DNA encodes instructions to make proteins. The DNA is first copied to make molecules of messenger ribonucleic acid (mRNA) that are then “translated” into proteins by large particles known as ribosomes. MicroRNAs are a type of very small RNA molecule that can reduce the amount of protein produced from mRNAs in animals and other eukaryotic organisms. However, the mechanism by which microRNAs achieve this has been unclear.

Many groups of researchers have shown that microRNAs promote the degradation of particular mRNAs. Others have shown that the mRNAs that are targeted by microRNAs are generally bound to active ribosomes. Since the degradation of mRNAs is widely believed to occur away from the ribosomes, these two sets of observations have been considered to be incompatible with each other. Tat et al. set out to resolve this paradox by studying how microRNAs work in fruit fly cells.

The experiments showed that microRNAs do indeed promote the degradation of the mRNAs they bind to and that these mRNAs are exclusively associated with active ribosomes. Furthermore, this process uses the same cellular machinery that is used for the normal destruction of mRNAs. MicroRNAs help to recruit this machinery to their target mRNAs and thereby enhance mRNA break down.

Tat et al.’s findings provide an explanation for a longstanding puzzle in microRNA research. However, although this mechanism is widely used, it does not appear to apply to all mRNAs targeted by microRNAs, so a future challenge is to understand how these other mRNAs are broken down.

microRNAs, a large family of regulatory molecules discovered over twenty years ago, have been shown to exert pervasive effects on a wide array of biological processes (reviewed in Ameres and Zamore, 2013; Ebert and Sharp, 2012). It has become clear that a majority of mRNAs are targeted by miRNAs via limited base pairing interactions between the miRNAs and miRNA responsive elements (MREs) (Bartel, 2009). Although there are exceptions, most MREs are located in 3’ UTRs (Bartel, 2009). The current consensus view of miRNA-mediated gene regulation is that it involves post-transcriptional down regulation of protein production from targeted mRNAs. Repression by miRNAs is largely attributable to mRNA destabilization and several lines of evidence indicate that this destabilization involves recruitment of factors which participate in normal mRNA decay pathways (Bagga et al., 2005; Guo et al., 2010; Eichhorn et al., 2014; Behm-Ansmant et al., 2006a; 2006b; Eulalio et al., 2008b; Jonas and Izaurralde, 2015). These factors include deadenylases and decapping enzymes (e.g. Braun et al., 2011; Chekulaeva et al., 2011; Rehwinkel et al., 2005).

Although much has been learned regarding the biological functions of mRNAs and their mechanism(s) of action, there is one aspect that has largely eluded explanation; i.e. the interplay between the translation machinery and miRNA-mediated repression. In very early studies, the first miRNA targeted mRNAs were found to be fully associated with polysomes (Olsen and Ambros, 1999; Seggerson et al., 2002; Wightman et al., 1993). These observations have been reproduced numerous times over the years; whenever the subcellular localization of mRNA targets has been examined, those mRNAs are found to be exclusively on polysomes and unambiguous evidence has indicated that these polysomes are engaged in active translation (Gu et al., 2009; Nottrott et al., 2006; Petersen et al., 2006). Complementing these studies are several studies indicating that a substantive fraction of miRNAs themselves is also associated with actively translating ribosomes (Maroney et al., 2006; Nelson et al., 2004; Kim et al., 2004). Other observations, including the fact that MREs are less effective when present in open reading frames and the presence of a so-called ribosome shadow (absence of MREs in the 3’ UTR just downstream of stop codons) are consistent with an interaction between miRNA-effector complexes and translating ribosomes (Bartel, 2009). In sum, this body of evidence has led to the development of the hypothesis, which still persists in the literature, that miRNAs regulate translation in some way during the elongation phase but this hypothesis has been recalcitrant to mechanistic proof.

It is difficult to reconcile the large body of evidence that shows that miRNA targeted mRNAs are associated with translating ribosomes with the equally compelling body of evidence that indicates that most miRNA-mediated downregulation occurs through mRNA destabilization. The conundrum is that the prevalent view of mRNA decay holds that decay occurs when mRNAs are in a ribosome free state, perhaps in subcellular aggregates known as P bodies, sites of concentrated RNA degradative activities (reviewed in Valencia-Sanchez et al., 2006). Here, we provide a solution to this longstanding puzzle by demonstrating that miRNA-mediated mRNA decay occurs cotranslationlly. Decay is initiated by mRNA decapping while targeted mRNAs are polysome bound and proceeds 5’ to 3’ following the last translating ribosome. These observations provide a unifying explanation for a large amount of what has been thought to be disparate experimental data.

We are not aware of any study in which both miRNA targeted mRNA stability and subcellular localization (i.e. polysome association) were analyzed. To address this gap and to avoid potential pitfalls associated with transient transfection approaches, we elected to study stable cell lines expressing miRNA targeted mRNAs and relied on endogenous miRNAs to repress them. Because miRNA targeted mRNAs are particularly well characterized in Drosophila (Stark et al., 2003), we chose to analyze Drosophila mRNAs encoding the proapoptotic proteins, reaper and hid. The reaper mRNA contains one microRNA recognition element (MRE) for the Drosophila miR-2 family of miRNA (miRs 2a, 2b, 2c, 6, 11, 13a, 13b, 308) in its 3’UTR (Figure 1A, Figure 1—figure supplement 1) (Jovanovic and Hengartner, 2006). The hid mRNA contains five MREs for the miRNA, bantam, in its 3’ UTR (Figure 1A, Figure 1—figure supplement 1) (Brennecke et al., 2003). To avoid the issue that overexpression of either the reaper or hid proteins would promote apopotosis, the entire 3’ UTRs of both mRNAs were appended to the firefly luciferase coding sequence and 5’ UTR (Figure 1—figure supplement 1). Two versions of each 3’ UTR, one containing wild type MREs and one containing mutant MREs were used in the construction of four stable Drosophila S2 cell lines. Cell lines were judged to be stable when no loss of cells was observed in the presence of the drugs used for their selection and when luciferase levels remained constant over several cell doublings. Reaper constructs were under control of the inducible metallothionine promoter (Bunch et al., 1988) while the hid constructs were under control of the constitutive actin promoter (Angelichio et al., 1991).

