Status lab-led Areas Small RNA Biology and the Limits of Antiviral Silencing
The dispute over mammalian antiviral silencing had been conducted in terms of products, over whether virus-derived small interfering RNAs of the right size and Dicer dependence could be found in infected cells. That framing assumes the only antiviral use of the silencing machinery is to make small RNAs. Two observations from the laboratory's own engineering work pointed elsewhere. Cytoplasmic processing of a primary microRNA transcript carried by an alphavirus was initially reported as microprocessor independent in Shapiro 2010, and Shapiro 2012 in RNA, using conditional deletion rather than inference, found Drosha absolutely required and Dicer needed only at the second cleavage, revising that earlier reading. The same study found endogenous Drosha redistributing from nucleus to cytoplasm after infection with parental as well as microRNA-expressing virus, so relocalisation responds to infection rather than to the presence of a substrate.
Shapiro 2014 separated the two RNase III enzymes genetically in primary fibroblasts. Loss of Drosha raised Sindbis and vesicular stomatitis virus titres and capsid protein while loss of Dicer had no significant effect. Infection with positive-sense, negative-sense and segmented nuclear RNA viruses, and transfection of double-stranded RNA alone, drove Drosha into the cytoplasm within six hours by CRM1-dependent export, without new protein synthesis and in cells lacking RIG-I, TBK1 or the type I interferon receptor, which is the basis for calling the activity interferon independent. A variant with serines 300 and 302 replaced by alanine was constitutively cytoplasmic and restricted Sindbis virus by more than two logs against about one log for wild type. The decisive negative result sits in the same paper, since more than 675,000 small RNA reads across the Sindbis genome showed no enrichment of the 21 nucleotide species, and loss of Drosha raised total viral small RNA reads without changing their profile.
Aguado 2017 established what the enzyme recognises. In cells lacking both Drosha and Dicer, and therefore any mature microRNA, Sindbis, Ross River and Langat viruses replicated better while influenza A and Sendai viruses did not, so enhancement tracked with positive genome polarity, reproduced in primary conditional Drosha mouse lung fibroblasts. Among six variants unable to process primary microRNAs, an RNA-binding mutant that is catalytically inactive and does not associate with DGCR8 still suppressed the virus, so the activity requires RNA binding and requires neither catalysis, nor DGCR8, nor microRNA processing. SELEX enriched RNAs with no conserved sequence that fold into unbranched hairpins, and the same protein bound a hairpin in the first 200 nucleotides of the Sindbis genome. Replicon work localised the defect to RNA synthesis, and a reconstituted minus-strand replicase assay showed polymerase output reduced by nearly half in fractions containing Drosha. RNase III proteins from bacteria, archaea, yeast and the urochordate Ciona intestinalis conferred the same activity against positive-strand viruses, which converts a Drosha observation into a statement about a protein fold.
This is a position neither side of the 2013 antiviral silencing dispute occupied. A component of the silencing machinery does contribute to antiviral defence in mammalian somatic cells, and it does so without the small interfering RNA products the dispute was about. The clamp model the authors use for steric occlusion is their interpretation, with no structural or single-molecule evidence, and the study does not test why encapsidated negative-strand genomes escape. The activity that converts a cytoplasmic primary transcript into a precursor was never identified in this corpus, the phosphatase implied by the serine data was not found, and both papers are bounded to cultured cells with no mammalian animal infection.
Substantiated by - Noncanonical cytoplasmic processing of viral microRNAs, establishes that a cytoplasmic virus can yield mature microRNA and reports the processing requirement as microprocessor independent - Evidence for a cytoplasmic microprocessor of pri-miRNAs, corrects that reading by conditional deletion and shows Drosha relocalising to the cytoplasm in response to infection - Drosha as an interferon-independent antiviral factor, shows Drosha but not Dicer restricting two RNA viruses without any small interfering RNA signature, independently of three sensing lesions - RNase III nucleases from diverse kingdoms serve as antiviral effectors, shows that RNA binding without catalysis suffices, identifies unbranched hairpins as the recognised structure, and reproduces the activity with RNase III proteins from three domains of life
RNase III nucleases, including human Drosha and homologues from bacteria, archaea, yeast and a urochordate, restrict positive-strand RNA viruses by recognizing unbranched RNA stem loops and impairing the viral polymerase, separably from microRNA biogenesis, catalysis and interferon.
Loss of the nuclear RNase III enzyme Drosha, but not of Dicer, increases RNA virus replication in mammalian fibroblasts, and diverse RNA viruses drive Drosha into the cytoplasm by CRM1-dependent export in a manner that does not require new protein synthesis, RIG-I, TBK1 or type I interferon signaling.
Primary microRNA transcripts generated in the cytoplasm by a recombinant Sindbis virus are cleaved without any nuclear involvement yet still require Drosha, which relocalises from nucleus to cytoplasm on infection while the endogenous microRNA profile of the cell remains largely unchanged.
Insertion of a primary microRNA locus into the exclusively cytoplasmic Sindbis virus genome yields mature, functional miR-124 through a Dicer-dependent but microprocessor- and Exportin-5-independent route, defining a cytoplasmic hairpin-processing activity in vertebrate cells that the authors term a virtron.