602 tethering assay and the binding of A602 to 77methylgua nosine triphosphate m7GTP7 Sepharose gr D melanogaster contains only one GWlSZ protein 136 but there are three GW182 paralogues in mammals known as TN RC6A r One related protein AIN 1 is expressed in Caenorhabditis elegansg39i NAGW MAGW and CAGW are regions enriched in glycine Ghtryptophan W dipeptidesr The NAGW domain or shorter GWArepeatsacontaining peptides were shown to mediate interaction of GWlSZ proteins with the PIWI domain of AGO proteins The region extending from the N terminusto the glutaminearich domain is responsible fortargeting GW182 to Pabodiesmr DUF domain of unknown function RBD RNA binding domain UBA ubiquitin associated domain Q glutaminer NATURE REVIEWS l CENEY 1C5 2008 Namre Publishing Group VOLUME 9 lFEBRUARY 2008 l101 REVIEWS or endogenous miRNAs55gt57 the repressed mRNAs associated with active polysomes 7 as demonstrated by sensitivity of the polysomes to different conditions that inhibit translation Moreover Peterson et al found that like the capidependent upstream ORF IREsimediated translation of the downstream ORF in the biicistronic reporter is sensitive to miRNAs Drawing on additional data the authors proposed a dropioff model in which miRNAs render ribosomes prone to premature terminai tion of translation Lytle et al also reported repression of IREsicontaining reporters35i The observation that three endogenous miRNAs and KRAS mRNA a known target of let77 miRNA cosedii ment with polysomes led Maroney et al to conclude that repression occurs at a postiinitiation stepi Because puromycin or hypertonic conditions 7 factors causing general inhibition of translation 7 shifted polysomei associated miRNAs towards the top of the gradient during polysome gradient analyses whereas the shift of KRAS mRNA was only partial the authors pro posed that miRNAs decelerate translation elongationi Cosedimentation of a significant fraction of cellular miRNAs or AGO proteins with polysomes has also been reported in other studies 71 and is often quoted in support of the postiinitiation mechanismi However it should be stressed that repression of mRNA targets by miRNAs is generally only partial and binding of a single miRNP to mRNA frequently has no significant effect see REFS 4351 for examples Hence cosedimentation of miRNPs with polysomes is not necessarily diagnostic of postiinitiation repression but might simply re ect the association of miRNPs with mRNAs undergoing productive translation How miRNAs could modulate the elongation or termination process remains unclear Apart from the proposed miRNAimediated control few other exam7 ples of regulation targeting postiinitiation steps have been reported Repression ofm mRNA by GLDl in C elega n5 seems to involve the stalling or slowing down of elongating ribosomes as does translational represi sion of unspliced HAC mRNA in yeast73i Other exam7 ples include m and m mRNAs in D melanoguster embryos74gt75 although the proposed mode of their regulation has recently been either reinterpreted or questioned 5775i Despite undeniable evidence that translational repression by miRNAs can occur by post7 initiation mechanisms the ndings do not demonstrate unequivocally that the initiation and postiinitiation mechanisms are mutually exclusive It is possible that inii tiation is always inhibited but when the elongation step is also repressed ribosomes would queue on the mRNA thereby masking the effect of an initiation block The association of repressed mRNAs with translai tionally competent polysomes has also fuelled speculai tions that proteins are continually synthesized from these mRNAs but do not accumulate because they are rapidly degraded by proteases recruited by miRNPs Pic 3 This possibility has been experimentally addressed in immunoprecipitation experiments nascent polypepi tides produced from the repressed reporter could not be detected57i Likewise in pulseilabelling experiments neither fullilength nor nascent polypeptides could be identified when the reporter mRNA was repressedssi On the other hand repression was not prevented when reporter proteins were targeted to the endoplasmic reticulum ER This excludes the possibility that nascent proteins are degraded in the cytosol In conclusion the proteolysis proposal is at present based on negative rather than positive datai Proteasome inhibitors had no effect on miRNAimediated repression 3gt55gt67 and other proteases have not been identi ed mRNA deadenylation and decay Although initial studies suggested that the levels of miRNAiinhibited mRNAs remain mostly unchanged more recent work has demonstrated that the represi sion of many miRNA targets is frequently associated