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Towards a molecular understanding of microRNA-mediated gene silencing

Key Points

  • MicroRNAs (miRNAs) silence gene expression by repressing translation and accelerating target mRNA degradation.

  • The current consensus is that miRNAs inhibit cap-dependent translation at initiation, but the precise molecular mechanism remains to be elucidated.

  • Degradation of miRNA targets is the dominant effect of miRNAs at steady state in cultured mammalian cells.

  • The degradation of miRNA targets is catalysed by enzymes involved in the 5′-to-3′ mRNA decay pathway. In this pathway, mRNAs are first deadenylated, then decapped and finally degraded from the 5′ end.

  • The GW182 proteins play a central part in silencing. They bridge the interaction between Argonaute proteins and downstream effector complexes, such as the cytoplasmic deadenylase complexes PAN2–PAN3 and CCR4–NOT.

  • Structural studies have revealed that GW182 proteins interact with their partners by inserting tryptophan residues into hydrophobic pockets exposed on the surface of Argonaute proteins, PAN3 and NOT9.

  • Current models for miRNA-mediated translational repression invoke displacement of eukaryotic translation initiation factor 4A1 (eIF4A1) or its paralogue eIF4A2, or the recruitment of the translational repressor and decapping activator DEAD box protein 6 (DDX6).

  • Remaining open questions include how miRNA-induced silencing complexes (miRISCs) displace eIF4A1 and eIF4A2, and how DDX6 represses translation.

Abstract

MicroRNAs (miRNAs) are a conserved class of small non-coding RNAs that assemble with Argonaute proteins into miRNA-induced silencing complexes (miRISCs) to direct post-transcriptional silencing of complementary mRNA targets. Silencing is accomplished through a combination of translational repression and mRNA destabilization, with the latter contributing to most of the steady-state repression in animal cell cultures. Degradation of the mRNA target is initiated by deadenylation, which is followed by decapping and 5′-to-3′ exonucleolytic decay. Recent work has enhanced our understanding of the mechanisms of silencing, making it possible to describe in molecular terms a continuum of direct interactions from miRNA target recognition to mRNA deadenylation, decapping and 5′-to-3′ degradation. Furthermore, an intricate interplay between translational repression and mRNA degradation is emerging.

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Figure 1: Overview of miRNA-mediated gene silencing in animals.
Figure 2: Structural insight into the interaction of AGO proteins with GW182 proteins.
Figure 3: Assembly and interaction of the PAN2–PAN3 complex with GW182 proteins.
Figure 4: Assembly and interaction of the CCR4–NOT complex with GW182 proteins.
Figure 5: Structure-based model of miRNA-mediated silencing.

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Acknowledgements

Research from the authors' laboratory is supported by the Max Planck Society and by the Gottfried Wilhelm Leibniz Program of the Deutsche Forschungsgemeinschaft (DFG) awarded to E.I.

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Correspondence to Elisa Izaurralde.

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Glossary

Deadenylation

Shortening of mRNA poly(A) tails. In eukaryotes, this process is catalysed by the consecutive but partially redundant action of two cytoplasmic deadenylase complexes: PAN2–PAN3 and CCR4–NOT.

Decapping

Hydrolysis of the 5′ cap structure on the mRNA. A major decapping enzyme in eukaryotes is decapping protein 2 (DCP2), which hydrolyses the cap structure, releasing 7-methyl-GDP and a 5′ monophosphorylated mRNA. This 5′ monophosphorylated mRNA is a substrate for 5′-to-3′ exoribonuclease 1 (XRN1), which rapidly degrades decapped mRNA.

Ribosome profiling

A method that allows the determination of the position of ribosomes on cellular mRNAs with high sequence resolution. Briefly, cells are treated with cycloheximide to stabilize ribosomes on mRNAs, then lysed and treated with nucleases to degrade mRNA regions not protected by ribosomes. Translating ribosomes protect RNA fragments of about 30 nucleotides in length (known as ribosome-protected fragments) that can be sequenced, generating millions of mRNA sequence tags.

Cooperativity

The changes that occur when the binding of a ligand to a binding site on one molecule increases (or decreases) the affinity for binding to a second ligand on another binding site on the same molecule.

Avidity

The phenomenon by which individual binding events increase the likelihood of other interactions occurring, for example, by increasing the local concentration of each binding partner in proximity to the binding site.

Cap-dependent translation

Initiation of translation that requires the ternary eukaryotic translation initiation factor 4F (eIF4F) complex, which consists of the cap-binding protein eIF4E, the adaptor protein eIF4G and the DEAD box RNA helicase eIF4A. This complex interacts with the cap structure at the 5′ end of an mRNA molecule and recruits the 43S pre-initiation complex.

Cap structure

Eukaryotic mRNA is modified by the addition of an m7G(5′)ppp(5′)N structure (7-methylguanosine attached, via its 5′ hydroxyl group, by a triphosphate group to the 5′ hydroxyl group of the first encoded nucleoside) at the 5′ terminus. Capping is essential for several important steps of gene expression, including mRNA stabilization, splicing, mRNA export from the nucleus and translation initiation.

Ribosome scanning

The 5′-to-3′ migration of the 43S pre-initiation complex towards the initiation codon. The 43S pre-initiation complex comprises a 40S ribosomal subunit, eukaryotic translation initiation factor 3 (eIF3), eIF1 and eIF1A, the ternary eIF2–GTP–Met-tRNAMeti complex and most likely eIF5.

Internal ribosome entry sites

(IRESs). Structured RNA elements, usually present in the 5′ untranslated region, that allow cap-independent association of ribosomes with mRNAs.

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Jonas, S., Izaurralde, E. Towards a molecular understanding of microRNA-mediated gene silencing. Nat Rev Genet 16, 421–433 (2015). https://doi.org/10.1038/nrg3965

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