Biology subjects

Glaunsinger, B.

Publications and source records attributed to Glaunsinger, B..

13 recordsLinked to original sources

bioRxiv2025

The Kaposi's sarcoma-associated herpesvirus TBP mimic uses a non-canonical DNA binding mode to promote viral late gene transcription

Kaposis sarcoma-associated herpesvirus (KSHV) orchestrates late gene transcription through viral transcriptional activators that hijack host RNA polymerase II machinery, maintaining selectivity for viral promoters. Among these, the KSHV protein ORF24 serves as a TATA-binding protein (TBP) mimic essential for recognizing viral late promoters, although the molecular mechanisms underlying its function remain poorly characterized. Here, we used AlphaFold3 to predict the structure of ORF24 in complex with DNA and validated key features in both transfected cells and during KSHV lytic replication. Structural modeling revealed that ORF24 employs a non-canonical DNA binding mode where the C-terminal domain (CTD) makes critical DNA contacts beyond the canonical TBP fold. Targeted mutagenesis confirmed that ORF24 requires conserved TBP-like phenylalanines alongside a polar-rich binding interface distinct from cellular TBP. During infection, both the TBP-like domain and CTD are essential for ORF24 occupancy at viral late promoters. Most surprisingly, we discovered that ORF24 pre-assembles with RNA polymerase II and the viral protein ORF34 to achieve stable promoter binding. This cooperative assembly mechanism represents a fundamental departure from stepwise eukaryotic transcription initiation, resembling a prokaryotic strategy within the eukaryotic nucleus. Summary Bullet pointsO_LIThe structure of the KSHV TBP mimic ORF24 binding DNA was modeled and experimentally tested. C_LIO_LIKSHV ORF24 uses an extended DNA-binding interface beyond the canonical TBP fold. C_LIO_LIORF24 requires cooperative pre-assembly with transcriptional machinery before DNA engagement C_LI

molecular biology↗

bioRxiv2025

Cleavage of the RNA polymerase II general transcription factor TFIIB tunes transcription during stress

Cellular stressors often cause widespread repression of RNA polymerase II (RNAP II) activity, which is thought to facilitate a focused transcriptional output towards stress resolution. In many cases, however, the underlying regulatory mechanisms remain unknown. Here, we demonstrate that stress-induced downregulation of the general transcription factor TFIIB tempers expression of specific stimulus response genes. Following a variety of stressors, TFIIB is proteolytically cleaved between its cyclin folds at conserved aspartic acid residue D207 by caspases- 3 and 7. Cleavage in this portion of the protein significantly reduces the ability of TFIIB to form a TBP-TFIIB-DNA promoter complex in vitro. Using both overexpression and endogenous base-editing, we find that B and T cells that are unable to cleave TFIIB upregulate expression of a select gene set during apoptosis. These TFIIB-sensitive genes are primarily short, stimulus-responsive and proto-oncogenic loci, and cleavage of TFIIB temporally restricts their expression. Failure to cleave TFIIB during stress leads to aberrant lymphocyte proliferation during chemical perturbation. Hence, caspase targeting of TFIIB destabilizes transcription to tune gene expression, allowing for proper stress resolution.

molecular biology↗

bioRxiv2025

Cytoplasmic mRNA decay by the anti-viral nuclease RNase L promotes transcriptional repression

Ribonuclease (RNase) L is an antiviral factor that promiscuously degrades viral and cellular RNA in the cytoplasm. This results in extensive translational reprogramming, altering mRNA processing and export. Here, we reveal that another major consequence of cytoplasmic RNase L activity is the repression of nascent RNA synthesis in the nucleus. This is not associated with altered nuclear RNA stability but instead results from transcriptional repression. For RNA polymerase II, repression is primarily associated with reduced occupancy of serine 2-phosphorylated polymerase in gene bodies, indicating an elongation defect. Prominent among the transcriptionally downregulated loci are immune-related genes, supporting a role for RNase L in tempering innate immune and inflammatory responses. RNase L activation also caused disruption of nucleoli and reduced RNA polymerase I and III transcription. Crosstalk between RNA decay and transcription thereby contributes to the large-scale modulation of gene expression in RNase L-activated cells.

molecular biology↗

bioRxiv2024

The SP140-RESIST pathway regulates interferon mRNA stability and antiviral immunity

