miRNA repression of translation in vitro takes place during 43S ribosomal scanning

Abstract

microRNAs (miRNAs) regulate gene expression at multiple levels by repressing translation, stimulating deadenylation and inducing the premature decay of target messenger RNAs (mRNAs). Although the mechanism by which miRNAs repress translation has been widely studied, the precise step targeted and the molecular insights of such repression are still evasive. Here, we have used our newly designed in vitro system, which allows to study miRNA effect on translation independently of deadenylation. By using specific inhibitors of various stages of protein synthesis, we first show that miRNAs target exclusively the early steps of translation with no effect on 60S ribosomal subunit joining, elongation or termination. Then, by using viral proteases and IRES-driven mRNA constructs, we found that translational inhibition takes place during 43S ribosomal scanning and requires both the poly(A) binding protein and eIF4G independently from their physical interaction.

mi RISC and the CCR 4– NOT complex silence mRNA targets independently of 43S ribosomal scanning

“…By contrast, Ricci et al (2013) failed to detect a direct correlation between silencing and 5′‐UTR secondary structure in rabbit reticulocyte lysates. Furthermore, it has been reported that 5′‐UTR features such as length and secondary structure do not reliably correlate with the extent of miRNA‐mediated repression (Agarwal et al , 2015).…”

“…Several studies reported that miRNAs silence gene expression by targeting eIF4A and interfering with ribosome scanning (Meijer et al , 2013; Ricci et al , 2013; Fukao et al , 2014; Fukaya et al , 2014). However, there were considerable disagreements between these studies.…”

“…eIF4A proteins are DEAD‐box RNA helicases that unwind secondary structures within mRNA 5′‐UTRs, allowing the 43S preinitiation complex (PIC) to scan the 5′‐UTR toward the start codon (Jackson et al , 2010). Several studies have indicated that miRISCs inhibit 43S scanning by interfering with eIF4A function (Meijer et al , 2013; Ricci et al , 2013; Fukao et al , 2014; Fukaya et al , 2014). How this interference is achieved remains unclear.…”

The Functions of MicroRNAs: mRNA Decay and Translational Repression

“…miRNA-Mediated Dissociation of eIF4A To date, many reports have shown that mRNAs lacking the 5 0 m 7 G-cap structure but instead bearing the nonphysiological A-cap structure or IRESes, which bypass the requirement for the 5 0 m 7 G-cap as well as one or more eIFs for translation initiation, are refractory to miRNA-mediated silencing [47,56,94,[104][105][106][107][108]. Supplementation or depletion of initiation factors attenuated miRNA-mediated repression [83,85,86,106,109].…”

The NS1 Protein from Influenza Virus Stimulates Translation Initiation by Enhancing Ribosome Recruitment to mRNAs

“…ACCEPTED MANUSCRIPT 20 The β-globin, GAPDH, PABP, cyclinD2 cellular 5'UTR, , FMDV, HAV, AEV, DCV, HCV and CrPV IRESes p0-renilla were previously described [50][51][52]. …”