with their destabilization55gt77 80 HG 3L Likewise microi array studies oftranscript levels in cells and tissues in which the miRNA pathway was inhibited78gt79gt8quot or in which miRNA levels were experimentally alteredBH7 revealed marked changes in the abundance of dozens of validated or predicted miRNA targets consistent with a role for miRNAs in mRNA destabilization In eukaryotes mRNA degradation can follow two pathways each of which is initiated by a gradual shorti ening of the mRNA polyA tail The mRNA body can then be degraded by progressive 3 7gt5 decay which is catalysed by the exosome or by the removal of the cap followed by 5 7gt3 degradation which is catalysed by the exonuclease XRNl REF 88L Levels of mRNA are controlled by mRNPs through the recruitment of decay machinery components leading to mRNA deadenylai tion and decapping The degradation or at least its final steps is thought to occur in Pibodies 7 cellular struc7 tures that are enriched in mRNAicatabolizing enzymes and translational repressors m BOX AL The mechanism of miRNAimediated mRNA destai bilization is best understood in D melanogasteri Studies in D melanoguster 52 cells demonstrated that the Pibody protein GW182 product of the gawky gene which interacts with the miRNP Argonaute1 the interaction also occurs between mammalian and worm orthoi loguesyo is a key factorthat marks mRNAs for decay78i The AGO PIWI domain and glycine7tryptophan GW dipeptideicontaining domains or peptides of GW182 family proteins are important for this interaction M HG 2L Consistent with its role in mediating mRNA degradation GW182 depletion leads to an upregulation of many mRNA targets that are also upregulated in cells that are depleted of Argonauteli Tethering of GW182 to the mRNA bypasses the Argonaute1 requirement for repression further demonstrating that GW182 func7 tions in the same pathway downstream of Argonauteli Depletion of the components of the CCR47NOT deadenylating complex prevents the decayipromoting activity of GW182 suggesting that it plays a part in recruiting CCR47NOT to repressed mRNAs Likewise the knockdown ofthe decappingicomplex proteins DCPl and DCPZ or different combinations of decapping activators prevents miRNAimediated degradation but leads to an accumulation of deadenylated mRNAsmi 108 FEBRUARY 2008 VOLUME 9 2008 Namre Publishing Group wwwnaturecomreviewsgenetics Deadenylation followed by decapprng and degradation Pbody mRNA storage or degradation Initiation block repressed cap recognition or 608 joining REVIEWS Proteolysis degradation of nascent peptide miRNP binding Hongation block slowed elongation or ribosome droproffj AAAAA Figure 3 Possible mechanisms ofthe microRNAmediated posttranscriptional gene repression in animal cells Binding of microiribonucleoproteins miRN Ps possibly complexed with accessory factors to mRNA 3 UTR can induce deadenylation and decay oftarget mRNAs56 7B 79 33 upper leftl Alternatively miRN Ps can repress translation initiation at either the capirecognition stage M quot5 orthe 60S subunit joining stageS7 bottom leftl mRNAs repressed by deadenylation or atthetranslatio 39tiation stage are moved to Pibodies foreitherdegradation orstoragei The repression can also occur at postiinitiation phases oftranslation a owing to eitherslowed elongation or ribosome dropioff bottom righti Proteolytic cleavage of nascent polypeptides was also proposed as a mechanism ofthe miRNAiinduced repression of protein production67 upperirightlA protease Xthat might be involved in the process has not been identifiediThe 7methylguanosine cap is represented bya red circle eF4E eukaryotic initiation factor4El Accelerated deadenylation also results in a reduced abundance of miRNAerepressed mRNAs in mammalian cellsssi Moreover knockdown experiments in C elegans and analysis of the decay intermediates originating from repressed mRNAs in worms77 and mammalian cells5m support the role of decapping and 5 gt3 exonucleolytic activities in these systems Widespread miRNAemediated deadenylation of mRNAs occurs during zebrafish embryogenesisi The miRNA miR7430 facilitates the removal of hundreds of maternal mRNAs by inducing their deadenylation and subsequent decay at the onset of zygotic transcription Interestingly some miR7430 targets such as nanos and tudorelike M mRNAs are repressed by miR7430 in somatic but not germ cells indicating that target destabie lization and0r repression can be tissue or cell specificgsl Likewise mRNA reporters targeted by 1amp7 miRNA are destabilized to different degrees in different mammalian cell lines Although many of the mRNAs that are targeted by miRNAs undergo substantial destabilization there are also numerous examples of