Type I interferons (IFN-Is) are essential for antiviral immunity but must be tightly regulated1-3. The conserved transcriptional repressor SP140 inhibits interferon beta (Ifnb1) expression via an unknown mechanism4,5. Here we report that SP140 does not directly repress Ifnb1 transcription. Instead, SP140 negatively regulates Ifnb1 mRNA stability by directly repressing the expression of a previously uncharacterized regulator we call RESIST (REgulated Stimulator of Interferon via Stabilization of Transcript, previously annotated as Annexin-2 Receptor). RESIST promotes Ifnb1 mRNA stability by counteracting Ifnb1 mRNA destabilization mediated by the Tristetraprolin (TTP) family of RNA-binding proteins and the CCR4-NOT deadenylase complex. SP140 localizes within nuclear bodies, punctate structures that play important roles in silencing DNA virus gene expression in the nucleus4. Consistent with this observation, we found that SP140 inhibits replication of the gammaherpesvirus MHV68. The antiviral activity of SP140 was independent of its ability to regulate Ifnb1. Our results establish dual antiviral and interferon regulatory functions for SP140. We propose that SP140 and RESIST participate in antiviral effector-triggered immunity6,7.

immunology↗

bioRxiv2024

The RNA polymerase II general transcription factor TFIIB is a target for transcriptome control during cellular stress and viral infection

Many stressors, including viral infection, induce a widespread suppression of cellular RNA polymerase II (RNAPII) transcription, yet the mechanisms underlying transcriptional repression are not well understood. Here we find that a crucial component of the RNA polymerase II holoenzyme, general transcription factor IIB (TFIIB), is targeted for post-translational turnover by two pathways, each of which contribute to its depletion during stress. Upon DNA damage, translational stress, apoptosis, or replication of the oncogenic Kaposis sarcoma-associated herpesvirus (KSHV), TFIIB is cleaved by activated caspase-3, leading to preferential downregulation of pro-survival genes. TFIIB is further targeted for rapid proteasome-mediated turnover by the E3 ubiquitin ligase TRIM28. KSHV counteracts proteasome-mediated turnover of TFIIB, thereby preserving a sufficient pool of TFIIB for transcription of viral genes. Thus, TFIIB may be a lynchpin for transcriptional outcomes during stress and a key target for nuclear replicating DNA viruses that rely on host transcriptional machinery. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=138 SRC="FIGDIR/small/575933v3_ufig1.gif" ALT="Figure 1"> View larger version (22K): org.highwire.dtl.DTLVardef@8c8db7org.highwire.dtl.DTLVardef@1ac2421org.highwire.dtl.DTLVardef@7828baorg.highwire.dtl.DTLVardef@b9de0a_HPS_FORMAT_FIGEXP M_FIG C_FIG Significance StatementTranscription by RNA polymerase II (RNAPII) synthesizes all cellular protein-coding mRNA. Many cellular stressors and viral infections dampen RNAPII activity, though the processes underlying this are not fully understood. Here we describe a two-pronged degradation strategy by which cells respond to stress by depleting the abundance of the key RNAPII general transcription factor, TFIIB. We further demonstrate that an oncogenic human gammaherpesvirus antagonizes this process, retaining enough TFIIB to support its own robust viral transcription. Thus, modulation of RNAPII machinery plays a crucial role in dictating the outcome of cellular perturbation.

cell biology↗

bioRxiv2021

A Two-tiered Functional Screen Identifies Herpesviral Transcriptional Modifiers and their Essential Domains

While traditional methods for studying large DNA viruses allow the creation of individual mutants, CRISPR/Cas9 can be used to rapidly create thousands of mutant dsDNA viruses in parallel. Here, we used this approach to study the human oncogenic Kaposis sarcoma-associated herpesvirus (KSHV). We designed a sgRNA library containing all possible ~22,000 guides targeting the genome of KSHV - one cut site approximately every 8 base pairs - enabling the pooled screening of the entire genome. We used this tool to phenotype all possible Cas9-targeted viruses for transcription of KSHV late genes, which is required to produce structural components of the viral capsid. By performing targeted deep sequencing of the viral genome to distinguish between knock-out and in-frame alleles created by Cas9, we discovered a novel hit, ORF46 - and more specifically its DNA binding domain - is required for viral DNA replication. Our pooled Cas9 tiling screen followed by targeted deep viral sequencing represents a two-tiered screening paradigm that may be widely applicable to dsDNA viruses.

genomics↗

bioRxiv2021

The N-terminal and central domains of CoV-2 nsp1 play key functional roles in suppression of cellular gene expression and preservation of viral gene expression

