Analysis of transcriptional and translational regulation during virus infection using RNA tagging and ribosomal profiling
Final Report Abstract
Advances in next-generation sequencing now allow studying real-time changes in transcriptional and translational regulation at nucleotide resolution. In this project, we employed 4sU-seq (=metabolic labeling of newly transcribed RNA (RNA tagging) combined with sequencing), sequencing of ribosome-protected mRNA fragments (ribosome profiling/Ribo-seq) and sequencing assays on chromatin accessibility (ATAC-seq) and RNA Polymerase II activity (ChIP-seq) to analyze regulatory processes in herpes simplex virus 1 (HSV-1) infection and T helper cell responses. Using 4sU-seq time-course analysis, we characterized disruption of transcription termination in HSV-1 infection resulting in read-through transcription for thousands of nucleotides beyond poly(A) sites and analyzed similarities and differences to a stress response. Using Ribo-seq and sequencing of nuclear RNA fractions, we showed that read-through transcripts are neither translated nor exported from the nucleus. Strikingly, read-through transcription in HSV-1 infection, but not stress responses, selectively increases chromatin accessibility downstream of genes. To characterize polyadenylation and exclude read-through transcription for viral transcripts, we developed a novel method for mapping reads containing part of poly(A)-tails, which is fully integrated into our RNA-seq mapping program ContextMap2. HSV-1 infection also leads to a global loss of host transcriptional activity as well as degradation of mRNAs by the HSV-1 virion host shutoff (vhs) protein. Using RNA-seq analysis of wild-type (WT) virus and a vhs-null mutant (Dvhs), we showed that both effects affect host mRNA abundances differentially depending on basal RNA turnover rates of genes. This makes it effectively impossible to distinguish gene-specific regulation from global effects in total RNA. On the positive side, it allowed estimating the kinetics of vhs activity and the loss of transcriptional activity during infection using mathematical modelling. Moreover, we showed that chromatin-associated RNA provides an unbiased picture of transcriptional regulation. In this way, we identified a previously unsuspected regulatory effect of vhs on transcription for a set of >150 genes. Many of these encode for components of the extracellular matrix and integrin adhesome. Quantitative proteomics showed a substantial impact of vhs-mediated transcriptional regulation on protein levels after only 8h of infection. Integrated 4sU- and Ribo-seq analysis was also performed on T helper cells activation to study the coupling of transcription and translation. This showed that for almost all genes changes in translation followed changes in de novo transcription with a delay depending on the RNA turnover rates of these genes. Upregulation of genes coincided with increased abundance of RNA Poymerase II at their promoters but not reduced pausing. Thus, rapid de novo recruitment of RNA Polymerase II was the dominant mechanism to ensure rapid transcriptional upregulation during the T cell response. Strikingly, this coincided with an initial drop in splicing rates in the first 1h of activation, which was followed by a gradual increase of splicing rates beyond levels in non-activated cells. Finally, to enable the required large-scale analyses for several sequencing assays, numerous samples and multiple replicates in this project, we developed the workflow management system Watchdog. The core features of Watchdog include straightforward processing of replicate data, support for and flexible combination of distributed computing or remote executors, customizable error detection, user notification on execution errors, and manual user intervention. Watchdog combines advantages of existing workflow management systems and provides a number of novel useful features for more flexible and convenient execution and control of workflows.
Publications
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Prediction of Poly(A) Sites by Poly(A) Read Mapping. PLoS One 12, e0170914 (2017)
Bonfert, T. & Friedel, C.C.
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Rapid Genome-wide Recruitment of RNA Polymerase II Drives Transcription, Splicing, and Translation Events during T Cell Responses. Cell Rep 19, 643-654 (2017)
Davari, K., Lichti, J., Gallus, C., Greulich, F., Uhlenhaut, N.H., Heinig, M., Friedel, C.C. & Glasmacher, E.
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HSV-1-induced disruption of transcription termination resembles a cellular stress response but selectively increases chromatin accessibility downstream of genes. PLoS Pathog 14, e1006954 (2018)
Hennig, T., Michalski, M., Rutkowski, A.J., Djakovic, L., Whisnant, A.W., Friedl, M.S., Jha, B.A., Baptista, M.A.P., L'Hernault, A., Erhard, F., Dölken, L. & Friedel, C.C.
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Watchdog - a workflow management system for the distributed analysis of large-scale experimental data. BMC Bioinformatics 19, 97 (2018)
Kluge, M. & Friedel, C.C.
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Dissecting HSV-1-induced host shut-off at RNA level. bioRxiv (2020)
Friedel, C.C., Whisnant, A.W., Djakovic, L., Rutkowski, A.J., Friedl, M.-S., Kluge, M., Williamson, J.C., Sai, S., Vidal, R.O., Sauer, S., Hennig, T., Prusty, B., Lehner, P.J., Matheson, N.J., Erhard, F. & Dölken, L.
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Watchdog 2.0: New developments for reusability, reproducibility, and workflow execution. GigaScience 9, giaa068 (2020)
Kluge, M., Friedl, M.-S., Menzel, A.L. & Friedel, C.C.
