Micromanagement: A Role for MicroRNAs in mRNA Stability Sarah F. Roush† and Frank J. Slack‡,* †Department of Molecular Biophysics and Biochemistry and ‡Department of Molecular, Cellular and
Developmental Biology, Yale University, P.O. Box 208103, New Haven, Connecticut 06520-8103
A b s t r a c t Small, inhibitory RNA molecules called microRNAs cause large decreases in target protein levels through a posttranscriptional mechanism. Until recently, it was believed this mechanism operated almost exclusively at a step in translation. However, new work has revealed that microRNAs have a second, post-transcriptional mechanism that accelerates the rate of deadenylation, the initial step of mRNA decay.
*To whom correspondence should be addressed. E-mail:
[email protected].
Published online April 21, 2006 10.1021/cb600138j CCC: $33.50 © 2006 by American Chemical Society
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ne of the most sought after answers in microRNA (miRNA) research is the mechanism by which these small, inhibitory molecules post-transcriptionally regulate their mRNA targets (for review, see ref 1 ). MiRNAs are ~22 nt, non-coding RNAs that are present in most eukaryotes and have the potential to post-transcriptionally regulate a large number of genes (2, 3 ). In animals, miRNAs bind to partially complementary sites in the 3´ UTRs of their target mRNAs, leading to significant decrease in the targets’ protein levels (4, 5 ). Early research indicated that miRNAs regulate their targets through translational inhibition without lowering mRNA levels (4, 6 ). The point in translation in which the regulation occurs is currently being debated, but appears to happen before the completion of the polypeptide chain (6–12 ). More recently, the idea that miRNAs function only through trans lational inhibition has been challenged by studies indicating that miRNAs can cause decreases in target mRNA levels (12–14 ). Two recent studies indicate that miRNA binding triggers target mRNA degradation through an increased rate of deadenylation from the poly(A) tail, normally the ratelimiting step in mRNA degradation (12, 14 ). In a report by Giraldez et al. (14 ), a miRNA expressed at the beginning of zebrafish zygotic transcription, miR-430, was shown to facilitate the clearing of many maternal mRNAs. These target mRNAs exhibited rapid deadenylation mediated by miR-430 and its target sites in the 3´ UTRs of the mRNAs. The deadenylation was not a result of blocked
transcription, as an antisense morpholino oligonucleotide hybridized to the translational start site of a GFP reporter did not cause as much mRNA deadenylation and decay in the absence of the miRNA as in the presence of the miRNA. The fact that the miRNA could still cause accelerated deadenyl ation while translation was inhibited indicated that the miRNA did not need translation to mediate the poly(A) tail removal. In a second report by Wu et al. (12 ), mammalian cell culture showed similar results to that of Giraldez et al. (14 ). However, the study conducted by Wu et al. (12 ) not only demonstrates rapid deadenylation and decay of the target mRNA but also provides evidence for the intriguing idea that miRNAs can use two independent mechanisms of post-transcriptional regulation: translational inhibition and mRNA degradation through accelerated deadenylation (Figure 1). To begin their work, Wu et al. (12 ) utilized an established promoter-reporter system to measure mRNA decay (15 ). The basic reporter contained an inducible c-fos promoter and β-globin gene (BG). Two copies of the miR-125b binding site, referred to as miRNA response element 1 (mRE1), from the 3´ UTR of miR-125b’s target mRNA, lin‑28 (16 ), were inserted into the 3´ untranslated region (UTR) of the basic reporter mRNA. This modified reporter (BG+2E1) and the miR-125b gene were transiently transfected into 293T human embryonic kidney cells, where it has previously been shown that miR-125b can down-regulate a luciferase reporter mRNA containing several copies of miR‑125b mRE 1 or 2 (16 ). In the present w w w. a c s c h e m i ca l biology.org
Figure 1. Two mechanisms of miRNA post-transcriptional regulation. A miRNA induced silencing complex (miRISC) loaded with its miRNA binds with imperfect complementarity to the 3´ UTR of its target where it performs one or both of the following roles: a) Translational inhibition. Recent studies show that parts of the miRNA repression complex are present in processing bodies (P body) (20 ). Generally, P bodies are areas of mRNA degradation that lack translational machinery (21, 22 ); however, it seems that miRNA target mRNAs do not appear to be degraded at the same rate as other mRNAs in the P bodies (11 ). Another study suggests that miRNAs cause ribosomes to fall off the target mRNA during translation (10 ). b) mRNA degradation. As described in Wu et al. (12 ), miRNAs lead to accelerated removal of the poly(A) tail and, as a consequence, lead to cap removal followed by target mRNA degradation by the nuclease Xrn1 and the exosome complex. The red circle represents an associated deadenylase whose identity is unknown.
