Biology subjects

Bass, B.

Publications and source records attributed to Bass, B..

3 recordsLinked to original sources

bioRxiv2025

Detection of Orsay viral replication intermediates reveals spatial and regulatory links to Caenorhabditis elegans innate immune responses

Orsay Virus (OV) is a positive-sense, single-stranded RNA (+ssRNA) virus that naturally infects C. elegans intestines. Like other +ssRNA viruses, the OV-encoded RNA-dependent RNA polymerase (oRdRP) synthesizes complementary antigenome for use as template for amplifying viral genome, but OV replication intermediates are underexplored. Using PCR, we observed viral genome in vast excess of antigenome, as for other +ssRNA viruses. Unlike interferon-based antiviral defense, C. elegans utilizes RNA interference (RNAi) for antiviral defense, producing sense and antisense small interfering RNAs (siRNAs) that cannot be distinguished from genome and antigenome with conventional hybridization protocols. Fluorescence-based imaging in C. elegans intestines using probes to antigenomic sequences revealed cytoplasmic as well as perinuclear localization patterns. The latter depended on factors required for generation of primary, but not secondary, siRNAs, connecting the antigenomic hybridization pattern to RNAi. We also observed cytoplasmic double-stranded RNA (dsRNA) associated with oRdRP, suggesting viral replication hubs, as well as infection-induced nuclear dsRNA, likely from endogenous dsRNA. Finally, using antibodies to oRdRP, we observed spherical structures of [~]1{micro}m in diameter with oRdRP at their surface, which decrease in animals lacking RDE-1. Our study defines features of OV replication intermediates, setting the stage for understanding their connection to host antiviral pathways. Significance StatementO_LIOrsay virus is a +ssRNA virus that infects C. elegans intestines. We advance understanding of viral replication intermediates and address the issue that for animals that use antiviral RNA interference, hybridization of probes occurs with both genome and antigenome and small interfering RNAs. C_LIO_LISingle-molecule fluorescence in-situ hybridization using antigenomic probes revealed cytoplasmic and perinuclear puncta, only upon denaturation, with perinuclear signal dependent on primary, but not secondary, siRNA biogenesis. Viral RNA-dependent RNA polymerase lined the perimeter of spherical structures of [~]1{micro}m diameter. C_LIO_LIThis study sets the stage for understanding the relationship between viral replication dynamics and antiviral RNA interference. C_LI

immunology↗

bioRxiv2025

Comprehensive Mapping of Human dsRNAome Reveals Conservation, Neuronal Enrichment, and Intermolecular Interactions

The human transcriptome contains millions of A-to-I editing sites arising from an unclear number of poorly characterized dsRNAs. Editing sites are often used to infer presence of dsRNA, but this method is limited by transcription levels, read depth, ADAR expression and cannot identify unedited dsRNA. To address these limitations, we developed dsRNAscan. Applying dsRNAscan to the human genome predicted 5 million dsRNAs. Genomic distribution of dsRNAs encompassing repetitive elements was widespread, but non-repetitive dsRNAs were sparse and enriched at chromosomal tips. dsRNAscan predicted hundreds of long, highly paired dsRNAs suspected to be immunogenic, but only one was in a 3'UTR, and thus likely to challenge cytoplasmic immune sensors. We observed several thousand editing enriched regions suspected to arise from intermolecular structures, and dozens of neuronally enriched dsRNAs conserved across vertebrates. This study offers the first comprehensive set of dsRNA annotations for the human genome, available as a resource at https://dsrna.chpc.utah.edu/.

genomics↗

bioRxiv2020

Binding of dsRNA by D. melanogaster Dicer-2 is substrate-dependent and regulated by Loquacious-PD

Drosophila melanogaster Dicer-2 is a large, multidomain protein that cleaves double-stranded RNA (dsRNA) into small interfering RNAs in a terminus-dependent manner as part of the RNA interference pathway. We characterize the local binding environment involved in this substrate-selective molecular recognition event by monitoring the time-resolved photophysics of a cyanine dye linked to the dsRNA terminus. We observe substantial changes in the molecular rigidity and local freedom of motion of the probe as a function of distinct conformations of the biomolecular complex between Dicer-2 and dsRNA as a function of dsRNA termini, the presence of regulatory proteins, and the addition of a biochemical energy source (ATP) or a non-hydrolysable equivalent (ATP-{gamma}S). With a clustering analysis based solely on these molecular-scale measures of the local binding environment at the dsRNA terminus, we identify sub-populations of similar conformations that define distinct modes of molecular recognition which are correlated with biochemical activity. These observations reveal the important role of substrate-selective molecular recognition properties for proteins with multiple domains that can bind RNA, regulatory proteins, and cofactors. STATEMENT OF SIGNIFICANCEThe molecular-scale determinants of protein-RNA binding remain elusive, particularly when different subunits of a single protein confer specificity toward small structural differences of their RNA partners. An important case is that of Drosophila melanogaster Dicer-2, a critical component of the antiviral RNA interference response. Dicer-2 discriminates between double stranded RNA with blunt or 3 overhang termini, a feature suggested to mediate recognition of "self" vs. "non-self" substrates. We study these interactions at the binding site with a fluorescent label at the RNA terminus, monitoring intramolecular and collective measures of flexibility to report on the local environment. Dicer-2 has distinct modes of molecular recognition which are regulated by accessory proteins and ATP, leading to different conformations and tuning biochemical activity.

biophysics↗