Figure 1 with 2 supplements see all

Download asset Open asset

Because experimental evidence indicates that mRNA destabilization is at least one component of miRNA-mediated repression in Drosophila (reviewed in Huntzinger and Izaurralde, 2011; Izaurralde, 2015; Eulalio et al., 2008a), we measured the stability of each of the four mRNAs. We first determined the half life of reaper reporter mRNAs. After transcriptional shut off, the reaper reporter mRNA with the wild type MRE decayed with a half life of approximately 8 min (Figure 1B). The half life of the reaper reporter mRNA containing the mutant MRE was approximately 31 min, about four fold longer than the mRNA with the wild type MRE (Figure 1B).

To confirm that this effect on mRNA stability was miRNA-mediated we designed a double stranded miRNA mimic. When loaded into an Ago protein the targeting strand contained bases complementary to the nucleotides changed in the mutant reaper MRE. While a control mimic had no effect on the half life of the reaper reporter containing the mutant MRE the mimic complementary to the mutant MRE markedly reduced the half life of the reporter to a level comparable to that of the reaper reporter containing the wild type MRE (Figure 1—figure supplement 2).

Figure 2 with 4 supplements see all

Download asset Open asset

To determine if the four fold effect on mRNA stability this was the maximal miRNA-mediated effect possible, expression of the reaper reporter mRNA was induced either ten-fold or one hundred fold with different concentrations of copper and half lives were again determined. Importantly the reaper reporter mRNA containing the wild type MRE had the same half life at all levels of expression; a result which indicated that miR-2 family miRNAs were well in excess of the targeted mRNA and that the reaper MRE was likely to be fully occupied at steady state (data not shown). Therefore a four fold enhancement of decay was the maximum effect that could be exerted by microRNAs on this mRNA target.

Parallel analysis of the hid constructs revealed a similar microRNA-mediated effect on mRNA stability (Figure 1C). The hid reporter mRNA with five wild type MREs had a half life of approximately 7.5 min whereas the mRNA with the mutant MREs had a half life longer than forty minutes (Figure 1C). A Bantam mimic analogous to the miR2 mimic described above caused marked destabilization of the reporter containing mutant Bantam sites, confirming that the observed effect on mRNA stability was miRNA-mediated (Figure 1—figure supplement 2). We concluded from those experiments that for these two targeted mRNAs, endogenous miRNAs caused substantive destabilization.

We then assessed the subcellular localization of all four mRNAs using sedimentation velocity sucrose gradients and Northern blotting (Figure 2). Remarkably, fractionation of cytoplasmic extracts in this manner revealed that all detectable mRNAs for all four of the constructs cosedimented with polysomes, an observation that suggested that the mRNAs were associated with translating ribosomes (Figure 2B and Figure 2—figure supplement 1). To ensure that polysome associated mRNA accounted for most or all mRNAs present in the cell, we performed an “accounting” experiment analogous to that we previously reported for miRNAs (Maroney et al., 2006) We found that the vast majority of reporter mRNAs was indeed associated with polysomes and that there was negligible mRNA loss during extract preparation or gradient fractionation (Figure 2—figure supplement 2) compare lanes total and gradient. To determine if polysome associated mRNAs were actually being translated, cells were treated with either emetine, an irreversible inhibitor of translation elongation (Akinboye and Bakare, 2011), or Harringtonine, an inhibitor of protein synthesis that binds to the A site of initiating ribosomes only and thereby prevents formation of the first peptide bond (Fresno et al., 1977). Because Harringtonine inhibits formation of only the first peptide bond; ribosomes that are already elongating continue to translocate through the open reading frame and eventually terminate and disassociate from the mRNA. As elongating ribosomes clear the mRNA, polysomes become smaller and the mRNA shifts to lighter fractions of the gradient; newly initiating messages accumulate in the monosome region (80S). Extracts prepared from Harringtonine treated cells were sedimented on sucrose gradients and fractions were analyzed by Northern blotting. These analyses revealed that all four mRNAs shifted to lighter fractions; a result which indicated that all four mRNAs were associated with actively translating ribosomes (Figure 2B and Figure 2—figure supplement 1). Consistent with other earlier observations (Maroney et al., 2006; Nelson et al., 2004) (Kim et al., 2004), both endogenous bantam and a representative member of the miR-2 family (miR-13b) showed similar sedimentation behavior as these targeted mRNAs; i.e. a substantive amount of each miRNA sedimented with polysomes and shifted to lighter fractions upon Harringtonine treatment (Figure 2B). Endogenous mRNAs encoding the ribosomal protein L32 (RP49) and polyA binding protein (PABP) were, as expected, also present on translating ribosomes (Figure 2C). At this point in our analysis it was clear that endogenous miRNAs were causing marked destabilization of targeted mRNAs, yet all detectable mRNAs were associated with actively translating ribosomes.

