scholarly journals RNA self-assembly contributes to stress granule formation and defining the stress granule transcriptome

2018 ◽  
Vol 115 (11) ◽  
pp. 2734-2739 ◽  
Author(s):  
Briana Van Treeck ◽  
David S. W. Protter ◽  
Tyler Matheny ◽  
Anthony Khong ◽  
Christopher D. Link ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Self Assembly ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Stress Granules ◽  
Stress Granule ◽  
Local Concentration ◽  
Granule Formation ◽  
In Cells

Stress granules are higher order assemblies of nontranslating mRNAs and proteins that form when translation initiation is inhibited. Stress granules are thought to form by protein–protein interactions of RNA-binding proteins. We demonstrate RNA homopolymers or purified cellular RNA forms assemblies in vitro analogous to stress granules. Remarkably, under conditions representative of an intracellular stress response, the mRNAs enriched in assemblies from total yeast RNA largely recapitulate the stress granule transcriptome. We suggest stress granules are formed by a summation of protein–protein and RNA–RNA interactions, with RNA self-assembly likely to contribute to other RNP assemblies wherever there is a high local concentration of RNA. RNA assembly in vitro is also increased by GR and PR dipeptide repeats, which are known to increase stress granule formation in cells. Since GR and PR dipeptides are involved in neurodegenerative diseases, this suggests that perturbations increasing RNA–RNA assembly in cells could lead to disease.

scholarly journals Distinct stages in stress granule assembly and disassembly

eLife ◽  
2016 ◽  
Vol 5 ◽  
Author(s):  
Joshua R Wheeler ◽  
Tyler Matheny ◽  
Saumya Jain ◽  
Robert Abrisch ◽  
Roy Parker
Keyword(s):  
Time Course ◽  
Binding Proteins ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Stress Granules ◽  
Stress Granule ◽  
Early Event ◽  
Intrinsically Disordered

Stress granules are non-membrane bound RNA-protein (RNP) assemblies that form when translation initiation is limited and contain a biphasic structure with stable core structures surrounded by a less concentrated shell. The order of assembly and disassembly of these two structures remains unknown. Time course analysis of granule assembly suggests that core formation is an early event in granule assembly. Stress granule disassembly is also a stepwise process with shell dissipation followed by core clearance. Perturbations that alter liquid-liquid phase separations (LLPS) driven by intrinsically disordered protein regions (IDR) of RNA binding proteins in vitro have the opposite effect on stress granule assembly in vivo. Taken together, these observations argue that stress granules assemble through a multistep process initiated by stable assembly of untranslated mRNPs into core structures, which could provide sufficient high local concentrations to allow for a localized LLPS driven by IDRs on RNA binding proteins.

scholarly journals Modulation of RNA condensation by the DEAD-box protein eIF4A

2019 ◽  
Author(s):  
Devin Tauber ◽  
Gabriel Tauber ◽  
Anthony Khong ◽  
Briana Van Treeck ◽  
Jerry Pelletier ◽  
...  
Keyword(s):  
Stress Granules ◽  
Protein Aggregates ◽  
Stress Granule ◽  
List Type ◽  
Dependent Manner ◽  
Granule Formation ◽  
Dead Box ◽  
The Dead ◽  
In Cells

SUMMARYStress granules are condensates of non-translating mRNAs and proteins involved in the stress response and neurodegenerative diseases. Stress granules form in part through intermolecular RNA-RNA interactions, although the process of RNA condensation is poorly understood. In vitro, we demonstrate that RNA is effectively recruited to the surfaces of RNA or RNP condensates. We demonstrate that the DEAD-box protein eIF4A reduces RNA condensation in vitro and limits stress granule formation in cells. This defines a purpose for eIF4A to limit intermolecular RNA-RNA interactions in cells, thereby allowing for proper RNP function. These results establish an important role for DEAD-box proteins as ATP-dependent RNA chaperones that can limit the intermolecular condensation and entanglement of RNA, analogous to the function of proteins like HSP70 in combatting protein aggregates.eTOC BlurbStress granules are formed in part by the process of RNA condensation, which is mediated by and promotes trans RNA-RNA interactions. The essential DEAD-box protein and translation initiation factor eIF4A limits stress granule formation by reducing RNA condensation through its function as an ATP-dependent RNA binding protein, behaving analogously to how protein chaperones like HSP70 combat protein aggregates.HighlightsRNA condensates promote intermolecular RNA-RNA interactions at their surfaceseIF4A limits the recruitment of RNAs to stress granules in cellseIF4A reduces the nucleation of stress granules in cellsRecombinant eIF4A1 inhibits the condensation of RNA in vitro in an ATP-dependent manner

scholarly journals Polyadenylated RNA and RNA-Binding Proteins Exhibit Unique Response to Hyperosmotic Stress

