Trends in Cell Biology
ReviewRepression of protein synthesis by miRNAs: how many mechanisms?
Introduction
MicroRNAs (miRNAs; see Glossary) are ∼21- nucleotide (nt)-long RNA regulators which control, at the post-transcriptional level, gene expression in metazoan animals and plants. Although 100–200 miRNAs are expressed in lower metazoa, 1000 or more are predicted to function in humans, possibly regulating ∼30% of human genes. Target mRNAs and biological function have been assigned to only a few dozen miRNAs but it is becoming apparent that miRNAs participate in the regulation of almost every process investigated. The expression of many miRNAs is specific to particular tissues or developmental stages, and miRNA profiles are altered in several human diseases 1, 2, 3.
miRNAs associate with their mRNA targets by base-pair complementarity. In plants, miRNAs generally show nearly perfect complementarity to target sequences positioned in either coding or 3′ untranslated (3′ UTR) regions of mRNAs. The perfect base pairing triggers mRNA degradation through a mechanism similar to that operating during RNA interference (RNAi) induced by small interfering RNAs (siRNAs). In animals, with very few exceptions, miRNAs regulate gene expression by base pairing imperfectly to the 3′ UTR of target mRNAs and inhibiting protein synthesis or causing mRNA degradation (Figure 1, Figure 2). Two major features of animal miRNA–mRNA interactions are the contiguous Watson–Crick pairing in the miRNA 5′ proximal seed region (usually positions 2–8) and a lack of complementarity in the central part of the miRNA (usually positions 10 and 11) that precludes the RNAi-like endonucleolytic cleavage of the target mRNA in the middle of the duplex. Although one perfectly complementary site is sufficient for the siRNA- or miRNA-induced cleavage of mRNA, studies using reporter mRNAs have indicated that effective translational repression usually requires multiple imperfect sites recognized by the same or several different miRNAs. The molecular basis of this apparent miRNP cooperativity remains unknown 4, 5, 6, 7.
miRNAs function as ribonucleoprotein particles, miRNA-induced silencing complexes (miRISCs) or miRNA ribonucleoprotein complexes (miRNPs), similar to RISCs operating in RNAi. Argonaute (Ago) proteins are the essential and best-characterized components of miRNPs. Of the four mammalian Argonautes, Ago1 to Ago4, only one, Ago2 (often referred to as a ‘Slicer’), functions in RNAi by endonucleolytically cleaving mRNA in the middle of the mRNA–siRNA duplex, whereas all four seem to participate in miRNA-mediated repression 8, 9, 10. In Drosophila, dAgo1 is dedicated to the miRNA pathway and dAgo2 functions in RNAi 11, 12.
Here, we review recent progress in understanding the mechanisms of translational repression and destabilization of target mRNAs by miRNAs. We also discuss a role for cytoplasmic processing bodies (P-bodies) in these reactions and the reversibility of the repression process. With our current knowledge, it is difficult to construct a unified model of precisely how miRNAs induce the inhibition of translation. This indicates that either miRNAs are able to interfere with protein synthesis in different ways or that the current experimental approaches have limitations that prevent us from gaining a comprehensive picture of the regulation. Our discussion is largely confined to the mode of action for animal miRNAs because little work has been performed so far on translational repression in plants. Mechanistic aspects of miRNA function are also discussed in two recent reviews 13, 14.