How was microRNA-mediated enhanced mRNA decay compatible with active translation of the targeted mRNAs? We considered two possible scenarios. In the first, translational initiation was somehow blocked on targeted mRNAs. After elongating ribosomes cleared the mRNA, the ribosome free mRNA was degraded so quickly that it could not be detected. The second scenario was that decay was occurring while the mRNAs were being translated; i.e. in a ribosome bound state. A clue to distinguish between these possibilities came from examination of long exposures of the Northern blot across polysome gradients shown in Figure 2. We observed a family of hybridizing species that were smaller than full length (Figure 2—figure supplement 3). We speculated that these species might have resulted from partial degradation of the mRNA. If this was the case, we predicted that such fragments would become enriched relative to full length mRNA if new synthesis of mRNA was prevented. Accordingly, we stopped ongoing transcription with Actinomycin D and incubated cells for approximately one mRNA half life in order to allow for partial decay of pre-existing mRNA; translation was then arrested with emetine (Figure 3B).

Figure 3 with 2 supplements see all

Download asset Open asset

Cells expressing the reaper reporter mRNA containing the wild type MRE were treated this way and extracts were fractionated on sucrose gradients (Figure 3—figure supplement 1). Upon analysis by Northern blotting, we observed a striking accumulation of truncated mRNA fragments (Figure 3C). These fragments were detected with a probe which hybridized to the 3’ region of the open reading frame but were not observed when a probe which hybridized to the 5’ region of the reading frame was used (Figure 3D). Comparable fragments were greatly underrepresented when the same analysis was performed on the reaper reporter mRNA containing the mutated MRE (Figure 3C). We concluded from this that the fragments represented mRNA decay intermediates degraded from the 5’ end of the mRNA. Because the decay intermediates were ribosome associated, as indicated by their sedimentation behavior, these data show that enhanced microRNA-mediated mRNA decay occurs cotranslationally.

Parallel analysis was carried out on the hid constructs (Figure 3—figure supplement 2). Because the hid 3’ UTR was so long (3kb) relative to the coding region the results were less clear cut than with the reaper constructs. Nevertheless, ribosome associated decay fragments derived from mRNAs containing wild type MREs accumulated in great excess to those derived from the mRNA containing mutant MREs.

Having shown that miRNA-mediated mRNA destabilization was cotranslational, we wished to determine the pathway by which this mRNA decay occurred. Because decay intermediates were truncated from the 5’ end it seemed likely that they were derived from decapped mRNAs. To determine if decapping was required for decay, the decapping factors DCP1 and 2 were knocked down by RNAi. The reaper reporter mRNA containing the wild type MRE was markedly stabilized in cells wherein decapping was compromised, a result that indicated that removal of the cap was an important step in initiating targeted mRNA degradation (Figure 5A).

Several lines of evidence indicate that decapping is contingent upon prior deadenylation of the mRNA (Braun et al., 2012; Eulalio et al., 2009; Giraldez et al., 2006). It has been shown in Drosophila that miRNAs bound to Ago1 recruit GW182; a protein that in turn recruits the CCR4-Not deadenylase complex (e.g. Braun et al., 2011; Chekulaeva et al., 2011). This complex deadenylates mRNA and in some way, perhaps by direct recruitment, facilitates mRNA decapping by Dcp1 and 2 (Behm-Ansmant et al., 2006a; 2006b; Rehwinkel et al., 2005). To determine if the cotranslational decay we observed followed this pathway, we examined the adenylation status of miRNA-targeted mRNAs. Using a polyA tail length assay (Rio et al., 2011) and Figure 4A, we observed what appeared to be “bands” for both reaper reporter and hid reporter tails. Since these “bands” migrated at the size expected for deadenylated mRNAs this result suggested that deadenylated species accumulated in polysome associated mRNAs (Figure 4B). To confirm this interpretation the “band” from the reaper reporter was excised and sequenced. The results confirmed this interpretation (Figure 4C). In contrast, control non-microRNA targeted mRNA displayed normal polyA tails. The presence of deadenylated mRNAs in polysome fractions strongly suggested that accelerated decapping was a consequence of accelerated deadenylation.

Figure 4

Download asset Open asset

Figure 5

Download asset Open asset

Following decapping, mRNA degradation is catalyzed by the 5’-3’ exonuclease XRN1; XRN1 leaves 5’ phosphorylated termini. To determine if the cotranslational decay we observed depended upon an enzyme that leaves 5’ phosphorylated termini, we used a ligation strategy designed to detect RNAs which terminated in a 5’ phosphate (Figure 5B). Unlike most splinted ligation assays which require precise juxtaposition of 5’ and 3’ ends and use DNA ligase as the sealing enzyme, we used a bridge oligonucleotide that brought a “reporter” oligonucleotide with a free 3’ hydroxyl into proximity of mRNA fragments with 5’phosphate termini and used RNA ligase 1 as the sealing enzyme (Figure 5B).

To validate this assay we sought to identify substrates that would provide both positive and negative controls. We reasoned that a fully capped mRNA could provide the negative control. We chose to analyze the mRNA encoding ribosomal protein L32, RP49 (Figure 5C). Because the RP49 mRNA has a very long half life (see Figure 1), we anticipated that it would be essentially all capped at steady state. Consistent with this, only trace amounts of ligation products diagnostic of decapped RP49 mRNA were present in total RNA. However, after treatment with tobacco acid pyrophosphatase (TAP), an enzyme that removes the cap, a robust signal for decapped RP49 was obtained (Figure 5C). We then used the ligation assay to analyze the reaper reporter mRNA. In this case we set up the assay to detect any 5’ phosphorylated mRNA fragments containing a 5’ terminal phosphate present in the region of the reaper reporter mRNA ranging from its transcription start site to just downstream of the AUG initiation codon. Here, we observed strong signals in the absence of TAP a result indicating that decapped mRNAs were present at steady state. Only the band corresponding to the mRNA start site was enhanced by TAP treatment (Figure 5C). Importantly, detection of these fragments was ligase dependent and just a trace of fragments were detected in RNA prepared from cells in which DCP 1 and 2 were knocked down (Figure 5D). When the assay was used to detect decay fragments across a polysome gradient we observed signal throughout the polysome region of the gradient (Figure 5C and see Discussion). Cloning of bands denoted by asterisks showed that their 5’ ends mapped at or near the transcription start site and near the translation initiation codon (Figure 5E). These results demonstrated that the decay fragments we observed had resulted from decapping of the reporter mRNA while it was polysome bound.