2021 ◽  
Vol 9 ◽  
Author(s):  
Benjamin L. Zaepfel ◽  
Jeffrey D. Rothstein
Keyword(s):  
Osmotic Stress ◽  
Binding Proteins ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Protein Localization ◽  
Stress Granules ◽  
Stress Granule ◽  
Granule Formation ◽  
Polyadenylated Rna ◽  
And Function

Stress granule formation is a complex and rapidly evolving process that significantly disrupts cellular metabolism in response to a variety of cellular stressors. Recently, it has become evident that different chemical stressors lead to the formation of compositionally distinct stress granules. However, it is unclear which proteins are required for the formation of stress granules under different conditions. In addition, the effect of various stressors on polyadenylated RNA metabolism remains enigmatic. Here, we demonstrate that G3BP1/2, which are common stress granule components, are not required for the formation of stress granules specifically during osmotic stress induced by sorbitol and related polyols. Furthermore, sorbitol-induced osmotic stress leads to significant depletion of nuclear polyadenylated RNA, a process that we demonstrate is dependent on active mRNA export, as well as cytoplasmic and subnuclear shifts in the presence of many nuclear RNA-binding proteins. We assessed the function of multiple shifted RBPs and found that hnRNP U, but not TDP-43 or hnRNP I, exhibit reduced function following this cytoplasmic shift. Finally, we observe that multiple stress pathways lead to a significant reduction in transcription, providing a possible explanation for our inability to observe loss of TDP-43 or hnRNP I function. Overall, we identify unique outcomes following osmotic stress that provide important insight into the regulation of RNA-binding protein localization and function.

Development of an RNA-protein crosslinker to capture protein interactions with diverse RNA structures in cells

RNA ◽  
2021 ◽  
pp. rna.078896.121
Author(s):  
Yan Han ◽  
Xuzhen Guo ◽  
Tiancai Zhang ◽  
Jiangyun Wang ◽  
Keqiong Ye
Keyword(s):  
Protein Interactions ◽  
Rna Structure ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
High Specificity ◽  
Ester Group ◽  
Yeast Cells ◽  
Primary Amines ◽  
Low Efficiency ◽  
In Cells

Characterization of RNA-protein interaction is fundamental for understanding metabolism and function of RNA. UV crosslinking has been widely used to map the targets of RNA-binding proteins, but is limited by low efficiency, requirement for zero-distance contact and biases for single-stranded RNA structure and certain residues of RNA and protein. Here, we report the development of an RNA-protein crosslinker (AMT-NHS) composed of a psoralen derivative and an N-hydroxysuccinimide ester group, which react with RNA bases and primary amines of protein, respectively. We show that AMT-NHS can penetrate into living yeast cells and crosslink Cbf5 to H/ACA snoRNAs with high specificity. The crosslinker induced different crosslinking patterns than UV and targeted both single- and double-stranded regions of RNA. The crosslinker provides a new tool to capture diverse RNA-protein interactions in cells.

A synthetic RNA editing factor edits its target site in chloroplasts and bacteria

2020 ◽  
Author(s):  
Santana Royan ◽  
Bernard Gutmann ◽  
Catherine Colas des Francs-Small ◽  
Suvi Honkanen ◽  
Jason Schmidberger ◽  
...  
Keyword(s):  
Rna Editing ◽  
Protein Interactions ◽  
Rna Binding ◽  
Catalytic Domain ◽  
Rna Binding Proteins ◽  
Cytidine Deaminase ◽  
Land Plant ◽  
Target Sequence ◽  
Pentatricopeptide Repeat

Abstract Targeted cytidine to uridine RNA editing is a widespread phenomenon throughout the land plant lineage. Members of the pentatricopeptide repeat (PPR) protein family act as the specificity factors in this process. These proteins consist of helix-turn-helix domains, each of which recognises a single RNA nucleotide following a well-elucidated code. A cytidine deaminase-like domain (present at the C-terminus of some PPR editing factors or provided in trans via protein-protein interactions) is the catalytic domain in the process. The huge expansion of the PPR superfamily in land plants provides the sequence variation required for design of novel consensus-based RNA-binding proteins. We used this approach to construct a synthetic RNA editing factor designed to target one of the two sites in the Arabidopsis chloroplast transcriptome naturally recognised by the RNA editing factor CHLOROPLAST BIOGENESIS 19 (CLB19). We show that this designed editing factor specifically recognises the target sequence in in vitro binding assays and can partially complement a clb19 mutant. The designed factor is specific for the target rpoA site and does not recognise or edit the other site recognised by CLB19 in the clpP1 transcript. We show that the designed editing factor can function equally specifically in the bacterium E. coli, and shows some activity even in the absence of the editing cofactors that are often required for natural editing factor activity in plants. This study serves as a successful pilot into the design and application of programmable RNA editing factors based on plant PPR proteins.