repression at the translae tional level with no or only a minimal effect on mRNA decay Supplementary information 51 table Studies using D melanogaster 52 cells identified some endoge enous or reporter miRNA targets for which repression could be entirely accounted for by either mRNA degradation or translational repression or by a com bination ofboth processesm It is not known what determines whether an mRNA follows the degradation or translationalerepression pathway Accessory proteins bound to the 3 UTR might be involved or structural sub tleties of imperfect miRNArmRNA duplexes particularly of their central regions could be importantmsi Whether the deadenylation and the ensuing decay are primary or secondary to the translational represe sion remains unknown Clearly the association of AGO instead of elF4E with the m7G cap would not only prevent effective recruitment of ribosomes but would also disrupt the circularization of the mRNA probably rendering the polyA tail more vulnerable to exonucleolytic degradationl Experiments that have been carried out to explore whether deadenylation is a primary or secondary event have not proved to be conclusive Reporter mRNAs that are repressed by either oligonucleotides that are complementary to the AUG codon or the 5 UTR hairpins do not undergo deadenylation unless they contain miRNA sitesmoi However it is unlikely that mRNA circularization is disrupted by the oligonucleotide or the hairpin both of which act at some distance from the cap By contrast the disruption could be effected by the miRNP AGO NATURE REVIEWS CENEY 1C5 2008 Namre Publishing Group VOLUME 9 FEBRUARY 2008 109 REVIEWS interacting with the cap Perhaps the strongest support for deadenylation as a primary event comes from the finding that the translationally inactive Appercapped mRNA which does not interact with arm and hence is unable to circularize is deadenylated when injected into zebrafish embryos only when it contains mTRr430 sites in its 3 UTR This and other experimentsWW indie cate that miRNArdependent mRNA deadenylation and decay is not dependent on active translation although examples of mRNA targets decay ofwhich requires ongoing translation have also been reported 80x4 l Pbodies and stress granules Prbodies also known as GWrbodies are discrete granules that are localized in the cytoplasm of eukaryotic cellsThey are enriched in proteinsthat are involved in mRNA catabolism deadenylation decapping and mRNA degradation and translational repressionmg gtThe core Prbody components conserved from budding yeast which are devoid of RNAcsilencing pathwaysto mammals includethe decapping enzyme complex DCPleDCPZ the decapping activators RCKp54 th 1 in yeast Pat1orthe Drosoplrila melanogaster orthologue ccszos also known as HPat RAPSS Scd6 in yeast and EDC3 Edc3 in yeast and the heptameric LSmH complex Metazoa contain yet another decapping activator Gerl orHedls Prbodies also contain other mRNA decay enzymes the deadenylase complex CAFleCCRAe NOT and the 5 exonuclease XRNl REFS 1789 Some proteins involved in nonsensermediated mRNA decay N MD and other mRNA degradation pathways are also enriched in Prbodies Prbodies lack ribosomes and alltranslation initiation fadors with the exception of eukaryotic initiation factor eIF 4E However eF4G and Pabl accumulate in Prbodies underspecific repressive conditions in yeast In metazoa Prbodies are enriched in proteins participating in miRNArepressioni Argonaute AGO proteins and W182 7 and miRNAs themselves Consistent with theirlocalizationAGO and GWlBZ proteins and miRNAs interact directly or indirectly with different Prbody components 7 91 93 1mm see figure The decapping activators RCKp54 and Patl and another Pcbodycresident protein 4EVT havethe abilityto represstranslationwith some affectingthe initiation step These proteins can contributetothe repressive function of miRNAs A K W DS DV DB Prbodies are highly dynamic structures fluctuating in size and number during the cell cycle and in response to changes in the translational status ofthe cell They require a continuous supply of repressed mRNAs and a globaltranslationcinitiation block leadsto an increase in Pcbodysize in yeast and metazoa inhibition of elongation by cycloheximide which retains mRNAs on polysomes results in their dispersion 7l 9 Likewise depletion of some Prbody components has a strong effect on their integrity at least as visualized by microscopy The mRNAs targeted to Prbodies either undergo degradation or are stored there forfuture use Stress granules 66s are anothertype of mRNAccontaining cytoplasmic aggregates formed in response to global repression of translation initiation or to various stress conditions Many