Manganese-dependent microRNA trimming by 3'→5' exonucleases generates 14-nucleotide or shorter tiny RNAs.

MicroRNAs (miRNAs) are about 22-nucleotide (nt) noncoding RNAs forming the effector complexes with Argonaute (AGO) proteins to repress gene expression. Although tiny RNAs (tyRNAs) shorter than 19 nt have been found to bind to plant and vertebrate AGOs, their biogenesis remains a long-standing question. Here, our in vivo and in vitro studies show several 3'→5' exonucleases, such as interferon-stimulated gene 20 kDa (ISG20), three prime repair exonuclease 1 (TREX1), and ERI1 (enhanced RNAi, also known as 3'hExo), capable of trimming AGO-associated full-length miRNAs to 14-nt or shorter tyRNAs. Their guide trimming occurs in a manganese-dependent manner but independently of the guide sequence and the loaded four human AGO paralogs. We also show that ISG20-mediated guide trimming makes Argonaute3 (AGO3) a slicer. Given the high Mn2+ concentrations in stressed cells, virus-infected cells, and neurodegeneration, our study sheds light on the roles of the Mn2+-dependent exonucleases in remodeling gene silencing.

Transcriptional and translational S-box riboswitches differ in ligand-binding properties.

There are a number of riboswitches that utilize the same ligand-binding domain to regulate transcription or translation. S-box (SAM-I) riboswitches, including the riboswitch present in the Bacillus subtilis metI gene, which encodes cystathionine γ-synthase, regulate the expression of genes involved in methionine metabolism in response to SAM, primarily at the level of transcriptional attenuation. A rarer class of S-box riboswitches is predicted to regulate translation initiation. Here we identified and characterized a translational S-box riboswitch in the metI gene from Desulfurispirillum indicum The regulatory mechanisms of riboswitches are influenced by the kinetics of ligand interaction. The half-life of the translational D. indicum metI RNA-SAM complex is significantly shorter than that of the transcriptional B. subtilis metI RNA. This finding suggests that, unlike the transcriptional RNA, the translational metI riboswitch can make multiple reversible regulatory decisions. Comparison of both RNAs revealed that the second internal loop of helix P3 in the transcriptional RNA usually contains an A residue, whereas the translational RNA contains a C residue that is conserved in other S-box RNAs that are predicted to regulate translation. Mutational analysis indicated that the presence of an A or C residue correlates with RNA-SAM complex stability. Biochemical analyses indicate that the internal loop sequence critically determines the stability of the RNA-SAM complex by influencing the flexibility of residues involved in SAM binding and thereby affects the molecular mechanism of riboswitch function.