Nonstructural protein 1 (nsp1) is the first viral protein synthesized during coronavirus (CoV) infection and is a key virulence factor that dampens the innate immune response. It restricts cellular gene expression through a combination of inhibiting translation by blocking the mRNA entry channel of the 40S ribosomal subunit and by promoting mRNA degradation. We performed a detailed structure-guided mutational analysis of CoV-2 nsp1 coupled with in vitro and cell-based functional assays, revealing insight into how it coordinates these activities against host but not viral mRNA. We found that residues in the N-terminal and central regions of nsp1 not involved in docking into the 40S mRNA entry channel nonetheless stabilize its association with the ribosome and mRNA, thereby enhancing its restriction of host gene expression. These residues are also critical for the ability of mRNA containing the CoV-2 leader sequence to escape translational repression. Notably, we identify CoV-2 nsp1 mutants that gain the ability to repress translation of viral leader-containing transcripts. These data support a model in which viral mRNA binding functionally alters the association of nsp1 with the ribosome, which has implications for drug targeting and understanding how engineered or emerging mutations in CoV-2 nsp1 could attenuate the virus.

molecular biology↗

bioRxiv2020

Cytoplasmic mRNA decay represses RNA polymerase II transcription during early apoptosis

RNA abundance is generally sensitive to perturbations in decay and synthesis rates, but crosstalk between RNA polymerase II transcription and cytoplasmic mRNA degradation often leads to compensatory changes in gene expression. Here, we reveal that widespread mRNA decay during early apoptosis represses RNAPII transcription, indicative of positive (rather than compensatory) feedback. This repression requires active cytoplasmic mRNA degradation, which leads to impaired recruitment of components of the transcription preinitiation complex to promoter DNA. Importin /{beta}- mediated nuclear import is critical for this feedback signaling, suggesting that proteins translocating between the cytoplasm and nucleus connect mRNA decay to transcription. We also show that an analogous pathway activated by viral nucleases similarly depends on nuclear protein import. Collectively, these data demonstrate that accelerated mRNA decay leads to the repression of mRNA transcription, thereby amplifying the shutdown of gene expression. This highlights a conserved gene regulatory mechanism by which cells respond to threats. IMPACT STATEMENTHuman cells respond to cytoplasmic mRNA depletion during early apoptosis by inhibiting RNA polymerase II transcription, thereby magnifying the gene expression shutdown during stress.

molecular biology↗

bioRxiv2020

The γ-herpesviral TATA box binding protein directly interacts with RNA Polymerase II to direct late gene transcription

{beta}- and {gamma}-herpesviruses include the oncogenic human viruses Kaposis sarcoma-associated virus (KSHV) and Epstein-Barr virus (EBV), and human cytomegalovirus (HCMV), which is a significant cause of congenital disease. Near the end of their replication cycle, these viruses transcribe their late genes in a manner distinct from host transcription. Late gene transcription requires six virally-encoded proteins, one of which is a functional mimic of host TATA-box-binding protein (TBP) that is also involved in recruitment of RNA polymerase II (Pol II) via unknown mechanisms. Here, we applied biochemical protein interaction studies together with electron microscopy-based imaging of a reconstituted human preinitiation complex to define the mechanism underlying Pol II recruitment. These data revealed that the herpesviral TBP, encoded by ORF24 in KSHV, makes a direct protein-protein contact with the C-terminal domain of host RNA polymerase II (Pol II), which is a unique feature that functionally distinguishes viral from cellular TBP. The interaction is mediated by the N-terminal domain (NTD) of ORF24 through a conserved motif that is shared in its {beta}- and {gamma}-herpesvirus homologs. Thus, these herpesviruses employ an unprecedented strategy in eukaryotic transcription, wherein promoter recognition and polymerase recruitment are facilitated by a single transcriptional activator with functionally distinct domains. SIGNIFICANCE STATEMENTThe {beta}- and {gamma}-herpesviruses mediate their late gene transcription through a set of viral transcriptional activators (vTAs). One of these vTAs, ORF24 in Kaposis sarcoma-associated herpesvirus (KSHV), is a mimic of host TATA-box-binding protein (TBP). We demonstrate that the N-terminal domain of ORF24 and its homologs from other {beta}- and {gamma}-herpesviruses directly bind the unstructured C-terminal domain (CTD) of RNA Pol II. This functionally distinguishes the viral TBP mimic from cellular TBP, which does not bind Pol II. Thus, herpesviruses encode a transcription factor that has the dual ability to directly interact with promoter DNA and the polymerase, a property which is unique in eukaryotic transcription and is conceptually akin to prokaryotic transcription factors.

