Biology subjects

Tucker, J. M.

Publications and source records attributed to Tucker, J. M..

6 recordsLinked to original sources

bioRxiv2026

Brf1-Mediated RNA Polymerase III Activity Limits Murine Gammaherpesvirus Spread

RNA polymerase III (Pol III) activity is upregulated during herpesvirus infection, yet the functional consequences of this response remain poorly understood. To investigate the role of host Pol III transcription during murine gammaherpesvirus 68 (MHV68) infection, we depleted the Pol III transcription factor Brf1, an essential component of the TFIIIB complex required for transcription from Type I and Type II Pol III promoters. Brf1 depletion enhanced MHV68 replication during low multiplicity of infection (MOI) conditions, resulting in increased viral gene expression, viral protein accumulation, infectious virion production, and extracellular viral genome copies. These effects were confirmed to be Brf1-specific through rescue with an siRNA-resistant Brf1 construct. In contrast, Brf1 depletion produced only modest effects during high-MOI infection, suggesting that Brf1-dependent antiviral activity is most important during multistep viral spread. Transcriptomic analysis revealed accelerated induction of interferon-responsive genes early during infection in Brf1-deficient cells, followed by enhanced host transcript depletion at later stages, consistent with amplified host shutoff. Genetic disruption of the RIG-I/MAVS signaling pathway failed to abolish Brf1 antiviral activity, indicating that this phenotype is independent of MAVS-dependent interferon signaling. Finally, plaque assays demonstrated that Brf1-deficient cells supported larger plaques and increased plaque numbers, suggesting enhanced viral spread and/or entry. Together, our results reveal an unexpected role for Brf1-dependent Pol III activity in controlling gammaherpesvirus spread and suggest that virus-induced Pol III activation contributes to host antiviral defense.

microbiology↗

bioRxiv2025

A thirty-year trend of increasing clinical orientation at the National Institutes of Health

It is widely recognized that funding for biomedical research supports the development of major medical advances. However, little systematic effort has been made to determine whether a link exists between the types of funding opportunities that are available to scientists and progress towards new treatments in the clinic. To better understand this relationship, we analyzed the funding opportunities offered by the National Institutes of Health (NIH) over a span of thirty-two years, together with the resulting portfolio of applications and awards. We found NIH funding opportunity announcements became more numerous and increasingly clinically oriented over that span, following a trend that parallels the increasing clinical and translational orientation of both NIH grant applications and NIH-funded publications. Surprisingly, this increase appears to be independent of the representation of clinician-scientists in the NIH workforce.

scientific communication and education↗

bioRxiv2024

Gammaherpesvirus infection triggers the formation of tRNA fragments from premature tRNAs

Transfer RNAs (tRNAs) are fundamental for both cellular and viral gene expression during viral infection. In addition, mounting evidence supports biological function for tRNA cleavage products, including in the control of gene expression during conditions of stress and infection. We previously reported that infection with the model murine gammaherpesvirus, MHV68, leads to enhanced tRNA transcription. However, whether this has any influence on tRNA transcript processing, viral replication, or the host response is not known. Here, we combined two new approaches, sequencing library preparation by Ordered Two Template Relay (OTTR) and tRNA bioinformatic analysis by tRAX, to quantitatively profile full-length tRNAs and tRNA fragment (tRF) identities during MHV68 infection. We find that MHV68 infection triggers both pre-tRNA and mature tRNA cleavage, resulting in the accumulation of specific tRFs. OTTR-tRAX revealed not only host tRNAome changes, but also the expression patterns of virally-encoded tRNAs (virtRNAs) and virtRFs made from the MHV68 genome, including their base modification signatures. Because the transcript ends of several host tRFs matched tRNA splice junctions, we tested and confirmed the role of tRNA splicing factors TSEN2 and CLP1 in MHV68-induced tRF biogenesis. Further, we show that CLP1 kinase, and by extension tRNA splicing, is required for productive MHV68 infection. Our findings provide new insight into how gammaherpesvirus infection both impacts and relies on tRNA transcription and processing. ImportanceDiverse conditions of infection and cellular stress incite the cleavage of transfer RNAs, leading to the formation of tRNA fragments which can directly regulate gene expression. In our study of gammaherpesviruses, such as the murine herpesvirus 68 and human oncogenic Kaposi Sarcoma associated Herpesvirus, we discovered that transfer RNA regulation and cleavage is a key component of gene reprogramming during infection. We present the first in-depth profile of tRNA fragment generation in response to DNA virus infection, using state-of-the-art sequencing techniques that overcome several challenges with tRNA sequencing. We present several lines of evidence that tRNA fragments are made from newly-transcribed premature tRNAs and propose that this may be a defining characteristic of tRNA cleavage in some contexts. Finally, we show that tRNA splicing machinery is involved with the formation of some MHV68-induced tRNA fragments, with a key regulator of splicing, CLP1, required for maximal viral titer. Together, we posit that tRNA processing may be integral to the elegant shift in gene expression that occurs during viral take-over of the host cell.