We conclude that cotranslational miRNA mediated mRNA destabilization employs the canonical mRNA decay machinery. This destabilization is accelerated relative to non-targeted mRNAs almost surely due to Ago1 recruitment of GW182 which in turn recruits the deadenylase machinery. Following deadenylation, mRNAs are decapped thereby yielding substrates for XRN1 (Figure 6).

Figure 6

Download asset Open asset

We have provided evidence that miRNAs promote enhanced mRNA decay while the mRNA targets are engaged with actively translating ribosomes. While we have analyzed only two miRNA targets, we think it likely that these observations will be broadly relevant. In cases where the subcellular localization of miRNA targets have been studied, they have invariably been found to be polysome associated (Olsen and Ambros, 1999; Gu et al., 2009; Seggerson et al., 2002; Wightman et al., 1993; Nottrott et al., 2006; Petersen et al., 2006). Moreover, miRNAs themselves have been shown to be polysome associated (Maroney et al., 2006; Nelson et al., 2004; Kim et al., 2004). Our observations are also consistent with a large body of transcriptome wide analyses obtained from studies primarily in mammalial systems that have shown that mRNA destabilization is the primary if not sole effect of miRNA-mediated down regulation of protein production from targeted mRNAs (Guo et al., 2010; Eichhorn et al, 2014). They are also consistent with studies in Drosophila S2 cells that have shown that miRNA effector complexes as well as general decay enzymes including deadenylases and decapping factors are ribosome associated (Antic et al., 2015). While cotranslational miRNA-mediated mRNA decay rationalizes a very large body of published results, there are some observations that are difficult to reconcile with this unifying model of miRNA-mediated action. For example one study in a C. elegans cell free system showed that miRNA-targeted mRNAs were deadenylated and translationally repressed but not degraded (Wu et al., 2010). Another example, also from C. elegans, used ribosome profiling in vivo to show that miRNA mediated regulation in some cases correlated with mRNA abundance but in other cases did not (Stadler et al., 2012). Finally, there are also examples of Drosophila miRNA targeted mRNAs which appear to be regulated at the level of translation and not stability (e.g. Huntzinger and Izaurralde, 2011 and Behm-Ansmant et al., 2006a; 2006b). These examples might indicate that some specific mRNPs are subject to distinct regulatory pathways. Future studies may elucidate mRNP specific factors that mediate these effects.

Cotranslational mRNA decay was shown several years ago for a selected group of mRNAs in budding yeast (Hu et al., 2009). More recently, studies have shown that such decay is the dominant if not sole pathway for normal mRNA decay in yeast (Pelechano et al., 2015). Our findings extend these observations to higher eukaryotes and suggest that cotranslational decay may be a widespread phenomenon.

Our results do not speak specifically to how mRNA decay is triggered. In that regard there has been a longstanding debate in the field as to whether inhibition of translation initiation is a prerequisite for miRNA-mediated enhanced decay (Bazzini et al., 2012; Djuranovic et al., 2012; Eichhorn et al., 2014; Subtelny et al., 2014). Indeed over the years a large variety of different inhibition scenarios have been proposed including mRNA sequestration from the translation apparatus and inactivation of an array of initiation factors (e.g. Valencia-Sanchez et al., 2006; Mathonnet et al., 2007; Chendrimada et al., 2007; Kiriakidou et al., 2007; Wang et al., 2008; Fukaya et al., 2014), the latest being eIF4AI and II (Fukao et al., 2014; Meijer et al., 2013). Unfortunately none of these mechanisms including eIF4AI and II (see Galicia-Vázquez et al., 2015) has received sustained experimental support and it is currently unclear how or if initiation of translation is truly impaired in a microRNA-mediated fashion.

Independent of miRNA-mediated mRNA decapping, mRNAs decap in general (Coller and Parker, 2004). For general mRNA decapping, translational initiation is also believed to be in competition with the DCP2/1 holoenzyme. Importantly, despite this well accepted notion that initiation and decapping compete, it remains to be established how, when, or even if dissolution of the translational initiation complex occurs prior to DCP2/1-mediated cleavage. Thus the events triggering decapping are still murky but may include recruitment of the RNA helicase DDX6 (Chen et al., 2014; Mathys et al., 2014; Rouya et al., 2014). Importantly, however, since eIF-4F requires a cap to stimulate translation, decapping itself clearly and dramatically limits any further ribosome association and thus can itself be thought of as a potent and irreversible inhibitor of translation initiation.

