Mechanism of Translational Inhibition by MicroRNA

Abstract

DoctorMicroRNAs (miRNAs) are 21~24-nt small noncoding RNAs that play a key role in the post-transcriptional gene silencing in metazoan animals, plants and protozoa. Many of the animal miRNAs are phylogenetically conserved, and ~55% of nematode miRNAs, for example, have homologues in humans, indicating that miRNAs have important roles throughout animal evolution. Indeed, mammalian miRNAs are bioinformatically predicted to control ~30% of the protein-coding genes, and participate in several biological processes such as development, differentiation, proliferation, apoptosis, metabolic control, metastasis, etc.miRNAs usually function as guidance in the post-transcriptional gene regulation by base-pairing with the 3-UTR of the target mRNAs. For this, Argonaute proteins, key factors in the small RNA-mediated gene silencing, form an effector protein complex called the RNA-induced silencing complex or the miRNA-containing ribonucleoprotein complex. Many miRNAs degrade the targeted mRNAs by promoting their deadenylation and/or decappig, resulting in the repression of gene expression. Also, many reports have indicated that miRNAs participate in the gene silencing by decreasing the translation of mRNAs. Several studies have suggested that miRNAs can reduce translation of their target mRNAs at the post-initiation stage (i.e., the elongation step), based on observations that miRNAs co-migrate with polyribosomes and their polysomal distributions are not altered during the gene repression. Recently, it has been suggested that the translational repression by miRNAs occurs at the initiation step of translation, and particularly that the 5 mRNA cap structure is critical for the miRNA-mediated translational gene silencing, probably via the competition of Ago2 with a cap-binding protein eIF4E for the mRNA cap structure. However, the molecular basis of this silencing is unclear.Here, I show that human Ago2 associates with the cap-binding protein complex and this association is mediated by human eIF4GI, a scaffold protein required for the translation initiation. Cap-pulldown assays show that Ago2 proteins form a complex with the cap-binding complex via association with the N-terminus of eIF4GI protein. Overexpression of the full-length of eIF4GI as well as of the N-terminus augments the Ago2-cap association, and the knock-down of eIF4GI by synthetic siRNAs decreases the Ago2-cap association in cells. Furthermore, the knock-down of eIF4GI de-represses the miRNA-mediated translational repression of capped poly(A)-tailed target mRNA. Finally, Cap photo-crosslinking assays confirm that Ago2 closely associates with cap structure, which is mediated by association with eIF4GI.In addition, I examine whether amino acid residues (Phe-470, Phe-505, Tyr-529, Lys-533 and Asp-537) previously suggested as requirements for the Ago2-cap association could be really involved in the Ago2-cap association. Cap-pulldown assays and cap-crosslinking assays demonstrate that the cap binding-like motif Ago2 (Phe-470 and Phe-505) is not critical for the Ago2-cap association. Similarly, the allosteric regulatory site Asp-537 equivalent to Asp-627 in Drosophila Ago2 does not influence both the Ago2-cap association, and the Ago2-mediated translational repression. Finally, I obtain the hypothetical structure of human Ago2 based on Aquifex aeolicus Ago through the PHYRE program, and find that Phe-294 and Tyr-805 can be potential players for regulation of the Ago2-cap association