microbiology↗

bioRxiv2024

Gammaherpesvirus infection alters transfer RNA splicing and triggers tRNA cleavage

Withdrawal statementThe authors have withdrawn this manuscript due to a duplicate posting of manuscript number BIORXIV/2024/592122. Therefore, the authors do not wish this work to be cited as reference for the project. If you have any questions, please contact the corresponding author. The correct preprint can be found at doi: https://doi.org/10.1101/2024.05.01.592122.

microbiology↗

bioRxiv2021

Manipulation of RNA Polymerase III by Herpes Simplex Virus-1

RNA Polymerase III (Pol III) transcribes noncoding RNA, including transfer RNA (tRNA), and acts as a pathogen sensor during the innate immune response. To promote enhanced proliferation, the Pol III machinery is commonly targeted during cancer and viral infection. Herein we employ DM-RNA-Seq, 4SU-Seq, ChIP-Seq, and ATAC-Seq to characterize how Herpes Simplex Virus-1 (HSV-1) perturbs the Pol III landscape. We find that HSV-1 stimulates tRNA expression 10-fold, with mature tRNAs exhibiting a 2-fold increase within 12 hours of infection. Perturbation of host tRNA synthesis requires nuclear viral entry, but not synthesis of specific viral transcripts, nascent viral genomes, or viral progeny. Host tRNA with a specific codon bias were not targeted--rather increased transcription was observed from euchromatic, actively transcribed loci. tRNA upregulation is linked to unique crosstalk between the Pol II and III transcriptional machinery. While viral infection is known to mediate host transcriptional shut off and lead to a depletion of Pol II on host mRNA promoters, we find that Pol II binding to tRNA loci increases. Finally, we report Pol III and associated factors bind the HSV genome, which suggests a previously unrecognized role in HSV-1 gene expression. These data provide insight into novel mechanisms by which HSV-1 alters the host nuclear environment, shifting key processes in favor of the pathogen.

molecular biology↗

bioRxiv2019

Alteration of the premature tRNA landscape by gammaherpesvirus infection

Transfer RNAs (tRNAs) are transcribed by RNA polymerase III (RNAPIII) and play a central role in decoding our genome, yet their expression and non-canonical function remain understudied. Many DNA tumor viruses enhance the activity of RNAPIII, yet whether infection alters tRNA expression is largely unknown. Here, we present the first genome-wide analysis of how viral infection alters the tRNAome. Using a tRNA-specific sequencing method (DM-tRNA-seq), we find that the murine gammaherpesvirus MHV68 induces global changes in pre-tRNA expression with 14% of tRNA genes upregulated more than 3-fold, indicating that differential tRNA gene induction is a characteristic of DNA virus infection. Elevated pre-tRNA expression corresponds to increased RNAPIII occupancy for the subset of tRNA genes tested; additionally, post-transcriptional mechanisms contribute to the accumulation of pre-tRNA species. We find increased abundance of tRNA fragments derived from pre-tRNAs upregulated by viral infection, suggesting that non-canonical tRNA cleavage is also affected. Further, pre-tRNA accumulation, but not RNAPIII recruitment, requires gammaherpesvirus-induced degradation of host mRNAs by the virally encoded mRNA endonuclease muSOX. We hypothesize that depletion of pre-tRNA maturation or turnover machinery contributes to robust accumulation of full-length pre-tRNAs in infected cells. Collectively, these findings reveal pervasive changes to tRNA expression during DNA virus infection and highlight the potential of using viruses to explore tRNA biology. SignificanceViral infection can dramatically change the gene expression landscape of the host cell, yet little is known regarding changes in non-coding gene transcription by RNA polymerase III (RNAPIII). Among these are transfer RNAs (tRNAs), which are fundamental in protein translation, yet whose gene regulatory features remain largely undefined in mammalian cells. Here, we perform the first genome-wide analysis of tRNA expression changes during viral infection. We show that premature tRNAs accumulate during infection with the model gammaherpesvirus MHV68 as a consequence of increased transcription, but that transcripts do not undergo canonical maturation into mature tRNAs. These findings underscore how tRNA expression is a highly-regulated process and that cells have strategies to balance tRNA pools during conditions of elevated RNAPIII activity.

molecular biology↗