The first report of inhibition of initiation of translation came from polysome analysis where it was shown that an miRNA targeted mRNA migrated on lighter polysomes than a control mRNA (Pillai et al., 2005). Notably, however, all of the targeted mRNA was associated with ribosomes; there was no mRNA sedimenting in the RNP region of the gradient (Pillai et al., 2005). These results were interpreted to mean that miRNAs repressed translation initiation. We too observe that targeted mRNAs migrate on lighter polysomes than untargeted mRNAs (Figure 2—figure supplement 4). However, we suggest an alternative explanation for this phenomenon. It seems possible that the act of decapping itself could account for the altered sedimentation of targeted mRNAs. If decapping is a prerequisite for but not rate limiting for mRNA decay, decapped but not degraded mRNAs could persist. Because already engaged ribosomes would continue to elongate and eventually terminate, decapped but intact mRNAs would shift to lighter polysomes as elongating ribosomes clear the mRNA. We note that this notion is not entirely speculative. We have observed decapped mRNAs with 5’ termini that map both near the start site of the mRNA and near the translational initiation codon. Importantly, these RNAs, which would appear to be full length, are detected throughout polysomes of different sizes both large and small (Figure 5D). These observations indicate that 5’ to 3’ exonucleolytic decay is not obligatorily coupled to decapping because if such coupling existed we would not observe full length decapped mRNA. It seems possible the concentration of degradation fragments near the initiation AUG could result from the buildup of ribosomes near the beginning of the open reading frame as observed in ribosome profiling studies (e.g. Ingolia et al., 2011). Collectively, these observations suggest that decapping while a prerequisite for mRNA decay is not likely to be rate limiting for decay itself; exonucleolytic digestion by XRN1 is likely to be rate limiting.

In sum, we have provided an explanation for a longstanding mystery in the miRNA field and have suggested a plausible model for miRNA action that is consistent with a large body of experimental results obtained in numerous laboratories.

1. 1. Akinboye E
   2. Bakare O

   (2011) Biological activities of emetine

   The Open Natural Products Journal 4:8–15.

   https://doi.org/10.2174/1874848101104010008

   • Google Scholar
2. 1. Ameres SL
   2. Zamore PD

   (2013) Diversifying microrna sequence and function

   Nature Reviews Molecular Cell Biology 14:475–488.

   https://doi.org/10.1038/nrm3611

   • Google Scholar
3. 1. Angelichio ML
   2. Beck JA
   3. Johansen H
   4. Ivey-Hoyle M

   (1991) Comparison of several promoters and polyadenylation signals for use in heterologous gene expression in cultured drosophila cells

   Nucleic Acids Research 19:5037–5043.

   https://doi.org/10.1093/nar/19.18.5037

   • Google Scholar
4. 1. Antic S
   2. Wolfinger MT
   3. Skucha A
   4. Hosiner S
   5. Dorner S

   (2015) General and microrna-mediated mrna degradation occurs on ribosome complexes in drosophila cells

   Molecular and Cellular Biology 35:2309–2320.

   https://doi.org/10.1128/MCB.01346-14

   • Google Scholar
5. 1. Bagga S
   2. Bracht J
   3. Hunter S
   4. Massirer K
   5. Holtz J
   6. Eachus R
   7. Pasquinelli AE

   (2005) Regulation by let-7 and lin-4 mirnas results in target mrna degradation

   Cell 122:553–563.

   https://doi.org/10.1016/j.cell.2005.07.031

   • Google Scholar
6. 1. Bartel DP

   (2009) Micrornas: Target recognition and regulatory functions

   Cell 136:215–233.

   https://doi.org/10.1016/j.cell.2009.01.002

   • Google Scholar
7. 1. Bazzini AA
   2. Lee MT
   3. Giraldez AJ

   (2012) Ribosome profiling shows that mir-430 reduces translation before causing mrna decay in zebrafish

   Science 336:233–237.

   https://doi.org/10.1126/science.1215704

   • Google Scholar
8. 1. Behm-Ansmant I
   2. Rehwinkel J
   3. Doerks T
   4. Stark A
   5. Bork P
   6. Izaurralde E

   (2006a) mRNA degradation by mirnas and GW182 requires both CCR4:NOT deadenylase and DCP1:DCP2 decapping complexes

   Genes & Development 20:1885–1898.

   https://doi.org/10.1101/gad.1424106

   • Google Scholar
9. 1. Behm-Ansmant I
   2. Rehwinkel J
   3. Izaurralde E

   (2006b) Micrornas silence gene expression by repressing protein expression and/or by promoting mrna decay

   Cold Spring Harbor Symposia on Quantitative Biology 71:523–530.

   https://doi.org/10.1101/sqb.2006.71.013

   • Google Scholar
10. 1. Braun JE
    2. Huntzinger E
    3. Fauser M
    4. Izaurralde E

    (2011) Gw182 proteins directly recruit cytoplasmic deadenylase complexes to mirna targets

    Molecular Cell 44:120–133.

    https://doi.org/10.1016/j.molcel.2011.09.007

    • Google Scholar
11. 1. Braun JE
    2. Huntzinger E
    3. Izaurralde E

    (2012) A molecular link between miriscs and deadenylases provides new insight into the mechanism of gene silencing by micrornas

    Cold Spring Harbor Perspectives in Biology 4:a012328.

    https://doi.org/10.1101/cshperspect.a012328

    • Google Scholar
12. 1. Brennecke J
    2. Hipfner DR
    3. Stark A
    4. Russell RB
    5. Cohen SM

    (2003) bantam encodes a developmentally regulated microrna that controls cell proliferation and regulates the proapoptotic gene hid in drosophila

    Cell 113:25–36.

    https://doi.org/10.1016/S0092-8674(03)00231-9

    • Google Scholar
13. 1. Bunch TA
    2. Grinblat Y
    3. Goldstein LSB

    (1988) Characterization and use of the drosophila metallothionein promoter in cultured drosophila melanogaster cells

    Nucleic Acids Research 16:1043–1061.

    https://doi.org/10.1093/nar/16.3.1043

    • Google Scholar
14. 1. Celotto AM
    2. Lee J-W
    3. Graveley BR

    (2005) Exon-specific RNA interference: A tool to determine the functional relevance of proteins encoded by alternatively spliced mrnas

    Methods in Molecular Biology 309:273–282.