Section snippets
Translational effects of miRNAs
The events during translation can be broadly divided into three stages: initiation, elongation and termination. Multiple protein factors are involved at each stage. Initiation is generally a rate-limiting step in translation and is frequently a subject of elaborate regulation. Translation of most cellular mRNAs requires a 5′ terminal m7G cap but a few mRNAs, and also many viral RNAs, are translated in a cap-independent manner (Box 1). Examples of miRNAs repressing translation of either
Repression at the initiation stage
Support for miRNAs inhibiting translation at the initiation step comes from experiments performed in mammalian cell cultures using either reporters or endogenous mRNAs. Polysome profile analysis of luciferase reporter mRNAs, expressed from transfected plasmid DNA and repressed by either endogenous let-7 miRNP or miRNA-independent tethering of Ago proteins to a reporter mRNA, showed a marked shift of the repressed mRNA towards the lighter fractions of a sucrose gradient, indicative of an effect
Repression at post-initiation steps
Evidence supporting a post-initiation, rather than initiation, mechanism of repression has been obtained in both Caenorhabditis elegans and mammalian cell cultures. The lin-4 miRNA, expression of which is induced late in the L1 stage of C. elegans larval development, represses translation of lin-14 and lin-28 mRNAs encoding two heterochronic regulators. The sucrose gradient and metrizamide-buoyant density gradients of the lin-4 target mRNAs remained largely unchanged under conditions of
Role of poly(A)
The poly(A) tail has an important role in mRNA stability [25] and also in translational initiation because the cytoplasmic poly(A)-binding protein PABPC1 interacts with eIF4G to bring the two ends of the mRNA together and increase the efficiency of translation [26]. Experiments to define the importance of poly(A) in miRNA-mediated repression have produced ambiguous results. Poly(A) was not essential for repression (although could increase its efficiency) of the in vitro-transcribed 5′ capped
Repressed mRNAs are enriched in P-bodies
Several recent reports indicated that Argonaute proteins, miRNAs and mRNAs repressed by miRNAs all accumulate in discrete cytoplasmic foci, the so-called P-bodies (also known as GW bodies) 20, 21, 28, 29, 30, 31. P-bodies have been established as sites of translational repression and mRNA decay, and they are enriched in factors involved in these pathways. They seem to be dynamic RNA–protein aggregates containing translationally repressed mRNAs but lacking ribosomes and most translation
miRNA-induced downregulation of mRNA levels
Repression by miRNAs is also frequently associated with a substantial degradation of target mRNAs. The first demonstration that miRNAs can downregulate levels of target mRNAs came from a microarray study in which introduction of an alien miRNA into human HeLa cells downregulated a large number of mRNAs [39]. Consistently, inhibition of miR-122 function in mouse liver using anti-miR oligonucleotides led to the stabilization of hundreds of mRNAs [40]. In another approach, depletion of the miRNA
Reversibility of miRNA-mediated repression and P-body localization of mRNAs
In yeast, the global inhibition of translation results in an increase in the number and size of P-bodies accumulating repressed mRNAs. Importantly, this process can be reversed (e.g. following return to a nutrient-rich medium), indicating that P-bodies, in addition to their role in mRNA degradation, can function as temporary storage sites for mRNAs not participating in protein synthesis. Can mRNAs accumulating in metazoan P-bodies as a result of the miRNA-mediated repression likewise exit these
Simplistic models and concluding remarks
We have presented evidence for different ways in which miRNAs can influence the expression of target mRNAs. They can affect either mRNA translation or its stability, and in many cases both effects are combined. Considering that the translational status of an mRNA can directly affect mRNA stability, it is possible that target mRNA degradation is a consequence of translational repression rather than a primary effect of miRNA. The mRNA degradation becomes more prominent in the event of accelerated
Update
Since writing this article, three papers have been published that address different aspects of translational regulation by miRNAs in mammalian cells 68, 69, 70. Nottrott et al. [68] show that a luciferase reporter mRNA, which carries the C. elegans lin-41 3′UTR with let-7a target sites, remains associated with translating polysomes under conditions that repress the accumulation of protein. Based on the inability to immunoprecipitate the repressed mRNA with an antibody that recognizes the
Acknowledgements
S.N.B. is a recipient of a long-term fellowship from the HFSPO. The Friedrich Miescher Institute is supported by the Novartis Research Foundation.
Glossary
- ApppG cap
- an unmethylated cap analog that is used to study the requirement of the m7G cap structure in translation. The mRNAs with an artificially introduced ApppG cap are translated inefficiently as they do not bind to eIF4E.
- A-form helix
- the double helix formed by two complementary RNA chains. It differs from the B-form DNA double helix. The major groove of the dsRNA A-form helix is deep but narrow, making it virtually inaccessible to proteins.
- Argonautes
- members of a highly conserved family of
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