scholarly journals Pseudouridine synthases modify human pre-mRNA co-transcriptionally and affect splicing

2020 ◽  
Author(s):  
Nicole M. Martinez ◽  
Amanda Su ◽  
Julia K. Nussbacher ◽  
Margaret C. Burns ◽  
Cassandra Schaening ◽  
...  
Keyword(s):  
Alternative Splicing ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Genetic Manipulation ◽  
Rna Modification ◽  
Messenger Rnas ◽  
Specific Expression ◽  
Pseudouridine Synthases ◽  
In Cells

AbstractEukaryotic messenger RNAs are extensively decorated with modified nucleotides and the resulting epitranscriptome plays important regulatory roles in cells 1. Pseudouridine (Ψ) is a modified nucleotide that is prevalent in human mRNAs and can be dynamically regulated 2–5. However, it is unclear when in their life cycle RNAs become pseudouridylated and what the endogenous functions of mRNA pseudouridylation are. To determine if pseudouridine is added co-transcriptionally, we conducted pseudouridine profiling 2 on chromatin-associated RNA to reveal thousands of intronic pseudouridines in nascent pre-mRNA at locations that are significantly associated with alternatively spliced exons, enriched near splice sites, and overlap hundreds of binding sites for regulatory RNA binding proteins. Multiple distinct pseudouridine synthases with tissue-specific expression pseudouridylate pre-mRNA sites, and genetic manipulation of the predominant pre-mRNA modifying pseudouridine synthases PUS1, PUS7 and RPUSD4 induced widespread changes in alternative splicing in cells, supporting a role for pre-mRNA pseudouridylation in alternative splicing regulation. Consistently, we find that individual pseudouridines identified in cells are sufficient to directly affect splicing in vitro. Together with previously observed effects of artificial pseudouridylation on RNA-RNA6–8 and RNA-protein 9–11 interactions that are relevant for splicing, our results demonstrate widespread co-transcriptional pre-mRNA pseudouridylation and establish the enormous potential for this RNA modification to control human gene expression.

scholarly journals Multivalent interactions with RNA drive RNA binding protein recruitment and dynamics in biomolecular condensates in Xenopus oocytes

2021 ◽  
Author(s):  
Sarah E Cabral ◽  
Kimberly Mowry
Keyword(s):  
Protein Interactions ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Binding Protein ◽  
Rna Binding Protein ◽  
Dependent Manner ◽  
Binding Domains ◽  
Multivalent Interactions

RNA localization and biomolecular condensate formation are key biological strategies for organizing the cytoplasm and generating cellular and developmental polarity. While enrichment of RNAs and RNA-binding proteins (RBPs) is a hallmark of both processes, the functional and structural roles of RNA-RNA and RNA-protein interactions within condensates remain unclear. Recent work from our laboratory has shown that RNAs required for germ layer patterning in Xenopus oocytes localize in novel biomolecular condensates, termed Localization bodies (L-bodies). L-bodies are composed of a non-dynamic RNA phase enmeshed in a more dynamic protein-containing phase. However, the interactions that drive the biophysical characteristics of L-bodies are not known. Here, we test the role of RNA-protein interactions using an L-body RNA-binding protein, PTBP3, which contains four RNA-binding domains (RBDs). We find that binding of RNA to PTB is required for both RNA and PTBP3 to be enriched in L-bodies in vivo. Importantly, while RNA binding to a single RBD is sufficient to drive PTBP3 localization to L-bodies, interactions between multiple RRMs and RNA tunes the dynamics of PTBP3 within L-bodies. In vitro, recombinant PTBP3 phase separates into non-dynamic structures in an RNA-dependent manner, supporting a role for RNA-protein interactions as a driver of both recruitment of components to L-bodies and the dynamics of the components after enrichment. Our results point to a model where RNA serves as a concentration-dependent, non-dynamic substructure and multivalent interactions with RNA are a key driver of protein dynamics.

scholarly journals m6A-binding YTHDF proteins promote stress granule formation by modulating phase separation of stress granule proteins

2019 ◽  
Author(s):  
Ye Fu ◽  
Xiaowei Zhuang
Keyword(s):  
Phase Separation ◽  
Binding Proteins ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Super Resolution ◽  
Stress Granule ◽  
Granule Formation ◽  
Intrinsically Disordered ◽  
Condensate Formation ◽  
Resolution Imaging

