Poster abstracts
Poster number 36 submitted by Allysan Nunes
The role of pseudouridine modification on mRNA stability during innate immunity
Allysan M. Nunes (The Ohio State University)
Abstract:
RNA modifications are present across all RNA species, including mRNA. Pseudouridine (ψ) is one of the most common modifications and is important for RNA stability, translation fidelity, and splicing. This modification is largely installed by the pseudouridine writer enzymes Pseudouridine Synthase 7 (PUS7) and TruB Pseudouridine Synthase Family Member 1 (TRUB1), which account for 70% of mammalian mRNA pseudouridylation. During viral infection, a subset of antiviral mRNAs involved in the interferon and inflammatory responses are upregulated by the host cell. These immune mRNAs show relatively longer mRNA half-lives compared to housekeeping transcripts. As part of the antiviral response, a non-selective endonuclease, Ribonuclease L (RNase L) is activated. When active, RNase L cleaves all host and viral single-stranded RNAs, including mRNAs, to help eliminate the viral infection. Despite global cleavage of host mRNAs, antiviral transcripts, such as interferon-β, display reduced cleavage by RNase L. It has been shown that pseudouridylation decreases RNA cleavage by RNase L in vitro. However, it is not known if this modification would influence mRNA cleavage by RNase L in cells and in vivo. Interestingly, some interferon-stimulated gene transcripts are enriched in ψ, suggesting that pseudouridylation of specific mRNAs during RNase L activity may be a potential mechanism to stabilize immune mRNAs during infection. We hypothesize that these transcripts are selectively enriched in ψ, reducing cleavage efficiency by RNase L, and aiding in their unique stability. To test this hypothesis, we use Nanopore direct RNA sequencing (DRS), which detects multiple pseudouridylation sites on each transcript. We found that activating RNase L alone without activation of other antiviral mechanisms (such as triggering the interferon response) does not elevate ψ levels in immune mRNAs. Therefore, we will further investigate the role of RNA modifications in RNA cleavage in the context of the broader antiviral response. Furthermore, we will test the role of the pseudouridine writers PUS7 and TRUB1 in this process by siRNA knockdowns followed by Nanopore DRS. These results can help elucidate critical post-transcriptional mechanisms involved in innate immunity during viral infection.
Keywords: RNase L, Pseudouridine, Nanopore