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Herpesvirus Effectors of RNA synthesis, Processing, Export and Stability

Periodic Reporting for period 2 - HERPES (Herpesvirus Effectors of RNA synthesis, Processing, Export and Stability)

Reporting period: 2018-11-01 to 2020-04-30

Herpes simplex virus 1 (HSV-1) is an important human pathogen, which intensively interacts with the cellular transcriptional machinery at multiple levels during lytic infection. Employing next-generation sequencing to study RNA synthesis, processing and translation in short intervals throughout lytic HSV-1 infection, my laboratory made the surprising observation that HSV-1 triggers widespread disruption of transcription termination of cellular but not viral genes. Transcription commonly extends for tens-of-thousands of nucleotides beyond poly(A)-sites and into downstream genes. In contrast to textbook knowledge, HSV-1 infection does not inhibit splicing but induces a broad range of aberrant splicing events associated with disruption of transcription termination. Furthermore, I found poly(A) read-through to be accompanied by a selective defect in histone repositioning downstream of genes resulting in the formation of extensive open chromatin regions. Exploring these fascinating phenomena will provide fundamental insights into RNA biology of human cells.
Within the frame of HERPES, I will elucidated the molecular mechanism by which HSV-1 disrupts transcription termination of cellular genes while maintaining transcription termination of viral genes. I will employ cutting-edge methodology ranging from large-scale omics approaches to single cell analysis. I will develop reporter assays to visualise and track poly(A) read-through in single cells. I will identify novel cellular proteins governing transcription termination using a genome-wide Cas9-knockout screen. I hypothesize that the alterations in RNA processing are depicted by specific changes in RNA Polymerase II CTD phosphorylation and in the associated proteins. I will characterise these dynamic changes using mNET-seq and quantitative proteomics. Finally, data-driven quantitative bioinformatic modelling will detail how the coupling of RNA synthesis, processing, export, stability and translation is orchestrated by HSV-1.
We found that poly(A) read-through is accompanied by a selective defect in histone repositioning downstream of genes resulting in the formation of extensive open chromatin regions (Hennig et al., PLoS Pathogens 2019). We identified the viral ICP22 protein to be responsible for this effect. We have performed extensive ChIP-seq analysis to elucidate the underlying molecular mechanism and are currently performing mechanistic studies to confirm and validate the obtained results.
Within the last 18 months, we elucidated the molecular mechanism by which HSV-1 disrupts transcription termination of cellular genes. Namely, the viral ICP27 protein directly targets and disrupts the cellular CPSF complex. This impairs pre-mRNA cleavage and thereby transcription termination. Viral transcription termination is rescued by ICP27 binding and recruitment of CPSF to GC-rich elements in the viral mRNAs (Wang, Hennig et al, Nature communications, resubmitted).
To study specific changes in RNA Polymerase II CTD phosphorylation, we established mNET-seq. This revealed that HSV-1 releases promoter-proximal pausing genome-wide. We are working on the underlying molecular mechanism.
We extensively tested the usefulness of the RNA aptamer Broccoli for live-cell RNA imaging and found it to be not sufficiently stable and bright even upon concatemerization. We found that it is not sufficiently stable and bright to serve as a reliable reporter. We now developed a new reporter based on a bicistronic reporter comprising two fluorophores separated by a candidate poly(A) site with translation of the downstream reporter reinitiating from an IRES element. This enables the visualization of impaired transcription termination at single cell level.
We pioneered metabolic RNA labeling combined with chemical nucleotide-conversion for single cell RNA sequencing (scSLAM-seq; Erhard et al., Nature 2019). This adds a temporal dimension to single cell RNA sequencing and now facilitates dose-response analyses at single cell level. This was only made possible due to the development of the computational approach GRAND-SLAM, which provides reliable new/total RNA ratios for thousands of genes in individual cells (Jürges et al, Bioinformatics 2018). We filed a patent on GRAND-SLAM.
The goal of this ERC Consolidator research program is to elucidate the molecular mechanisms responsible for the profound alterations in RNA processing I observed in HSV-1 infection and utilise this model to decipher the regulation of the transcriptional machinery in human cells. The key objectives are:
O1: Completed (Wang and Hennig, Nature communications, manuscript resubmitted).
We elucidated the molecular mechanisms by which HSV-1 both disrupts and rescues transcription termination.
O2: To detail the molecular mechanisms by which RNA processing is coordinated in human cells.
RNA polymerase II (Pol II) transcriptional activity as well as coupled pre-mRNA processing are orchestrated by complex changes in the phosphorylation of the 52 heptad repeats in its C-terminal domain (CTD) (10). I hypothesized that the alterations in RNA processing in HSV-1 infection are depicted by both specific changes in Pol II CTD phosphorylation and in the associated proteins. We now found that HSV-1 infection results in a global loss of Serine-7 phosphorylation of the Pol II CTD thereby explaining impaired transcription termination of canonical histones and other genes that rely on Integrator for 3'-end processing. Work is ongoing to characterise these dynamic changes in the HSV-1 model using mNET-seq and quantitative proteomics. In particular, we are currently setting up new tools to determine post-translational modifications of all 52 heptad repeats by mass spec.
This will clarify whether HSV-1 affects the “writers” or the “readers” of the Pol II CTD molecular code and will comprehensively detail the functional role of the complex regulatory mechanisms in RNA processing.
O3: Identify new host factors that governing transcription termination
We have developed new dual-color reporters that enable us to visualize poly(A) read-through at single cell level. This should enable us to perform a Cas9 screen to identify novel cellular factors governing transcription termination. However, before we initiate this work we need to complete the work on objective 2, namely (i) elucidate how poly(A) read-through results in open chromatin formation and how HSV-1 disrupts and exploits the Pol II CTD to impair RNA processing of cellular and aid processing of viral RNAs.
O4: Identify viral RNA elements that govern viral RNA processing and export.
We have set up the reporter system to now screen for novel viral RNA elements that govern RNA export. We will now generate the respective screening library and perform the screen. I expect that this will reveal the viral RNA elements that mediate both ICP27-dependent and -independent effects o RNA export and detail the underlying molecular mechanisms.
O5: To elucidate how the coupling of RNA synthesis, processing, export, stability and translation is modulated throughout HSV-1 infection using data-driven quantitative bioinformatic modelling:
We have just completed a comprehensive reannotation of the HSV-1 coding capacity based on our large datasets (Whisnant et al., Nature communications, resubmitted). I expect integrative analysis of the obtained big data to provide a plethora of new findings which need to be experimentally validated.
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