Heat Shock Causes a Reversible Increase in RNA Polymerase II Occupancy Downstream of mRNA Genes, Consistent with a Global Loss in Transcriptional Termination

Cardiello, Joseph F; Goodrich, James A.; Kugel, Jennifer F.

doi:10.1128/mcb.00181-18

Cited by 35 publications

(34 citation statements)

References 40 publications

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“…While other mechanisms may be also at play, protein sequestration away from the site of their function provides a straightforward biophysical explanation for many of these effects ( Figure 6). In fact, such a mechanism may also explain the defects in transcription termination observed in cells exposed to prolonged heat shock (Cardiello et al, 2018), suggesting that protein sequestration might be a common mechanism across multiple stress responses. Future studies will help better understand the connection between MLO formation and protective cellular mechanisms heralded here.…”

Section: Hops May Serve As a Rapid Cellular Sensor Of Volume Compressionmentioning

confidence: 99%

Multivalent proteins rapidly and reversibly phase-separate upon osmotic cell volume change

Jalihal

Pitchiaya

Xiao

et al. 2019

Preprint

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GRAPHICAL ABSTRACT IN BRIEF Cells constantly experience osmotic variation. These external changes lead to changes in cell volume, and consequently the internal state of molecular crowding. Here, Jalihal and Pitchiaya et al. show that multimeric proteins respond rapidly to such cellular changes by undergoing rapid and reversible phase separation. HIGHLIGHTS  DCP1A undergoes rapid and reversible hyperosmotic phase separation (HOPS)  HOPS of DCP1A depends on its trimerization domain  Self-interacting multivalent proteins (valency ≥ 2) undergo HOPS  HOPS of CPSF6 may explain transcription termination defects during osmotic stress SUMMARY Processing bodies (PBs) and stress granules (SGs) are prominent examples of subcellular, membrane-less granules that phase-separate under physiological and stressed conditions, respectively. We observe that the trimeric PB protein DCP1A rapidly (within ~10 s) phase-separates in mammalian cells during hyperosmotic stress and dissolves upon isosmotic rescue (over ~100 s) with minimal impact on cell viability even after multiple cycles of osmotic perturbation. Strikingly, this rapid intracellular hyperosmotic phase separation (HOPS) correlates with the degree of cell volume compression, distinct from SG assembly, and is exhibited broadly by homo-multimeric (valency ≥ 2) proteins across several cell types. Notably, HOPS leads to nuclear sequestration of pre-mRNA cleavage factor component CPSF6, rationalizing hyperosmolarity-induced global impairment of transcription termination. Together, our data suggest that the multimeric proteome rapidly responds to changes in hydration and molecular crowding, revealing an unexpected mode of globally programmed phase separation and sequestration that adapts the cell to volume change.

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Section: Hops May Serve As a Rapid Cellular Sensor Of Volume Compressionmentioning

confidence: 99%

Multivalent proteins rapidly and reversibly phase-separate upon osmotic cell volume change

Jalihal

Pitchiaya

Xiao

et al. 2019

Preprint

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“…In addition, we note that these cellular stresses and viral infections also led to a broad repression of transcription and gene expression, as observed here upon splicing disruption. Heat shock, for instance, caused decreased RNAPII occupancy across most protein-coding genes (Cardiello et al, 2018), accumulation of promoter-proximal paused RNAP, and broad gene down-regulation (Gressel et al, 2019;Mahat et al, 2016). Our findings therefore suggest that splicing complexes may play an important role in sensing and responding to a variety of environmental and infectious stresses through the modulation of gene expression programs.…”

Section: Pladb-induced Transcriptional Alterations Resemble Other Strmentioning

confidence: 71%

“…DoG genes showed significant enrichments for specific gene ontologies (GOs), including translation initiation and ribosome biogenesis, viral process, response to stress, mitochondrial gene expression, and chromatin organization ( Figure 3D, Figure S3B). The appearance of stress-related and viral-related GOs is notable given precedents for DoG transcription triggered upon environmental stresses and viral infections (Bauer et al, 2018;Cardiello et al, 2018;Hennig et al, 2018;Vilborg et al, 2015;Vilborg et al, 2017).…”

Section: Dog Transcription Affects a Specific Subset Of Stress-responmentioning

confidence: 99%

“…DoG genes were enriched for GOs related to protein translation and response to cellular stress, consistent with our observation of a broad cellular response characterized by increased ribosome biogenesis, protein translation, mitochondrial respiration, and ATP synthesis ( Figure 6). Interestingly, DoG transcription has been observed in response to a range of cellular stresses including oxidative stress (Giannakakis et al, 2015;Vilborg et al, 2017), heat shock (Cardiello et al, 2018;Vilborg et al, 2017), and osmotic stress (Vilborg et al, 2015;Vilborg et al, 2017), in addition to viral infections (Bauer et al, 2018;Heinz et al, 2018;Hennig et al, 2018). However, the mechanisms underlying DoG transcription are not clear.…”

Section: Pladb-induced Transcriptional Alterations Resemble Other Strmentioning

confidence: 99%

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SF3B1-targeted Splicing Inhibition Triggers Global Alterations in Transcriptional Dynamics and R-Loop Metabolism

