Department of Microbiology & Immunology Seminar: “What Makes Circles Go Round: Cis- and Trans-acting Factors Coordinating Herpesvirus Backsplicing”
Speaker:
Sarah Dremel, PhD
Assistant Professor of Microbiology, Immunology, and Cancer Biology
School of Medicine, University of Virginia
Presentation: “What Makes Circles Go Round: Cis- and Trans-acting Factors Coordinating Herpesvirus Backsplicing”
Location: Med-Dent NE301 and via Zoom
Abstract:
Circular RNAs (circRNA) are alternative splicing products which form a continuous loop due to a 5’ to 3’ covalent linkage. Circularity can imbue a distinct set of molecular characteristics including altered localization, binding partners, immunogenicity, and half-lives. CircRNAs have been historically overlooked due to the almost complete sequence identity they share with linear counterparts derived from the same gene. To overcome this complication, we developed a custom bioinformatic pipeline (CHARLIE) tailored for de novo annotation and quantitation of viral circRNAs. We performed RNA-Seq and profiled circRNAs expressed during lytic, latent, and reactivation models of Herpes Simplex Virus-1 (HSV-1) and Kaposi sarcoma herpesvirus (KSHV) infection. Using this approach we identified dozens of high confidence viral circRNAs, some of which approach abundance of the housekeeping geneGAPDH. Ours is the first report of HSV-1 circRNAs, including species derived from the middle exon of ICP0 and the lariat intron of the latency-associated transcript. Next, we characterized cis- and trans-acting factors which promote circRNA biogenesis. We found that >90% of viral circRNA species lacked canonical splice donor-acceptor sites. Unlike host circRNA, those derived from the virus had elevated GC-content in regions flanking the BSJ site. Viral circRNA synthesis was subsequently found refractory to spliceosome inhibition or depletion. Thus, we tested the potential contribution of alternative machinery, namely RNA ligases. Of the eight viral circRNAs tested, five were significantly decreased by RNA ligase depletion. These data support RNA ligases as novel trans-acting factors in viral circRNA synthesis, with loci specific dependencies. Finally, using eCLIP and Nascent (4sU) RNA-Seq, we determined that the KSHV RNA binding protein (ORF57) enhanced circRNA synthesis for a subset of viral and host transcripts. Our work identifies dozens of novel herpesvirus circRNAs with contrasting cis- and trans-acting features for those derived from the host versus viral genome.