proteins found in Prbodies are absent from 56s and vice versa Howevertheyshare some proteins and Prbodies and 56s are frequently located adjacent to each other possibly exchanging their cargo material m Compartmentalilation of miRNA repression Translationally inactive eukaryotic mRNAs gener ally assemble into repressive mRNPs that accumulate in discrete cytoplasmic foci known as Prbodies or GWrbodies m Another type of aggregate that contains repressed mRNAs are stress granules SGs which accur mulate in response to various stressconditionsW BOX 4 Originally considered as being primarily involved in mRNA degradationm Prbodies are now known to also be temporary sites ofstorage for repressed mRNAs in yeast and mammals s gwg The demonstration that AGO proteins miRNAs and mRNAs repressed by miRNAs are all enriched in lxbodieswmwmum implir cated Prbodies in miRNA repression and in the fate of repressed mRNAs Relevant data are emerging although their interpretation is sometimes difficult owing to the lack ofaprecise definition oflxbodies microscopicallyr visible versus submicroscopic and limited information on the distribution of miRNP components and other factors between Prbodies and the cytosol There is a good correlation between miRNAr mediated translational repression and accumulation omeNAs in visible Prbodies f i m m Moreover there is an inverse relationship between Prbody localization and polysome association oftarget mRNAs in mamr malian cellswm The endogenous CATI mRNA a target ofrmRVJZZ localizes to Prbodies when transla tion is repressed but not when it is reversed by stress In addition transfection of mTRVIZZ into cells that normally do not express it is sufficient to concentrate CATI mRNA in Prbodies s So far quantitative data on the cytosolic distribution oflxbodies are available only for Zetr7 miRNA and its reporter mRNA target both ofwhich are ectopically expressed in HeLa cells Approximately 20 ofeach RNA was localized to visible Prbodies indicating that the repression either involves submicroscopic Prbodies or occurs outside them Note also that the knockdown of some Prbody components such as LSMl or LSM3 which results in dispersion ofmicroscopic Prbodies has no effect on miRNA fuch tion w Hence the microscopically visible Prbodies are not essential for repression and their formation is a consequence rather than the cause ofsilencing m These data are consistent with the recent analysis ofyeast cells that demonstrate that submicroscopic mRNPsconr taining a set ofcore Prbody components are sufficient for basic control oftranslation repression and mRNA decay In contrast to knockdowns ofLSMl and LSM3 depletion ofother Pebodycomponents such as DCPl or DCPZ GW182 and various decapping activators either individually forexampleRCIltp54 or in combinations preventsefficient inhibition oftarget mRNAs in cultured Cdlsrsusrsarunw Notwithstanding the above findings a functional miRNA pathway is clearly essential for the formation oflarge Prbody aggregates Global inhibition of miRNA biogenesis or depletion ofthe proteins that are involved in miRNA repression such as GW182 orArgonauteI results in strong dispersal ofvisible Prbodies in mamr malian and D melanagastef sz cellslm Interestingly depletion ofDicerz or Argonautez which are involved 110 l FEBRUARY 2008 l VOLUME 9 ZEIEIE Nature Publishing Group wwwnarnrecomreviewsgenerics Figure 4 i Possible interplay between RNA binding proteins and micro ribonucleoproteins interacting with the mRNAs 3 UTR A single mRNA can have severalcisiacting motifs interacting with different RNA binding proteins RBPs and microiribonucleoproteins miRNPswhich togetherwilldetermine mRNA translatabilityor stability The suppressive effect ofthe 3 UTRibinding protein ELAV1 on the miRNAimediated repression not shown has recently been documented However it is possiblethat RBPs willalso interact with miRNPs to augment their repressive function and that miRNPs will have a positive or negative effect on the activity of RBPs bound at the 3 UTR The 7methylguanosine cap is represented by a red circle in the figure in RNAi also results in dispersion oflarge Pebodies in D melanogaster cells arguing for a role of both RNAesilencing pathways in Pebody formation Most Pebody components including AGO proteins are also found throughout the cytosol 7l Hence it isprobe able that repression by miRNPs is initiated in the cytosol or at least outside Pebodies and that the repressed mRNAs form Pebody aggregates either small or large upon runoff from the ribosomes Pebodyeresident proteins such as RCKp54 and the yeast orthologue thl gt 9quot 4E7T Zgt 3 Patl and the D melanogaster orthologue HPatl 3gt 9 and RAPSS REF H4 have an