    https://doi.org/10.1385/1-59259-935-4:273

    • Google Scholar
15. 1. Chekulaeva M
    2. Mathys H
    3. Zipprich JT
    4. Attig J
    5. Colic M
    6. Parker R
    7. Filipowicz W

    (2011) miRNA repression involves gw182-mediated recruitment of CCR4–NOT through conserved w-containing motifs

    Nature Structural & Molecular Biology 18:1218–1226.

    https://doi.org/10.1038/nsmb.2166

    • Google Scholar
16. 1. Chen Y
    2. Boland A
    3. Kuzuoğlu-Öztürk D
    4. Bawankar P
    5. Loh B
    6. Chang C-T
    7. Weichenrieder O
    8. Izaurralde E

    (2014) A DDX6-CNOT1 complex and w-binding pockets in CNOT9 reveal direct links between mirna target recognition and silencing

    Molecular Cell 54:737–750.

    https://doi.org/10.1016/j.molcel.2014.03.034

    • Google Scholar
17. 1. Chendrimada TP
    2. Finn KJ
    3. Ji X
    4. Baillat D
    5. Gregory RI
    6. Liebhaber SA
    7. Pasquinelli AE
    8. Shiekhattar R

    (2007) Microrna silencing through RISC recruitment of eif6

    Nature 447:823–828.

    https://doi.org/10.1038/nature05841

    • Google Scholar
18. 1. Coller J
    2. Parker R

    (2004) Eukaryotic mrna Decapping

    Annual Review of Biochemistry 73:861–890.

    https://doi.org/10.1146/annurev.biochem.73.011303.074032

    • Google Scholar
19. 1. Djuranovic S
    2. Nahvi A
    3. Green R

    (2012) miRNA-mediated gene silencing by translational repression followed by mrna deadenylation and decay

    Science 336:237–240.

    https://doi.org/10.1126/science.1215691

    • Google Scholar
20. 1. Ebert MS
    2. Sharp PA

    (2012) Roles for micrornas in conferring robustness to biological processes

    Cell 149:515–524.

    https://doi.org/10.1016/j.cell.2012.04.005

    • Google Scholar
21. 1. Eichhorn SW
    2. Guo H
    3. McGeary SE
    4. Rodriguez-Mias RA
    5. Shin C
    6. Baek D
    7. Hsu Shu-hao
    8. Ghoshal K
    9. Villén J
    10. Bartel DP

    (2014) mRNA destabilization is the dominant effect of mammalian micrornas by the time substantial repression ensues

    Molecular Cell 56:104–115.

    https://doi.org/10.1016/j.molcel.2014.08.028

    • Google Scholar
22. 1. Eulalio A
    2. Huntzinger E
    3. Izaurralde E

    (2008a) Getting to the root of mirna-mediated gene silencing

    Cell 132:9–14.

    https://doi.org/10.1016/j.cell.2007.12.024

    • Google Scholar
23. 1. Eulalio A
    2. Huntzinger E
    3. Izaurralde E

    (2008b) Gw182 interaction with argonaute is essential for mirna-mediated translational repression and mrna decay

    Nature Structural & Molecular Biology 15:346–353.

    https://doi.org/10.1038/nsmb.1405

    • Google Scholar
24. 1. Eulalio A
    2. Huntzinger E
    3. Nishihara T
    4. Rehwinkel J
    5. Fauser M
    6. Izaurralde E

    (2009) Deadenylation is a widespread effect of mirna regulation

    RNA 15:21–32.

    https://doi.org/10.1261/rna.1399509

    • Google Scholar
25. 1. Fresno M
    2. Jimenez A
    3. Vazquez D

    (1977) Inhibition of translation in eukaryotic systems by harringtonine

    European Journal of Biochemistry 72:323–330.

    https://doi.org/10.1111/j.1432-1033.1977.tb11256.x

    • Google Scholar
26. 1. Fukao A
    2. Mishima Y
    3. Takizawa N
    4. Oka S
    5. Imataka H
    6. Pelletier J
    7. Sonenberg N
    8. Thoma C
    9. Fujiwara T

    (2014) Micrornas trigger dissociation of eif4ai and eif4aii from target mrnas in humans

    Molecular Cell 56:79–89.

    https://doi.org/10.1016/j.molcel.2014.09.005

    • Google Scholar
27. 1. Fukaya T
    2. Iwakawa Hiro-oki
    3. Tomari Y

    (2014) Micrornas block assembly of eif4f translation initiation complex in drosophila

    Molecular Cell 56:67–78.

    https://doi.org/10.1016/j.molcel.2014.09.004

    • Google Scholar
28. 1. Galicia-Vázquez G
    2. Chu J
    3. Pelletier J

    (2015) eIF4AII is dispensable for mirna-mediated gene silencing

    RNA 21:1826–1833.

    https://doi.org/10.1261/rna.052225.115

    • Google Scholar
29. 1. Giraldez AJ

    (2006) Zebrafish mir-430 promotes deadenylation and clearance of maternal mrnas

    Science 312:75–79.

    https://doi.org/10.1126/science.1122689

    • Google Scholar
30. 1. Gu S
    2. Jin L
    3. Zhang F
    4. Sarnow P
    5. Kay MA

    (2009) Biological basis for restriction of microrna targets to the 3′ untranslated region in mammalian mrnas

    Nature Structural & Molecular Biology 16:144–150.

    https://doi.org/10.1038/nsmb.1552

    • Google Scholar
31. 1. Guo H
    2. Ingolia NT
    3. Weissman JS
    4. Bartel DP

    (2010) Mammalian micrornas predominantly act to decrease target mrna levels

    Nature 466:835–840.

    https://doi.org/10.1038/nature09267

    • Google Scholar
32. 1. Hu W
    2. Sweet TJ
    3. Chamnongpol S
    4. Baker KE
    5. Coller J