AbstractDiverse RNAs and RNA-binding proteins form phase-separated, membraneless granules in cells under stress conditions. However, the role of the prevalent mRNA methylation, m6A, and its binding proteins in stress granule (SG) assembly remain unclear. Here, we show that m6A-modified mRNAs are enriched in SGs, and that m6A-binding YTHDF proteins are critical for SG formation. Depletion of YTHDF1/3 inhibits SG formation and recruitment of m6A-modified mRNAs to SGs. Both the N-terminal intrinsically disordered region and the C-terminal m6A-binding YTH domain of YTHDF proteins are crucial for SG formation. Super-resolution imaging further reveals that YTHDF proteins are in a super-saturated state, forming clusters that reside in the periphery of and at the junctions between SG core clusters, and promote SG phase separation by reducing the activation energy barrier and critical size for condensate formation. Our results reveal a new function and mechanistic insights of the m6A-binding YTHDF proteins in regulating phase separation.

scholarly journals The pseudophosphatase MK-STYX interacts with G3BP and decreases stress granule formation

2010 ◽  
Vol 427 (3) ◽  
pp. 349-357 ◽  
Author(s):  
Shantá D. Hinton ◽  
Michael P. Myers ◽  
Vincent R. Roggero ◽  
Lizabeth A. Allison ◽  
Nicholas K. Tonks
Keyword(s):  
Rna Binding ◽  
Binding Protein ◽  
Active Form ◽  
Protein Tyrosine Phosphatases ◽  
Stress Granules ◽  
Stress Granule ◽  
Mitogen Activated Protein Kinase ◽  
Granule Formation ◽  
Dual Specificity ◽  
Catalytically Active

MK-STYX [MAPK (mitogen-activated protein kinase) phospho-serine/threonine/tyrosine-binding protein] is a pseudophosphatase member of the dual-specificity phosphatase subfamily of the PTPs (protein tyrosine phosphatases). MK-STYX is catalytically inactive due to the absence of two amino acids from the signature motif that are essential for phosphatase activity. The nucleophilic cysteine residue and the adjacent histidine residue, which are conserved in all active dual-specificity phosphatases, are replaced by serine and phenylalanine residues respectively in MK-STYX. Mutations to introduce histidine and cysteine residues into the active site of MK-STYX generated an active phosphatase. Using MS, we identified G3BP1 [Ras-GAP (GTPase-activating protein) SH3 (Src homology 3) domain-binding protein-1], a regulator of Ras signalling, as a binding partner of MK-STYX. We observed that G3BP1 bound to native MK-STYX; however, binding to the mutant catalytically active form of MK-STYX was dramatically reduced. G3BP1 is also an RNA-binding protein with endoribonuclease activity that is recruited to ‘stress granules’ after stress stimuli. Stress granules are large subcellular structures that serve as sites of mRNA sorting, in which untranslated mRNAs accumulate. We have shown that expression of MK-STYX inhibited stress granule formation induced either by aresenite or expression of G3BP itself; however, the catalytically active mutant MK-STYX was impaired in its ability to inhibit G3BP-induced stress granule assembly. These results reveal a novel facet of the function of a member of the PTP family, illustrating a role for MK-STYX in regulating the ability of G3BP1 to integrate changes in growth-factor stimulation and environmental stress with the regulation of protein synthesis.

scholarly journals RNA-Binding Proteins Tia-1 and Tiar Link the Phosphorylation of Eif-2α to the Assembly of Mammalian Stress Granules

1999 ◽  
Vol 147 (7) ◽  
pp. 1431-1442 ◽  
Author(s):  
Nancy L. Kedersha ◽  
Mita Gupta ◽  
Wei Li ◽  
Ira Miller ◽  
Paul Anderson
Keyword(s):  
Environmental Stress ◽  
Binding Proteins ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Rna Binding Protein ◽  
Plant Cells ◽  
Stress Granules ◽  
Binding Domains ◽  
Translational Arrest ◽  
In Cells

In response to environmental stress, the related RNA-binding proteins TIA-1 and TIAR colocalize with poly(A)+ RNA at cytoplasmic foci that resemble the stress granules (SGs) that harbor untranslated mRNAs in heat shocked plant cells (Nover et al. 1989; Nover et al. 1983; Scharf et al. 1998). The accumulation of untranslated mRNA at SGs is reversible in cells that recover from a sublethal stress, but irreversible in cells subjected to a lethal stress. We have found that the assembly of TIA-1/R+ SGs is initiated by the phosphorylation of eIF-2α. A phosphomimetic eIF-2α mutant (S51D) induces the assembly of SGs, whereas a nonphosphorylatable eIF-2α mutant (S51A) prevents the assembly of SGs. The ability of a TIA-1 mutant lacking its RNA-binding domains to function as a transdominant inhibitor of SG formation suggests that this RNA-binding protein acts downstream of the phosphorylation of eIF-2α to promote the sequestration of untranslated mRNAs at SGs. The assembly and disassembly of SGs could regulate the duration of stress- induced translational arrest in cells recovering from environmental stress.

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