Biology subjects

Guydosh, N. R.

Publications and source records attributed to Guydosh, N. R..

10 recordsLinked to original sources

bioRxiv2026

RSV competes with the host for translational machinery without a host shutoff strategy

RNA viruses often enhance ribosome recruitment to their own mRNAs through non-canonical sequence elements or by degrading host mRNA. Respiratory syncytial virus (RSV) produces mRNAs with host-like features, including 5'-cap and poly(A) tail. Therefore, the virus lacks an obvious mechanism to preferentially protect its own mRNAs or recruit ribosomes. Furthermore, it remains unknown how RSV interacts with antiviral defense pathways that would reduce cap-dependent translation. Using spike-in normalized sequencing of total and ribosome-associated RNA, we found that RSV does not appear to evoke any host shutoff mechanisms to limit the expression of host genes. These findings show that RSV manages to make use of available ribosomes by competing effectively with host mRNAs and any translational shutoff mechanism would be detrimental. Consistent with this, we found that following activation of antiviral host pathways that reduce cap-dependent translation, translation of RSV mRNAs is decreased to the same extent as host mRNAs. Furthermore, we found that RSV infection does not trigger the dsRNA-activated kinase PKR (which initiates the ISR) and OAS (activates endonuclease RNase L) pathways. These data support a model in which RSV achieves viral protein production, not though inhibiting the host, but by successfully competing with host mRNAs and avoiding activation of antiviral pathways.

molecular biology↗

bioRxiv2026

Quantitative profiling of basal and stress-induced ribosome collisions

Ribosomes stall when they encounter problematic codons or cellular stress that perturbs translation. Stalled ribosomes can lead to the formation of ribosome collisions, also known as disomes, that engage cellular surveillance and stress signaling pathways. How many disomes form during basal conditions and how disome levels change under stress remain poorly understood. Here, we used spike-in normalized Ribo-seq and Disome-seq to quantify transcriptome-wide disome levels. Applying this approach in yeast and human cells, we found that disomes comprise approximately 2-10% of translating ribosomes under basal conditions. A high-resolution Disome-seq experiment in human cells identified reproducible disome-forming sites that contribute to the basal level of disome formation in the cell. Exposure of yeast cells to methyl methanesulfonate and human cells to anisomycin increased disome abundance up to four-fold and changed the distribution of collisions in a stress-specific and context-dependent manner. Overall, these data provide a quantitative, transcriptome-wide framework for measuring disome levels and reveal how translational stress reshapes the landscape of ribosome collisions in cells.

molecular biology↗

bioRxiv2025

Identification of translation events that drive nonsense-mediated mRNA decay reveals functional roles for noncoding RNAs

The nonsense-mediated mRNA decay (NMD) pathway targets mRNAs undergoing premature translation termination for degradation. Previously, RNA-seq of yeast lacking NMD revealed that most genes targeted by NMD lack obvious premature termination codons (PTCs). We developed a combined approach using RNA-seq and a novel 40S ribosome profiling strategy to identify cryptic premature termination events that could account for NMD on nearly all these transcripts, including many non-coding RNA transcript isoforms associated with annotated genes. Many NMD-targeted transcripts appear to be involved in two-promoter gene regulatory systems and share properties with long un-decoded transcript isoforms (LUTIs). In particular, we show that the DAL5 LUTI regulates expression of the DAL5 protein-coding mRNA in response to changes to environmental nitrogen. Our work expands the functional roles for LUTIs and establishes the importance of NMD in their regulation. HIGHLIGHTSO_LI40S ribosome profiling reveals a comprehensive catalog of NMD substrates in yeast. C_LIO_LIMost NMD targets have short uORFs or iORFs, resulting in premature termination. C_LIO_LIMany long undecoded transcript isoforms (LUTIs) contain NMD-triggering uORFs. C_LIO_LILUTIs, such as DAL5, play a role in metabolic regulation (i.e. nitrogen and thiamine). C_LI

molecular biology↗

bioRxiv2025

Stalled translation on transcripts cleaved by RNase L activates signaling important for innate immunity

