Poster abstracts
Poster number 134 submitted by Ankita Srivastava
SARS-CoV-2 Infection Alters CPSF73 Expression, Modulating Immune Response via Alternative Polyadenylation
Ankita Srivastava (Tufts University), Salwa Mohd Mustafa, Wendy Puryear, Jonathan Runstadler, (Tufts University), Luyang Wang, Bin Tian (The Wistar Institute), Claire Moore (Tufts University)
Abstract:
The emergence of SARS-CoV-2 in 2019 sparked a global pandemic, leading to extensive research into its impact on the host immune system, key to understanding viral replication and disease severity. mRNA maturation requires 3' end cleavage and poly(A) (pA) tail addition, which stabilize mRNA and promote translation. Approximately 70% of human genes undergo alternative polyadenylation (APA), generating diverse mRNA isoforms. Previous studies have shown that viral infections, such as Influenza and HIV-1, modify APA to enhance immune responses or support viral replication. We hypothesize that SARS-CoV-2 modulates APA to facilitate its infection.
To test this, we analyzed 3' end processing of ACTB and MYC mRNAs in SARS-CoV-2-infected samples using RT-qPCR. We observed a significant increase in unprocessed ACTB (1.8-fold) and MYC (2.5-fold), suggesting disrupted mRNA processing. Next, we conducted 3' mRNA sequencing to assess pA site usage and found notable activation of intronic polyadenylation (IPA) (834 instances) versus 116 3' UTR modifications. Genes undergoing IPA were enriched in pathways related to DNA damage response and RNA metabolism. Gene Set Enrichment Analysis (GSEA) further highlighted APA alterations in TNF-alpha signaling pathways.
To understand why mRNA 3' end processing is altered, we quantified cleavage/polyadenylation (C/P) proteins post-infection and found reduced levels of CPSF73 (the pA site nuclease) and CPSF6 (an RNA-binding protein in the C/P complex). RNA-seq analysis from CPSF73 knockdown and SARS-CoV-2 infection showed a 40-fold increase in read-through at the EEF1A2 pA site, suggesting impaired processing. Additionally, we noted a 2.1-fold increase in UTR lengthening of CPSF6 mRNA in both conditions.
Ectopic expression of SARS-CoV-2 nonstructural protein NSP12 disrupted 3' end processing of MYC and ACTB and reduced CPSF73 levels via the ubiquitin-proteasome pathway. Restoring CPSF73 levels reversed the processing defects. Our findings suggest that SARS-CoV-2 targets the C/P complex, inducing APA and influencing viral pathogenesis, which could offer new therapeutic targets.
Keywords: cleavage, polyadenylation, APA, CPSF73, mRNA, SARS-Cov2