COSAM News Articles 2021 March Gan’s award-winning abstract

Gan’s award-winning abstract

Published: 03/21/2021

Abstract: Towards elucidation of the mechanism guiding pseudouridylation of Kaposi sarcoma-associated herpesvirus long non-coding PAN RNA.

Gan, Huachen (graduate student)
Gabriella Toomer
Joanna Sztuba-Solinska
Department of Biological Sciences, Auburn University
e-mail: hzg0021@auburn.edu

Long non-coding RNAs (lncRNAs) are an important class of regulatory RNAs, that fulﬁll their function via structure-mediated interactions with effectors. Recently, post-transcriptional modifications to RNA have been shown to govern RNAs structure, function, and metabolism. Kaposi's sarcoma-associated herpesvirus (KSHV) encodes polyadenylated nuclear (PAN) lncRNA that governs lytic reactivation of viral genes and mod-ulates cellular immune response. We found that during the latent-to-lytic reactivation of KSHV replication, specific uridines on PAN RNA undergo isomerization to pseudouridine (Ψ). Using CMC-RT (N-Cyclohexyl-Nʹ-(2 -morpholinoethyl) carbodiimide (CMC) reverse transcription) and Ligation Assisted PCR analysis (CLAP), we identified the position and frequency of Ψs and showed that the PAN pseudouridylation status is dynamic during KSHV replication, with some sites being modified with higher frequency than others. We performed the RNA antisense purification and mass spectrometry to affinity capture PAN-protein complexes. We identi-fied three candidate proteins that can guide PAN pseudouridylation, with pseudouridine synthase 7 (PUS7) further confirmed by immuno-Western blotting to associate with PAN expressed during lytic stages of KSHV replication. The confocal microscopy analysis in KSHV-infected BCBL-1 cells verified the temporal and spatial co-localization of PAN RNA and PUS7 in the nucleus, during the lytic stage of viral replication. Ongoing re-search focuses on dissecting 1) the mechanistic aspects of PAN pseudouridylation, i.e., kinetics of the pro-cess, 2) the effects of Ψ modifications on PAN RNA biology, i.e., stability, structure, interactions with effec-tors, 3) the influence of PAN Ψs on KSHV replication, viral gene expression, progeny virion production, and infectivity. Our studies contribute to the more in-depth understanding of viral lncRNA plasticity and will con-tribute to an underrepresented body of knowledge regarding the host-pathogen interaction.