RNase L is an endonuclease that responds to infections by cleaving most host- and pathogen-derived single-stranded RNAs. This widespread RNA cleavage can lead to death of the infected cell via the ribotoxic stress response (RSR). An ongoing challenge is to understand how RNase Ls endonuclease activity triggers cell death to benefit the host. To address this question, we used nanopore-based long-read sequencing to show that 3 mRNA fragments in the cell were not fully degraded after RNase L activation and that these fragments were translated by ribosomes. We further asked whether ribosomes on mRNA fragments stall when they reach 3 ends created by RNase L. We used ribosome profiling to capture footprints protected by these ribosomes, which can be identified by their short length (15-18 nt). We found that RNase L activation increased the number of stalled ribosomes at RNase L cleavage sites. Loss of the ribosome rescue factor PELO increased the number of short footprints derived from stalled ribosomes and augmented the RSR. Our work therefore establishes a role for fragmented mRNA in causing ribosome stalling that promotes innate immunity via the RSR. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=84 SRC="FIGDIR/small/658914v1_ufig1.gif" ALT="Figure 1"> View larger version (40K): org.highwire.dtl.DTLVardef@1f08b5forg.highwire.dtl.DTLVardef@14e2cd1org.highwire.dtl.DTLVardef@160814corg.highwire.dtl.DTLVardef@c8ddb4_HPS_FORMAT_FIGEXP M_FIG Stalled translation of mRNAs that are fragmented by RNase L leads to ribosome stalling and potentially collisions. Stalled ribosomes are rescued by PELO or activate innate immune signaling via ZAK. Renderings based on PDB 4o1o and 3jag. C_FIG HighlightsO_LIActivation of RNase L leads to accumulation and translation of mRNA fragments C_LIO_LIRibosomes stall at the 3 end of the RNase L cleaved mRNA fragments C_LIO_LIPELO rescues ribosomes stalled due to RNase L activation C_LI

molecular biology↗

bioRxiv2024

Respiratory Syncytial Virus (RSV) optimizes the translational landscape during infection

Viral infection often triggers eukaryotic initiator factor 2 (eIF2) phosphorylation, leading to global 5-cap-dependent translation inhibition. RSV encodes messenger RNAs (mRNAs) mimicking 5-cap structures of host mRNAs and thus inhibition of cap-dependent translation initiation would likely also reduce viral translation. We confirmed that RSV limits widespread translation initiation inhibition and unexpectedly found that the fraction of ribosomes within polysomes increases during infection, indicating higher ribosome loading on mRNAs during infection. We found that AU-rich host transcripts that are less efficiently translated under normal conditions become more efficient at recruiting ribosomes, similar to RSV transcripts. Viral transcripts are transcribed in cytoplasmic inclusion bodies, where the viral AU-rich binding protein M2-1 has been shown to bind viral transcripts and shuttle them into the cytoplasm. We further demonstrated that M2-1 is found on polysomes, and that M2-1 might deliver host AU-rich transcripts for translation. ImportanceViruses strongly rely on the hosts translational machinery to produce viral proteins required for replication. However, it is unknown how viruses that do not globally inhibit cap-dependent translation compete with abundant host transcripts for ribosomes. In this study, we found that respiratory syncytial virus (RSV) infection results in redistribution of 80S monosomes into the polysomes. High-throughput sequencing of translating transcripts revealed that low translation efficiency transcripts become more efficient at ribosome recruitment which are virus-resembling AU-rich host transcripts. Finally, we also uncover that AU-rich RNA binding protein RSV-M2-1 interacts with polysomes through contacts to mRNA. These findings revealed that RSV optimizes the translational landscape rather than inhibiting host translation.

molecular biology↗

bioRxiv2023

Endonucleolytic RNA cleavage drives changes in gene expression during the innate immune response

Viral infection triggers several dsRNA sensors that lead to changes in gene expression in the cell. One of these sensors activates an endonuclease, RNase L, that cleaves single stranded RNA. However, how the resultant widespread RNA fragmentation affects gene expression is not fully understood. Here we show that this fragmentation induces the Ribotoxic Stress Response via ZAK, potentially through ribosome collisions. The p38 and JNK pathways that are activated as part of this response promote outcomes that inhibit the virus, such as programmed cell death. We also show that RNase L limits the translation of stress-responsive genes, including antiviral IFIT mRNAs and GADD34 that encodes an antagonist of the Integrated Stress Response. Intriguingly, we found the activity of the generic endonuclease, RNase A, recapitulates many of the same molecular phenotypes as activated RNase L, demonstrating how widespread RNA cleavage can evoke an antiviral program. HighlightsO_LIActivated RNase L acts with dsRNA-sensing pathways to promote cell signaling C_LIO_LIRNA fragmentation induces transcription through ZAK signaling C_LIO_LIActivation of RNase L modulates levels of eIF2 phosphorylation C_LIO_LITranslation of the GADD34 and IFIT mRNAs is inhibited by active RNase L C_LI

molecular biology↗

bioRxiv2023

mRNA Location and Translation Rate Determine Protein Targeting to Dual Destinations