established inhibitory activity on translation some at the initiation step These proteins as well as GW182 GW182 functions as a translational repreor in addition to recruiting the CAFerCRAtrNOT deadenylase can assist miRNPs in initiating the repression Whereas RCKp54 and GW182 can be enrolled directly through their interaction with AGO proteins7ggt94gt104 recruitment of others might occur through RCKp54 or GW182 Surprisingly only 13 of enhanced GFP EGFP7 tagged AGOZ localized to Pebodies in HeLa cells O l Moreover the Pebodyeassociated EGFPrAGOZ exchanged with the cytoplasm at a much slower rate than DCPerCPZ or LSM6 the Pebody components involved in decapping gt 3 GW182 also exchanges slowly at Pebodies Rationalization of these observa tions is difficult at present Pebodies could consist of compartments with differing component dynamicsW Alternatively miRNPs and associated proteins such as GW182 could be anchored to some cytoplasmic structures and not be readily available for diffusion into the preeexisting photoebleached Pebodiesl In support of this model most cellular AGO proteins fractionate with the ER or Golgi m gl Moreover fol lowingpermeabilization of the plasma membrane only a small fraction of AGOZ is readily extractable and is probably cytosolic However the observation that the EGFPrAGOZ that accumulates in SGs following stress or treatment with initiation inhibitors exchanges rape idly with the cytosolic AGOZ pool101 is at odds with the REVIEWS anchoring model Association of AGO proteins with mRNAs stored in Pebodies but not those undergoing degradation could be another explanation for their low enrichment in these structures Leung et al101 found that in addition to AGO pro teins miRNA mimics and the repressed reporter mRNA accumulate in SGsl Moreover the localization of AGO proteins to SGs but not Pebodies was miRNAedependantl Because SGs are now known to form not only in response to stress but also following general inhibie tion of translational initiation gm SGs like Pebodies might have a role in the miRNAemediated regula tion oftranslationmo l Alternatively localization of miRNP components to SGs might re ect dragging of the mRNAeassociated but not necessarily inhibitory miRNPs to SGs that are formed in response to general translational inhibition This scenario could also explain why the localization of AGO to SGs but not to Pebodies is miRNA dependent AGO proteins directly interact with other Pebody components7ggt gt but their localizae tion to SGs might require assembly into miRNP to allow association with mRNA by base pairing Reversibility of miRNAmediated repression Recent findings indicate that under certain conditions or in specific cells miRNAemediated repression can be effectively reversed or prevented 5gt 5gtmgt z and miRNPs or their components can even act as translational actie vators7 l The ability to disengage miRNPs from the repressed mRNA or render them stimulatory makes miRNA regulation much more wideeranging and dynamic In human hepatoma cells CAT mRNA is trans lationally repressed by the liverespecific miR7122 and accumulates in Pebodiesl Following aminoeacid starvae tion or other types of stress CAT mRNA is released from Pebodies and recruited to polysomes in a proce ess that depends on binding of ELAVLl also known as HuR a member of the embryonic lethal abnormal vision ELAV protein family to the CAT 3 UTR APOBECSG apolipoprotein B mRNAeediting enzyme catalytic polypeptideelike 3G also interferes with the miRNA action possibly by altering the distribution of target messages between Pebodies and polysomesml Other examples of the reversible action of miRNAs have been reported in neuronal cells In neurons some mRNAs are transported along the dendrites as repressed mRNPs to become translated at dendritic spines upon synaptic activation miR7134 is implicated in the regue lation of LIMKI a protein kinase that is important for the development of the spine In response to extracele lular stimuli miR7134emediated repression of Limk mRNA is partially relieved at dendritic spines of rat neurons In D melanogaster stimulation of olface tory neurons is associated with proteolysis of the armitage armi protein which is essential for the assembly of miRNPs Following armi degradation mRNAs that are normally repressed by miRNAs become translated at the synapse z l Given that many miRNAs are specifically expressed in the brain1 23 and that three of the four mam malian ELAV proteins 7 ELAVZ ELAV3 and ELAV4 NATURE REVIEWS l CENEY 1C5 2008 Nature Publishing Group VOLUME 9 iFEBRUARY 2008 i111 REVIEWS also known as HuB HuC and HuD 7 are restricted to neurons reversible miRNA regulation might have a general role in