    (2009) Co-translational mrna decay in saccharomyces cerevisiae

    Nature 461:225–229.

    https://doi.org/10.1038/nature08265

    • Google Scholar
33. 1. Huntzinger E
    2. Izaurralde E

    (2011) Gene silencing by micrornas: Contributions of translational repression and mrna decay

    Nature Reviews Genetics 12:99–110.

    https://doi.org/10.1038/nrg2936

    • Google Scholar
34. 1. Ingolia NT
    2. Lareau LF
    3. Weissman JS

    (2011) Ribosome profiling of mouse embryonic stem cells reveals the complexity and dynamics of mammalian proteomes

    Cell 147:789–802.

    https://doi.org/10.1016/j.cell.2011.10.002

    • Google Scholar
35. 1. Izaurralde E

    (2015) Breakers and blockers--mirnas at work

    Science 349:380–382.

    https://doi.org/10.1126/science.1260969

    • Google Scholar
36. 1. Jonas S
    2. Izaurralde E

    (2015) Towards a molecular understanding of microrna-mediated gene silencing

    Nature Reviews Genetics 16:421–433.

    https://doi.org/10.1038/nrg3965

    • Google Scholar
37. 1. Jovanovic M
    2. Hengartner MO

    (2006) miRNAs and apoptosis: Rnas to die for

    Oncogene 25:6176–6187.

    https://doi.org/10.1038/sj.onc.1209912

    • Google Scholar
38. 1. Kim J
    2. Krichevsky A
    3. Grad Y
    4. Hayes GD
    5. Kosik KS
    6. Church GM
    7. Ruvkun G

    (2004) Identification of many micrornas that copurify with polyribosomes in mammalian neurons

    Proceedings of the National Academy of Sciences of the United States of America 101:360–365.

    https://doi.org/10.1073/pnas.2333854100

    • Google Scholar
39. 1. Kiriakidou M
    2. Tan GS
    3. Lamprinaki S
    4. De Planell-Saguer M
    5. Nelson PT
    6. Mourelatos Z

    (2007) An mrna m7g cap binding-like motif within human ago2 represses translation

    Cell 129:1141–1151.

    https://doi.org/10.1016/j.cell.2007.05.016

    • Google Scholar
40. 1. Kunkel TA
    2. Roberts JD
    3. Zakour RA

    (1987) Rapid and efficient site-specific mutagenesis without phenotypic selection

    Methods in Enzymology 154:367–382.

    https://doi.org/10.1016/0076-6879(87)54085-X

    • Google Scholar
41. 1. Maroney PA
    2. Yu Y
    3. Fisher J
    4. Nilsen TW

    (2006) Evidence that micrornas are associated with translating messenger rnas in human cells

    Nature Structural & Molecular Biology 13:1102–1107.

    https://doi.org/10.1038/nsmb1174

    • Google Scholar
42. 1. Maroney PA
    2. Chamnongpol S
    3. Souret F
    4. Nilsen TW

    (2007) A rapid, quantitative assay for direct detection of micrornas and other small rnas using splinted ligation

    RNA 13:930–936.

    https://doi.org/10.1261/rna.518107

    • Google Scholar
43. 1. Mathonnet G
    2. Fabian MR
    3. Svitkin YV
    4. Parsyan A
    5. Huck L
    6. Murata T
    7. Biffo S
    8. Merrick WC
    9. Darzynkiewicz E
    10. Pillai RS
    11. Filipowicz W
    12. Duchaine TF
    13. Sonenberg N

    (2007) Microrna inhibition of translation initiation in vitro by targeting the cap-binding complex eif4f

    Science 317:1764–1767.

    https://doi.org/10.1126/science.1146067

    • Google Scholar
44. 1. Mathys H
    2. Basquin J
    3. Ozgur S
    4. Czarnocki-Cieciura M
    5. Bonneau F
    6. Aartse A
    7. Dziembowski A
    8. Nowotny M
    9. Conti E
    10. Filipowicz W

    (2014) Structural and biochemical insights to the role of the CCR4-NOT complex and DDX6 atpase in microrna repression

    Molecular Cell 54:751–765.

    https://doi.org/10.1016/j.molcel.2014.03.036

    • Google Scholar
45. 1. Meijer HA
    2. Kong YW
    3. Lu WT
    4. Wilczynska A
    5. Spriggs RV
    6. Robinson SW
    7. Godfrey JD
    8. Willis AE
    9. Bushell M

    (2013) Translational repression and eif4a2 activity are critical for microrna-mediated gene regulation

    Science 340:82–85.

    https://doi.org/10.1126/science.1231197

    • Google Scholar
46. 1. Nelson PT
    2. Hatzigeorgiou AG
    3. Mourelatos Z

    (2004) miRNP:mRNA association in polyribosomes in a human neuronal cell line

    RNA 10:387–394.

    https://doi.org/10.1261/rna.5181104

    • Google Scholar
47. 1. Nottrott S
    2. Simard MJ
    3. Richter JD

    (2006) Human let-7a mirna blocks protein production on actively translating polyribosomes

    Nature Structural & Molecular Biology 13:1108–1114.

    https://doi.org/10.1038/nsmb1173

    • Google Scholar
48. 1. Olsen PH
    2. Ambros V

    (1999) The lin-4 regulatory RNA controls developmental timing in caenorhabditis elegans by blocking LIN-14 protein synthesis after the initiation of translation

    Developmental Biology 216:671–680.

    https://doi.org/10.1006/dbio.1999.9523

    • Google Scholar
49. 1. Pelechano V
    2. Wei W
    3. Steinmetz LM

    (2015) Widespread co-translational RNA decay reveals ribosome dynamics

    Cell 161:1400–1412.