Numerous proteins are targeted to two or multiple subcellular destinations where they exert distinct functional consequences. The balance between such differential targeting is thought to be determined post-translationally, relying on protein sorting mechanisms. Here, we show that protein targeting can additionally be determined by mRNA location and translation rate, through modulating protein binding to specific interacting partners. Peripheral localization of the NET1 mRNA and fast translation lead to higher cytosolic retention of the NET1 protein, through promoting its binding to the membrane-associated scaffold protein CASK. By contrast, perinuclear mRNA location and/or slower translation rate favor nuclear targeting, through promoting binding to importins. This mRNA location-dependent mechanism is modulated by physiological stimuli and profoundly impacts NET1 function in cell motility. These results reveal that the location of protein synthesis and the rate of translation elongation act in coordination as a partner-selection mechanism that robustly influences protein distribution and function.

cell biology↗

bioRxiv2022

The ubiquitin conjugase Rad6 mediates ribosome pausing during oxidative stress

Oxidative stress causes K63-linked ubiquitination of ribosomes by the E2 ubiquitin conjugase, Rad6. How Rad6-mediated ubiquitination of ribosomes affects global translation, however, is unclear. We therefore performed Ribo-seq and Disome-seq in Saccharomyces cerevisiae, and found that oxidative stress caused ribosome pausing at specific amino acid motifs, and this also led to ribosome collisions. However, these redox pausing signatures were lost in the absence of Rad6 but did not depend on the ribosome-associated quality control (RQC) pathway. We also found that Rad6 is needed to inhibit overall translation in response to oxidative stress and its deletion leads to increased expression of antioxidant genes. Finally, we observed that the lack of Rad6 leads to changes during translation initiation that affect activation of the integrated stress response (ISR) pathway. Our results provide a high-resolution picture of the gene expression changes during oxidative stress and unravel an additional stress response pathway affecting translation elongation. HIGHLIGHTSO_LIRad6 is required for sequence-specific ribosome pausing under oxidative stress. C_LIO_LIRad6 affects translation independently of the RQC pathway. C_LIO_LICells lacking Rad6 show dysregulated translational repression upon oxidative stress. C_LIO_LILoss of Rad6 leads to altered activation of the ISR pathway. C_LI

molecular biology↗

bioRxiv2021

ribofootPrinter: A precision python toolbox for analysis of ribosome profiling data

Ribosome profiling is a valuable methodology for measuring changes in a cells translational program. The technique can report how efficiently mRNA coding sequences are translated and pinpoint positions along mRNAs where ribosomes slow down or arrest. It can also reveal when translation takes place outside coding regions, often with important regulatory consequences. While many useful software tools have emerged to facilitate analysis of these data, packages can become complex and challenging to adapt to specialized needs. We therefore introduce ribofootPrinter, a suite of Python tools designed to offer an accessible and modifiable set of code for analysis of data from ribosome profiling and related types of small RNA sequencing experiments. Read alignments are made to a simplified transcriptome to keep the code intuitive. Multiple normalization options help facilitate interpretation of data, particularly outside coding regions. We also demonstrate how the length of reads that map to the transcriptome affects the frequency of matches to multiple sites and we provide multimapper identifier files to highlight these regions. Overall, this tool has the capability to carry out sophisticated analyses while maintaining enough simplicity to make it readily understandable and adaptable.

genomics↗

bioRxiv2020

Activation of the antiviral factor RNase L triggers translation of non-coding mRNA sequences

Ribonuclease L (RNase L) is activated as part of the innate immune response and plays an important role in the clearance of viral infections. When activated, it endonucleolytically cleaves both viral and host RNAs, leading to a global reduction in protein synthesis. However, it remains unknown how widespread RNA decay, and consequent changes in the translatome, promote the elimination of viruses. To study how this altered transcriptome is translated, we assayed the global distribution of ribosomes in RNase L activated human cells with ribosome profiling. We found that RNase L activation leads to a substantial increase in the fraction of translating ribosomes in ORFs internal to coding sequences (iORFs) and ORFs within 5 and 3 UTRs (uORFs and dORFs). Translation of these alternative ORFs was dependent on RNase Ls cleavage activity, suggesting that mRNA decay fragments are translated to produce short peptides that may be important for antiviral activity.

molecular biology↗