Castillo-Guzman

Hartono

Sanz

et al. 2020

Preprint

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Efficient co-transcriptional splicing is thought to suppress the formation of genome-destabilizing R-loops upon interaction between nascent RNA and the DNA template. Inhibition of the SF3B splicing complex using Pladienolide B (PladB) in human K562 cells caused widespread intron retention and nearly 2,000 instances of R-loops gains. However, only minimal overlap existed between these events, arguing that unspliced introns do not cause excessive R-loops. R-loop gains were instead driven by readthrough transcription resulting from loss of transcription termination over a subset of stress-response genes, defining a new class of aberrant "downstream of genes" (DoG) R-loops. Such DoG R-loops were temporally and spatially uncoupled from loci experiencing DNA damage. Unexpectedly, the predominant response to splicing inhibition was a global R-loop loss resulting from accumulation of promoter-proximal paused RNA polymerases and defective elongation. Thus, SF3B1-targeted splicing inhibition triggered profound alterations in transcriptional dynamics, leading to unexpected disruptions in the global R-loop landscape. HIGHLIGHTS • Intron retention caused by SF3B1 inhibition does not lead to excessive R-loops • A subset of genes shows readthrough transcription and accompanying R-loop gains • SF3B1 inhibition causes broad reduction in nascent transcription and R-loop loss • R-loop gains and DNA damage are temporally and spatially uncoupled RNAPII RNA +PladB PAS zZz Promoter pausing Elongation R-loops downstream of gene PAS zZz R-loops Txn read through DoG R-loops Promoter pausing Elongation No R-loop PAS zZz

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“…Pro-seq analysis) will be required to definitively determine the relative contribution of these cellular processes. Published studies demonstrate that loci susceptible to heat shock-induced downstream-of-gene transcription are marked by open chromatin before heat shock [16] and are depleted of the transcriptional termination factor CPSF-73 after heat shock [33]. These results suggest that altered transcriptional processing itself leads to the altered transcript accumulation after heat shock.…”

Section: Discussionmentioning

confidence: 99%

Heat Shock in C. elegans Induces Downstream of Gene Transcription andAccumulation of Double-Stranded RNA

Melnick

Gonzales

Cabral

et al. 2018

Preprint

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43Abstract 44 We observed that heat shock of Caenorhabditis elegans leads to the formation of nuclear 45 double-stranded RNA (dsRNA) foci, detectable with a dsRNA-specific monoclonal antibody.46 These foci significantly overlap with nuclear HSF-1 granules. To investigate the molecular 47 mechanism(s) underlying dsRNA foci formation, we used RNA-seq to globally characterize total 48 RNA and immunoprecipitated dsRNA from control and heat shocked worms. We find antisense 49 transcripts are generally increased after heat shock, and a subset of both sense and antisense 50 transcripts enriched in the dsRNA pool by heat shock overlap with dsRNA transcripts enriched 51 by deletion of tdp-1, which encodes the C. elegans ortholog of TDP-43. Interestingly, transcripts 52 involved in translation are over-represented in the dsRNAs induced by either heat shock or 53 deletion of tdp-1. Also enriched in the dsRNA transcripts are sequences downstream of 54 annotated genes (DoGs), which we globally quantified with a new algorithm. To validate these 55 observations, we used fluorescence in situ hybridization (FISH) to confirm both antisense and 56 downstream of gene transcription for eif-3.B, one of the affected loci we identified. 57 58 Introduction 59 Cytoplasmic proteotoxic stress induced by temperatures outside of the optimal range for 60 cells or organisms triggers the heat shock response (HSR) [1]. The response to heat shock is 61 multi-faceted and regulation of both transcription and translation occurs. Transcriptional 62 responses include formation of stress granules, alternative splicing, and aberrant transcriptional 63 termination [2-5]. The HSR is a highly conserved transcriptional response and is driven largely 64 by the heat shock transcription factor HSF1 [6]. Under basal level conditions, HSF1 is a 65 monomer in the cytoplasm and nucleus. Upon stress, HSF1 undergoes homotrimerization and 66 binds to DNA heat shock elements (HSE) and initiates the transcription of heat shock protein 3 67 genes [7,8]. In addition, translation of non-heat shock mRNAs is reduced through pausing of 68 translation elongation as well as inhibition of translation initiation [9-11]. Regulation and 69 clearance of misfolded proteins by heat shock proteins has been implicated in neurodegenerative 70 diseases such as Huntington's disease (HD), Parkinson's disease (PD), Alzheimer's disease 71 (AD), and amyotrophic lateral sclerosis (ALS) [12]. 72 Aside from the canonical binding of HSF1 to HSE loci, heat shock can cause HSE-73 independent transcriptional changes [2]. In mammalian cells, HSF1 granules co-localize with 74 markers of active transcription where HSF1 binds at satellite II and III repeat regions [13]. In the 75 worm Caenorhabditis elegans, HSF-1 granules also show markers of active transcription but the 76 putative sites of HSF-1 stress granule binding are unknown [14]. 77 In addition to formation of HSF1 stress granules, heat shock can cause reduced efficiency 78 of transcription termination and the accumulation of normally untransc...

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Heat Shock Causes a Reversible Increase in RNA Polymerase II Occupancy Downstream of mRNA Genes, Consistent with a Global Loss in Transcriptional Termination

Cited by 35 publications

References 40 publications

Multivalent proteins rapidly and reversibly phase-separate upon osmotic cell volume change

Multivalent proteins rapidly and reversibly phase-separate upon osmotic cell volume change

SF3B1-targeted Splicing Inhibition Triggers Global Alterations in Transcriptional Dynamics and R-Loop Metabolism

Heat Shock in C. elegans Induces Downstream of Gene Transcription andAccumulation of Double-Stranded RNA

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