Non-Coding RNAs Play Significant Roles in Host-Virus Interactions

Author(s): Li, Lichao | Advisor(s): Gu, Weifeng | Abstract: Previous Dicer immunoprecipitation (IP) discovered an RNA polyphosphatase PIR-1 interacting with Dicer, may participate in RNAi but the mechanism is unrevealed. Here we demonstrate that C. elegans PIR-1 is involved in the RNAi-mediated silencing of Orsay virus via promoting the biogenesis of 23-mer RNAs and the loading of 23-mer RNAs to RDE-1. We also showed that PIR-1 acts as a de facto RNA phosphatase in vivo to regulate triphosphorylated RNAs (ppp-RNAs). Thus, PIR-1 is a conserved master regulator of ppp-RNAs and plays important roles in silencing viral ppp-RNAs and modifying cellular ppp-RNAs.Next we apply PIR-1 in our small RNA cloning strategy. The high-throughput sequencing has become a standard tool for analyzing RNA and DNA. We have developed a new strategy to clone modified/unmodified small RNA in an all-liquid-based reaction carried out in a single PCR tube with as little as 16 ng total RNA. The 7-hour cloning process only needs ~1-hour labor. Moreover, this method can also clone mRNA, simplifying the need to prepare two cloning systems for small RNA and mRNA.At last, we study the function of non-coding RNA in influenza A virus. It utilizes a special process, cap-snatching, to obtain a host capped small RNA for priming viral mRNA synthesis, generating hybrid capped mRNA for translation. Previous studies have been focusing on cap-snatching at thevii5' end of viral mRNA. Here we report two non-canonical cap-snatching regions: one 300-nt upstream of the 3' end of each mRNA generating capped mRNA/ncRNA, and the other in the 5' region of vRNA and mapped primarily at the 2-nt, likely generating ncRNA. We also demonstrate that the influenza virus snatches virus-derived capped RNA in addition to host capped RNA. These findings expand our understanding of the cap-snatching mechanism and suggest that the influenza A virus may utilize this process to diversify its mRNA/ncRNA.