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↗

bioRxiv2019

Xrn1 activity broadly represses RNA polymerase II occupancy at mammalian but not viral promoters during herpesvirus infection

In mammalian cells, widespread acceleration of cytoplasmic mRNA degradation is linked to impaired RNA polymerase II (Pol II) transcription. This mRNA decay-induced transcriptional repression occurs during infection with gammaherpesviruses including Kaposis sarcoma-associated herpesvirus (KSHV) and murine gammaherpesvirus 68 (MHV68), which encode an mRNA endonuclease that initiates widespread RNA decay. Here, we show that MHV68-induced mRNA decay leads to a genome-wide reduction of Pol II occupancy at mammalian promoters. Viral genes, despite the fact that they require Pol II for transcription, escape this transcriptional repression. Protection is not governed by viral promoter sequences; instead, location on the viral genome is both necessary and sufficient to escape the transcriptional repression effects of mRNA decay. We hypothesize that the ability to escape from transcriptional repression is linked to the localization of viral DNA in replication compartments, providing a means for these viruses to counteract decay-induced viral transcript loss.

microbiology↗

bioRxiv2019

An integrative approach identifies direct targets of the late viral transcription complex and an expanded promoter recognition motif in Kaposi's sarcoma-associated herpesvirus

The structural proteins of DNA viruses are generally encoded by late genes, whose expression relies on recruitment of the host transcriptional machinery only after the onset of viral genome replication. {beta} and {gamma}-herpesviruses encode a unique six-member viral pre-initiation complex (vPIC) for this purpose, although how the vPIC directs specific activation of late genes remains largely unknown. The specificity underlying late transcription is particularly notable given that late gene promoters are unusually small, with a modified TATA-box being the only recognizable element. Here, we explored the basis for this specificity using an integrative approach to evaluate vPIC-dependent gene expression combined with promoter occupancy during Kaposis sarcoma-associated herpesvirus (KSHV) infection. This approach distinguished the direct and indirect targets of the vPIC, ultimately revealing a novel promoter motif critical for KSHV vPIC binding. Additionally, we found that the KSHV vPIC component ORF24 is required for efficient viral DNA replication. Together, these results identify an elusive element that contributes to vPIC specificity and suggest novel links between KSHV DNA replication and late transcription. Author summaryGene expression in DNA viruses often occurs in temporal waves, with expression of essential structural proteins occurring late in infection, after viral genome replication has begun. Strategies underlying expression of these viral late genes are often sophisticated; for example, the {beta}- and {gamma}-herpesviruses encode a six-component viral complex that directs late gene transcription, largely by unknown mechanisms. Here, we evaluated how this complex specifically recognizes late promoters during infection with the oncogenic human {gamma}-herpesvirus Kaposis sarcoma-associated herpesvirus (KSHV). We found that one of the components of the late transcription complex was required for robust viral DNA replication, suggesting new links between KSHV replication and transcription. Combined measurements of late gene expression and promoter occupancy then revealed which KHSV genes are directly controlled by the late gene transcription complex, leading to identification of a key new regulatory element in KSHV late promoters. Together, these data help explain how the late gene transcription complex is able to bind seemingly minimal promoters with high specificity, ensuring robust expression of viral factors necessary for assembly of progeny virions.

microbiology↗

bioRxiv2018

Global changes in mRNA abundance drive differential shuttling of RNA binding proteins, linking cytoplasmic RNA degradation to transcription

Alterations in global mRNA decay broadly impact multiple stages of gene expression, although signals that connect these processes are incompletely defined. Here, we used tandem mass tag labeling coupled with mass spectrometry to reveal that changing the mRNA decay landscape, as frequently occurs during viral infection, results in subcellular redistribution of RNA binding proteins (RBPs) in human cells. Accelerating Xrn1-dependent mRNA decay through expression of a gammaherpesviral endonuclease drove nuclear translocation of many RBPs, including poly(A) tail-associated proteins. Conversely, cells lacking Xrn1 exhibited changes in the localization or abundance of numerous factors linked to mRNA turnover. Using these data, we uncovered a new role for relocalized cytoplasmic poly(A) binding protein in repressing recruitment of TATA binding protein and RNA polymerase II to promoters. Collectively, our results show that changes in cytoplasmic mRNA decay can directly impact protein localization, providing a mechanism to connect seemingly distal stages of gene expression.

molecular biology↗