Buchta, D. | bioRxiv Science

bioRxiv2026

A negatively charged unstructured loop autoinhibits mammalian Dicer and supports fidelity of miRNA biogenesis.

Dicer RNases produce small RNAs for RNA interference (RNAi) and microRNA (miRNA) pathways. Although vertebrate Dicers share a conserved overall architecture with other eukaryotic Dicers, they are uniquely adapted for the production of gene-regulating miRNAs. We report here this adaptation extends beyond the structured core of the enzyme and relies on an intrinsically disordered region (IDR) conserved across jawed vertebrates. This negatively charged IDR supports fidelity of miRNA biogenesis while inhibiting RNAi. The IDR projects into a positively charged substrate-binding groove of Dicer and stabilizes a pre-dicing state, which effectively licenses authentic miRNA precursors. Electron cryo-microscopy shows that removal of the IDR shifts the equilibrium from the pre-dicing to the dicing state, thus increasing enzymatic activity and broadening substrate accessibility. In cells, deletion, replacement or variations of the IDR affect fidelity of microRNA biogenesis, change substrate specificity, and enhance RNAi. Altogether, our work highlights functional relevance of unstructured protein parts, which escape attention in structural analyses, and introduces an autoinhibitory IDR element mimicking negative RNA charge as a component of the molecular grammar governing IDRs in RNA-binding proteins.

molecular biology↗

bioRxiv2023

Cryo-electron tomography of enterovirus cell entry and endosome escape

Enveloped viruses deliver their genomes into the cell cytoplasm by membrane fusion; in contrast, membrane penetration by non-enveloped viruses is more diverse and less well understood. Enteroviruses, one of the largest groups of non-enveloped viruses, cause diseases ranging from the common cold to life-threatening encephalitis. To initiate infection, most enteroviruses enter cells by endocytosis. However, how enterovirus particles or RNA genomes cross the endosome membrane into the cytoplasm remains unknown. Here we used cryo-electron tomography of infected cells to show that endosomes containing rhinovirus 2, echovirus 18, echovirus 30, or enterovirus 71 deform, rupture, and release their content into the cytoplasm. Blocking endosome acidification with bafilomycin A1 reduced the number of enterovirus particles that released their genomes in endosomes, but did not prevent them from reaching the cytoplasm. Inhibiting N-WASP-mediated post-endocytic membrane remodeling with wiskostatin promoted abortive enterovirus genome release in endosomes. We show that the rupture of endosomes also occurs in uninfected cells. In summary, our results indicate that cellular membrane remodeling disrupts enterovirus-containing endosomes and thus releases the virus genomes and particles into the cytoplasm. Since the studied enteroviruses employ different receptors for cell entry but are all delivered into the cytoplasm by cell-mediated endosome disruption, it is possible that many other enteroviruses utilize endosome rupture to infect cells.

microbiology↗