MicroRNA (miRNA) are well known to target 3’ untranslated regions (3’UTR) in mRNAs to silence gene expression at post-transcriptional levels. Multiple reports have also indicated the capability of miRNAs to target protein-coding sequences (CDS); however, miRNAs have been generally believed to function in a similar mechanism(s) regardless of the location of their action sites. We herein report a class of miRNA recognition elements (MREs) that exclusively function in CDS regions in humans. Through functional and mechanistic characterization of these “unusual” MREs, we demonstrate that CDS-targeted miRNAs require extensive base pairings in the 3’ side rather than the 5’ seed; cause gene silencing in an Argonaute-dependent, but GW182-independent manner; and repress translation by inducing transient ribosome stalling instead of mRNA destabilization. These findings reveal distinct mechanisms and functional consequences for miRNAs to target CDS versus 3’UTR and suggest that CDS-targeted miRNAs may enlist a translational quality control (QC)-related mechanism to regulate translation in mammalian cells.

During translation, elongation factor G (EF-G) catalyzes the translocation of tRNA2-mRNA inside the ribosome. Translocation is coupled to a cycle of conformational rearrangements of the ribosomal machinery, and how EF-G initiates translocation remains unresolved. Here we performed systematic mutagenesis of Escherichia coli EF-G and analyzed inhibitory single-site mutants of EF-G that preserved pretranslocation (Pre)-state ribosomes with tRNAs in A/P and P/E sites (Pre-EF-G). Our results suggest that the interactions between the decoding center and the codon-anticodon duplex constitute the barrier for translocation. Catalysis of translocation by EF-G involves the factor's highly conserved loops I and II at the tip of domain IV, which disrupt the hydrogen bonds between the decoding center and the duplex to release the latter, hence inducing subsequent translocation events, namely 30S head swiveling and tRNA2-mRNA movement on the 30S subunit.

During translation, elongation factor G (EF-G) plays a catalytic role in tRNA translocation and a facilitative role in ribosome recycling. By stabilizing the rotated ribosome and interacting with ribosome recycling factor (RRF), EF-G was hypothesized to induce the domain rotations of RRF, which subsequently performs the function of splitting the major intersubunit bridges and thus separates the ribosome into subunits for recycling. Here, with systematic mutagenesis, FRET analysis and cryo-EM single particle approach, we analyzed the interplay between EF-G/RRF and post termination complex (PoTC). Our data reveal that the two conserved loops (loop I and II) at the tip region of EF-G domain IV possess distinct roles in tRNA translocation and ribosome recycling. Specifically, loop II might be directly involved in disrupting the main intersubunit bridge B2a between helix 44 (h44 from the 30S subunit) and helix 69 (H69 from the 50S subunit) in PoTC. Therefore, our data suggest a new ribosome recycling mechanism which requires an active involvement of EF-G. In addition to supporting RRF, EF-G plays an enzymatic role in destabilizing B2a via its loop II.