study, the presence of miR-125b led to an acceleration of reporter mRNA poly(A) tail deadenylation and decay. RT-PCR revealed that the degradation did not occur through a siRNA-like endonucleolytic cleavage within the mRE1 site, as occurred in a reporter containing a synthetic, perfectly complementary mIR-125b binding site. Using susceptibility to a 5´ exonuclease as an assay, it was then shown that deadenylation preceded removal of the 5´ cap. In similar experiments, when the let-7a gene and a luciferase reporter containing lin-28 mRNA’s let-7 binding elements (L7) were transiently transfected into 293T cells, there were also accelerated reporter mRNA deadenylation and decay. To identify other possible miR-125b targets, a mouse genome microarray was performed using P19 cells. Undifferentiated P19 cells, which produce miR-125b when they differentiate into neurons, were mocktransfected or transfected with chemically synthesized miR-125b. Cytoplasmic RNA from these cells was used to probe the www.acschemicalbiolog y.o rg
microarray. Twenty-two putative targets of miR-125b were identified, all containing at least one possible miR-125b binding element. Possible miR-125b binding elements were selected from the 3´ UTRs of two putative targets, Ajuba and MAPK kinase 7, and inserted into reporter vectors. These elements were found to accelerate the rate of reporter mRNA deadenylation and decay in the same mRNA decay and deadenylation assays used for mRE1 and L7. The most intriguing experiments indicate that miRNAs may have two independent mechanisms by which they can downregulate target expression. The first set of experiments demonstrated that a loss of translation was not what led to the accelerated target poly(A) tail loss and mRNA decay observed in the earlier miR-125b and BG+2E1 experiments. A large, 40 nt stem-loop was added into the 5´ UTR of BG+2E1, which eliminated translation by specifically blocking translation initiation. Blocking translation in the absence of
miR‑125b caused a small increase in the target mRNA’s deadenylation and decay rates. However, rapid rates of deadenylation and decay were observed with both a block in translation and the presence of miR-125b. Importantly, the rapid rates of the mRNA decay were close to the rates observed when miR-125b was present and translation was not blocked, thus, indicating translation was not necessary for miR‑125b’s ability to accelerate deadenylation (Figure 2, panel b). Reciprocal experiments showed that translational repression by miR-125b was not dependent on the presence of a poly(A) tail. These experiments used a luciferase reporter containing multiple mRE1 sites in its 3´ UTR and a poly(A) tail or a histonederived stem-loop in place of the poly(A) tail (Figure 2, panel c). Histone stem-loops mediate translation by using the stem-loop binding protein (SLBP) instead of a poly(A) tail and poly(A)-binding protein (PABP) (17 ). When these reporters were transfected with the miR-125b gene, no decrease in mRNA levels was seen, compared to the polyadenylated reporter, which had a drop in mRNA levels, as expected. However, a key observation was made upon calculation of the actual levels of luciferase protein, which showed that the level of translational inhibition by the miRNA was the same for both reporters. Therefore, even though removal of the poly(A) tail and the subsequent drop in mRNA levels was an important component of miR-125b regulation through mRE1, a large portion of the regulation was also due to translational repression that was independent of the poly(A) tail. Together, both of these regulatory mechanisms produced an even greater effect than either alone could do (Figure 2, panel d). The idea that miRNAs may have two distinct functional mechanisms helps to reconcile previous results where evidence from different studies pointed to different types of post-transcriptional regulation, such as in the cases of lin-4 and let-7 (6, 13, 18 ). VOL.1 NO. 3 • 132—134 • 2 0 0 6
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Figure 2. Additive effects of miR-125b repression. a) Major pathway of mRNA decay. The pathway begins with removal of the poly(A) tail and releasing of poly(A)-binding protein (PABP) followed by dissociation of the cap-binding proteins, eIF4E and eIF4G, which prevents further translation and leads to mRNA decay (19 ). b) Blocked translation. Blocking translation initiation by inserting a strong stem-loop structure reveals that the accelerated rate of deadenylation is not due to impaired translation, and hypothetically prevents any repression through translational inhibition. c) Poly(A) tail removed. Replacing the poly(A) tail with a histone-derived stem-loop prevents miRNA-mediated deadenylation and mRNA decay but does not prevent repression by translational inhibition. The triangle represents a hypothetical interacting partner. d) Two additive miRNA mechanisms. The two mechanisms can act additively to enhance repression of the target.
However, it also makes defining the exact nature of how miRNAs inhibit translation more complex. Recently, other reports have suggested that miRNAs may inhibit translation at initiation (10, 11 ) and/or elongation (10 ). If so, it could be expected that translational inhibition could be due to the loss of PABP binding as would result from the replacement of the poly(A) tail with a histone stem-loop (19 ). PABP binds to the poly(A) tail and interacts with the 5´ cap binding protein eIF4G, resulting in mRNA circularization and aiding translation initiation (Figure 2, panel a) (19 ). However, the results of Wu et al. (12 ) indicate that miRNA translational inhibition is independent of the poly(A) tail and PABP loss or at a step when PABP and SLBP function similarly (12 ). Continued research into miRNA cap and poly(A) tail dependence and their associated proteins, along with the development of novel inhibitors for specific translational states, such as hippuristanol (10 ), will further elucidate possible miRNA mechanisms of action. Perhaps when the two mechanisms of miRNA are completely separated and understood, synthesized 1 34
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miRNAs can be produced to specifically inhibit translation initiation or deadenylation. Conceivably, miRNAs could use multiple combinations of additional, as yet unknown, mechanisms to regulate different targets. Though, in the end, it may be that the mechanism of every miRNA is finetuned for its specific target(s). REFERENCES 1. Bartel, D. P. (2004) MicroRNAs: Genomics, biogenesis, mechanism, and function, Cell 116, 281–297. 2. Krek, A., Grun, D., Poy, M. N., Wolf, R., Rosenberg, L., Epstein, E. J., MacMenamin, P., da Piedade, I., Gunsalus, K. C., Stoffel, M., and Rajewsky, N. (2005) Combinatorial microRNA target predictions, Nat. Genet. 37, 495–500. 3. Lewis, B. P., Shih, I. H., Jones-Rhoades, M. W., Bartel, D. P., and Burge, C. B. (2003) Prediction of mammalian microRNA targets, Cell 115, 787–798. 4. Wightman, B., Ha, I., and 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. 5. Reinhart, B. J., Slack, F. J., Basson, M., Pasquinelli, A. E., Bettinger, J. C., Rougvie, A. E., Horvitz, H. R., and Ruvkun, G. (2000) The 21nucleotide let-7 RNA regulates developmental timing in Caenorhabditis elegans, Nature 403, 901–906. 6. Olsen, P. H., and 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, Dev. Biol. 216, 671–680.
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