brain development and function It is likely that RNAabinding proteins RBPs other than ELAV proteins act as modi ers of miRNAamediated repressionl MR 430 repression of nanosl and tdrd7 mRNAs in somatic but not germline cells can be attrib uted to a specific 3 UTRabinding protein that prevents miRa430 function in germline cellsgsl lntriguingly together with the FMRParelated protein AGOZ possibly as a part of an miRNP acts as an activator of translation when bound to the 3 UTR of tumour necroa sis factoraOL mRNA in serumastarved human cells7 l This finding possibly reveals one of many potential combina tions of the interplay between the miRNPs and RBPs that are interacting with mRNA 3 UTRs Because RBPs such as ELAVl can act as translational activators by interfere ing with the miRNPamediated repression of translation it is also possible that miRNPs might act as translational activators by either displacing or modulating inhibitory RBPs bound at the 3 UTR Likewise in other circuma stances miRNPs and RBPs might act synergistically to either repress or activate mRNA translation Flc 4J1 Conclusions and prospects Perhaps the paramount open question is whether miRNAs inhibit protein synthesis by a primary single mechanism or by different mechanisms In otherwords is it possible that miRNAs trigger an initial event that is then amplified by different mechanisms On the basis of the many lines of evidence it is widely believed that miRNAs suppress protein synthesis by a bevy of mechanisms Although this could be the case it is too early to draw this conclusion with certainty A simple alternative mechanistic model posits that the earliest event in proteinasynthesis repression is the inhibition of capadep endent translation through the binding of AGO to the cap structurel Secondary effects of this inhibition could then be manifested at other steps such as mRNA degradation or proteolysis of the nascent polypeptide chains It is conceivable that the different outcomes of the miRNA repression experiments occur partially because of the different experimental systems and methodologies Although the use of in vitro systems allows identification and biochemical characterization of early events during repression the reporter mRNAs lack a nuclear history that could involve deposition of RBPs that modify the mRNA properties and affect the response to miRNAs The same applies to the in vitro transcribed mRNAs that were transfected into cultured cells lndeed differences in the outcome of miRNAa mediated repression have been reported depending on whether RNA or DNA was used for transfection or even on the method of transfection351 Finally it should be recognized that the steps that limit protein expression can differ among different transfected reporter genes or in vitro transcribed mRNAs ol It will be crucial to understand the regulation of miRNA function through modulation of the activity of RISC components and associated factors possibly by phosphorylation and other protein modifications Thus the involvement of different signalling pathways in the control of miRNA function should be studied It is also highly likely that the mechanisms that control translation initiation will have a significant impact on miRNAaregulated gene expression It will also be impora tant to determine the precise contributions of different cellular structures such as Pabodies and SGs to mRNA mediated repression of translation The fact that miRNA function can be recapitulated in cellafree extracts argues against a primary and essential role of Pabodies and SGs in miRNA repression inasmuch as these microscopic struca tures are unlikely to exist in cellafree extracts However pseudoapolysomes that are formed in extracts from D melanogaster embryos might contain constituents of Pabodies and it will be interesting to find out if this is indeed the case The availability of cellafree systems to study miRNA function is a signi cant development It is hoped that these systems will generate a detailed and precise mechanistic picture of the miRNAamediated inhibition of protein synthesis as has been accomplished for transcription translation and splicingl Finally a complete and accurate understanding of the mechanism of miRNA function will require elucidation of threeadimensional structures of animal AGO proteins their complexes with the miRNA and the cap structure and ultimately the structure of miRNP bound to mRNA Structural information would help validate or refute the current models for miRNA functionl Note added in proof Two papers have recently appeared which add new information about the miRNAamediated repressionl Vasudevan et all 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