Alteration of the premature tRNA landscape by gammaherpesvirus infection

Abstract

12Transfer RNAs (tRNAs) are transcribed by RNA polymerase III (RNAPIII) and play a 13 central role in decoding our genome. Many DNA tumor viruses enhance the activity of RNAPIII, 14 yet whether infection alters tRNA expression is largely unknown. Here, we present the first 15 genome wide analysis of how viral infection alters the tRNAome. Using a tRNA-specific 16 sequencing method (DM-tRNA-seq), we find that the murine gammaherpesvirus MHV68 17 induces global changes in pre-tRNA expression, indicating that differential tRNA gene induction 18 is a characteristic of DNA virus infection. Elevated pre-tRNA expression corresponds to 19 increased RNAPIII occupancy, but post-transcriptional mechanisms also contribute. Pre-tRNA 20 accumulation, but not RNAPIII recruitment, requires the virally encoded mRNA endonuclease 21 muSOX, suggesting interplay between the different facets of virus-induced gene regulation. 22Collectively, these findings reveal pervasive changes to tRNA expression during DNA virus 23 infection and highlight the potential of using viruses to explore tRNA biology. 24 25 Impact 26 The first genome-wide analysis of virus-induced tRNA expression changes reveals that 27 tRNA transcription and processing are perturbed by the gammaherpesvirus MHV68. 28 29 30The elegant design of the tRNA decoder, an adaptor molecule which converts genetic 31 information into protein, was proposed over 60 years ago (1). Although tRNAs were the first 32 non-coding RNA described, our understanding of tRNA biology, especially their functions 33 outside of protein translation and what dictates their expression, remains limited. This is partly 34 due to the difficulties in applying RNA sequencing and analysis platforms to tRNAs, but recent 35 methodological advances have resulted in a surge of new studies that help to resolve these 36 complexities (2-4). Non-canonical functions of tRNA transcripts have been described, namely 37 the discovery that both premature and mature tRNAs undergo fragmentation into shorter RNAs 38 with numerous regulatory functions in response to stress, cancer, and viral infection (reviewed 39 in (5, 6)). Given the evidence that the ~400 predicted tRNA genes in mammalian genomes are 40 differentially expressed across cell lines (7-9) and that viral infection can lead to post-41 transcriptional modulation of tRNAs (10, 11), defining the tRNAome under varying conditions will 42 be central to our understanding of tRNA function, canonical and otherwise. 43RNA polymerase III (RNAPIII) transcribes tRNA genes and exhibits enhanced activity 44 during infection with DNA viruses (12-17), many of which encode their own RNAPIII genes. 45Though RNAPIII activation stimulates expression of viral RNAPIII genes, concomitant 46 accumulation of host RNAPIII transcripts can trigger antiviral immune responses. For example, 47 the induction or misprocessing of RNAPIII-generated host 5S RNA pseudogene and vault RNA 48 transcripts during Herpes Simplex Virus-1 (HSV-1) and Kaposi's sarcoma-associated 49 herpesvirus (KSHV) infection...