Dynamic recruitment of single RNAs to processing bodies depends on RNA functionality

Pitchiaya, Sethuramasundaram; Mourao, Marcio Duarte Albasini; Jalihal, Ameya P.; Xiao, Lanbo; Jiang, Xia; Chinnaiyan, Arul M.; Schnell, Santiago; Walter, Nils G.

doi:10.1101/375295

Cited by 6 publications

(8 citation statements)

References 42 publications

(51 reference statements)

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“…In a previous study (Pitchiaya et al, 2019), we observed that osmotic stress leads to phase separation of DCP1A, a non-catalytic protein component of the eukaryotic decapping complex and conserved PB marker (Anderson and Kedersha, 2009). To study the intracellular kinetics of PB and SG formation in response to stress more broadly, we subjected U2OS cells to osmotic and oxidative stressors, and performed fixed-cell protein immunofluorescence (IF) or combined IF and RNA fluorescent in situ hybridization (RNA-FISH) before and after the stressors (Figure 1).…”

Section: Changes In Extracellular Tonicity Induce Rapid and Reversiblmentioning

confidence: 88%

“…Since fixed-cell experiments revealed that the rapid phase separation of DCP1A condensates was distinct from SG formation over minutes/hours, we decided to probe the sub-cellular dynamics at greater temporal resolution. To this end, we subjected the previously developed UGD cell line (a U2OS cell line that stably expresses GFP-DCP1A; (Pitchiaya et al, 2019) to a systematic set of hypertonic conditions. We chose this cell line because it contains a similar number of DCP1A foci as the parental U2OS cells, and each of these foci compositionally resembles endogenous PBs (Liu et al, 2005).…”

Section: Hypertonicity Rapidly Induces the Formation Of Immobile Dcp1mentioning

confidence: 99%

“…Both rapid and prolonged HOPS are reversible ( Figure 1) and can mediate widespread effects, including impairment of transcription termination ( Figure 6) (Vilborg et al, 2015), YAP-programmed transcription initiation (Cai et al, 2018), inhibition of ribosomal translocation (Wu et al, 2019), and modulation of RNA silencing (Pitchiaya et al, 2019). 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).…”

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

confidence: 99%

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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: Changes In Extracellular Tonicity Induce Rapid and Reversiblmentioning

confidence: 88%

Section: Hypertonicity Rapidly Induces the Formation Of Immobile Dcp1mentioning

confidence: 99%

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

confidence: 99%

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

Jalihal

Pitchiaya

Xiao

et al. 2019

Preprint

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“…Fortunately, single-molecule tracking has recently emerged as a powerful tool for examining LLPS in vivo (51,111,112). Indeed, the strongest evidence for bacterial condensates to date comes from single-molecule tracking of proteins in polar microdomains in Caulobacter (113).…”

Section: Single-molecule Tracking As a Tool For Examining Llps In Vivomentioning

confidence: 99%

Clusters of bacterial RNA polymerase are biomolecular condensates that assemble through liquid-liquid phase separation

Ladouceur

Parmar

Biedzinski

et al. 2020

Preprint

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Once described as mere "bags of enzymes", bacterial cells are in fact highly organized, with many macromolecules exhibiting non-uniform localization patterns. Yet the physical and biochemical mechanisms that govern this spatial heterogeneity remain largely unknown. Here, we identify liquid-liquid phase separation (LLPS) as a mechanism for organizing clusters of RNA polymerase (RNAP) in E. coli. Using fluorescence imaging, we show that RNAP quickly transitions from a dispersed to clustered localization pattern as cells enter log phase in nutrientrich media. RNAP clusters are sensitive to hexanediol, a chemical that dissolves liquid-like compartments in eukaryotic cells. In addition, we find that the transcription antitermination factor NusA forms droplets in vitro and in vivo, suggesting that it may nucleate RNAP clusters. Finally, we use single-molecule tracking to characterize the dynamics of cluster components.Our results indicate that RNAP and NusA molecules move inside clusters, with mobilities faster than a DNA locus but slower than bulk diffusion through the nucleoid. We conclude that RNAP clusters are biomolecular condensates that assemble through LLPS. This work provides direct evidence for LLPS in bacteria and suggests that this process serves as a universal mechanism for intracellular organization across the tree of life. SignificanceBacterial cells are small and were long thought to have little to no internal structure. However, advances in microscopy have revealed that bacteria do indeed contain subcellular compartments. But how these compartments form has remained a mystery. Recent progress in larger, more complex eukaryotic cells has identified a novel mechanism for intracellular organization known as liquid-liquid phase separation. This process causes certain types of molecules to concentrate within distinct compartments inside the cell. Here, we demonstrate that the same process also occurs in bacteria. This work, together with a growing body of literature, suggests that liquidliquid phase separation is a universal mechanism for intracellular organization that extends across the tree of life.

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“…The biochemical function of P-bodies and stress granules on mRNA decay is less well, understood. P-bodies have been found to stimulate mRNA decay (Sheth and Parker, 2003) and recent, mathematical models have found that smaller P-bodies might act as more efficient sites of mRNA decay, (Pitchiaya et al, 2019), yet P-bodies are not necessary for mRNA decay (Eulalio et al, 2007). P-bodies, can also store untranslated mRNAs (Hubstenberger et al, 2017; Sheth and Parker, 2003), suggesting, that the mRNA fate may depend on the specific mRNA identity and context of cellular conditions (Wang et al, 2018).…”

Section: Introductionmentioning

confidence: 99%

BR-bodies provide selectively permeable condensates that stimulate mRNA decay and prevent release of decay intermediates

Al-Husini

Tomares

Pfaffenberger

et al. 2019

Preprint

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AbstractBiomolecular condensates play a key role in organizing RNAs and proteins into membraneless organelles. Bacterial RNP-bodies (BR-bodies) are a biomolecular condensate containing the RNA degradosome mRNA decay machinery, but the biochemical function of such organization remains poorly defined. Here we define the RNA substrates of BR-bodies through enrichment of the bodies followed by RNA-seq. We find that long, poorly translated mRNAs, small RNAs, and antisense RNAs are the main substrates, while rRNA, tRNA, and other conserved ncRNAs are excluded from these bodies. BR-bodies stimulate the mRNA decay rate of enriched mRNAs, helping to reshape the cellular mRNA pool. We also observe that BR-body formation promotes complete mRNA decay, avoiding the build-up of toxic endo-cleaved mRNA decay intermediates. The combined selective permeability of BR-bodies for both, enzymes and substrates together with the stimulation of the sub-steps of mRNA decay provide an effective organization strategy for bacterial mRNA decay.

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Dynamic recruitment of single RNAs to processing bodies depends on RNA functionality

Cited by 6 publications

References 42 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

Clusters of bacterial RNA polymerase are biomolecular condensates that assemble through liquid-liquid phase separation

BR-bodies provide selectively permeable condensates that stimulate mRNA decay and prevent release of decay intermediates

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