    https://doi.org/10.1016/j.cell.2015.05.008

    • Google Scholar
50. 1. Petersen CP
    2. Bordeleau M-E
    3. Pelletier J
    4. Sharp PA

    (2006) Short rnas repress translation after initiation in mammalian cells

    Molecular Cell 21:533–542.

    https://doi.org/10.1016/j.molcel.2006.01.031

    • Google Scholar
51. 1. Pillai RS
    2. Bhattacharyya SN
    3. Artus CG
    4. Zoller T
    5. Cougot N
    6. Basyuk E
    7. Bertrand E
    8. Filipowicz W

    (2005) Inhibition of translational initiation by let-7 microrna in human cells

    Science 309:1573–1576.

    https://doi.org/10.1126/science.1115079

    • Google Scholar
52. 1. Rehwinkel J

    (2005) A crucial role for GW182 and the DCP1:DCP2 decapping complex in mirna-mediated gene silencing

    RNA 11:1640–1647.

    https://doi.org/10.1261/rna.2191905

    • Google Scholar
53. Book

    1. Rio DC
    2. Ares JM
    3. Hannon GJ
    4. Nilsen TW

    (2011)

    RNA: A Laboratory Manual

    Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press.

    • Google Scholar
54. 1. Rouya C
    2. Siddiqui N
    3. Morita M
    4. Duchaine TF
    5. Fabian MR
    6. Sonenberg N

    (2014) Human DDX6 effects mirna-mediated gene silencing via direct binding to CNOT1

    RNA 20:1398–1409.

    https://doi.org/10.1261/rna.045302.114

    • Google Scholar
55. 1. Seggerson K
    2. Tang L
    3. Moss EG

    (2002) Two genetic circuits repress the caenorhabditis elegans heterochronic gene lin-28 after translation initiation

    Developmental Biology 243:215–225.

    https://doi.org/10.1006/dbio.2001.0563

    • Google Scholar
56. 1. Stadler M
    2. Artiles K
    3. Pak J
    4. Fire A

    (2012) Contributions of mrna abundance, ribosome loading, and post- or peri-translational effects to temporal repression of C. elegans heterochronic mirna targets

    Genome Research 22:2418–2426.

    https://doi.org/10.1101/gr.136515.111

    • Google Scholar
57. 1. Stark A
    2. Brennecke J
    3. Russell RB
    4. Cohen SM

    (2003) Identification of drosophila microrna targets

    PLoS Biology 1:e60.

    https://doi.org/10.1371/journal.pbio.0000060

    • Google Scholar
58. 1. Subtelny AO
    2. Eichhorn SW
    3. Chen GR
    4. Sive H
    5. Bartel DP

    (2014) Poly(A)-tail profiling reveals an embryonic switch in translational control

    Nature 508:66–71.

    https://doi.org/10.1038/nature13007

    • Google Scholar
59. 1. Valencia-Sanchez MA
    2. Liu J
    3. Hannon GJ
    4. Parker R

    (2006) Control of translation and mrna degradation by mirnas and sirnas

    Genes & Development 20:515–524.

    https://doi.org/10.1101/gad.1399806

    • Google Scholar
60. 1. Wang B
    2. Yanez A
    3. Novina CD

    (2008) Microrna-repressed mrnas contain 40S but not 60S components

    Proceedings of the National Academy of Sciences of the United States of America 105:5343–5348.

    https://doi.org/10.1073/pnas.0801102105

    • Google Scholar
61. 1. Wightman B
    2. Ha I
    3. Ruvkun G

    (1993) Posttranscriptional regulation of the heterochronic gene lin-14 by lin-4 mediates temporal pattern formation in C. elegans

    Cell 75:855–862.

    https://doi.org/10.1016/0092-8674(93)90530-4

    • Google Scholar
62. 1. Wu E
    2. Thivierge C
    3. Flamand M
    4. Mathonnet G
    5. Vashisht AA
    6. Wohlschlegel J
    7. Fabian MR
    8. Sonenberg N
    9. Duchaine TF

    (2010) Pervasive and cooperative deadenylation of 3′utrs by embryonic microrna families

    Molecular Cell 40:558–570.

    https://doi.org/10.1016/j.molcel.2010.11.003

    • Google Scholar

Author details

1. Trinh To Tat

   Center for RNA Molecular Biology, Case Western Reserve University, Cleveland, United States

   Contribution

   TTT, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article

   Competing interests

   No competing interests declared.

2. Patricia A Maroney

   Center for RNA Molecular Biology, Case Western Reserve University, Cleveland, United States

   Contribution

   PAM, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article

   Competing interests

   No competing interests declared.

3. Sangpen Chamnongpol

   Affymetrix, Inc., Cleveland, United States

   Contribution

   SC, Conception and design, Analysis and interpretation of data, Drafting or revising the article

   Competing interests

   No competing interests declared.

4. Jeff Coller

   Center for RNA Molecular Biology, Case Western Reserve University, Cleveland, United States

   Contribution

   JC, Conception and design, Analysis and interpretation of data, Drafting or revising the article

   Competing interests

   No competing interests declared.

5. Timothy W Nilsen

   Center for RNA Molecular Biology, Case Western Reserve University, Cleveland, United States

   Contribution

   TWN, Conception and design, Analysis and interpretation of data, Drafting or revising the article

   For correspondence

   twn@case.edu

   Competing interests

   TWN: Reviewing editor, eLife.

   "This ORCID iD identifies the author of this article:" 0000-0002-7276-3571

• 3,989

  views

• 1,010

  downloads

• 36

  citations

Views, downloads and citations are aggregated across all versions of this paper published by eLife.