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 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 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.

scholarly journals Principles of mRNA targeting and regulation via the Arabidopsis m6A-binding proteins ECT2 and ECT3

2021 ◽  
Author(s):  
Laura Arribas-Hernández ◽  
Sarah Rennie ◽  
Tino Köster ◽  
Michael Schon ◽  
Carlotta Porcelli ◽  
...  
Keyword(s):  
Binding Proteins ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Target Identification ◽  
Super Resolution ◽  
Binding Activity ◽  
Intrinsically Disordered ◽  
Mrna Targets ◽  
Yth Domain

AbstractGene regulation dependent on N6-methyladenosine (m6A) in mRNA involves RNA-binding proteins that recognize m6A through a YTH domain. The Arabidopsis YTH-domain protein ECT2 is thought to influence mRNA 3’-end formation via binding to URU(m6A)Y sites, an unexpected conclusion given that ECT2 functions require its m6A binding activity, and that RR(m6A)CH is the m6A consensus site in all eukaryotes. Here, we apply the orthogonal techniques individual nucleotide-resolution UV-crosslinking and immunoprecipitation (iCLIP) and HyperTRIBE to define high-quality target sets of the YTH-domain proteins ECT2 and ECT3. The results show that in vivo, ECT2 does in fact bind to RR(m6A)CH. URUAY and other pyrimidine-rich motifs are enriched around, but not at m6A-sites, reflecting a preference for N6-adenosine methylation of RRACH islands in pyrimidine-rich regions. Such regions may also be implicated in ECT2-binding. In particular, a series of properties unique to the URUAY motif suggest that URUAY-type sequences act as sites of competition between unknown RNA-binding proteins and the intrinsically disordered region of ECT2. We also show that the abundance of many ECT2/3 mRNA targets is decreased in meristematic cells devoid of ECT2/3/4-activity. In contrast, loss of ECT2/3/4 activity has no effect on polyadenylation site usage in ECT2/3 targets, consistent with the exclusive cytoplasmic localization of ECT2 observed by super-resolution confocal microscopy. Our study reconciles conflicting results between genetic observations on N6-adenosine methylation and ECT2/3/4 function on the one side, and ECT2 target identification on the other, and point to regulation of cytoplasmic mRNA function, including abundance, as a mechanism of plant YTHDF action.

scholarly journals PARylation regulates stress granule dynamics, phase separation, and neurotoxicity of disease-related RNA-binding proteins

Cell Research ◽  
2019 ◽  
Vol 29 (3) ◽  
pp. 233-247 ◽  
Author(s):  
Yongjia Duan ◽  
Aiying Du ◽  
Jinge Gu ◽  
Gang Duan ◽  
Chen Wang ◽  
...  
Keyword(s):  
Phase Separation ◽  
Binding Proteins ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Stress Granule

O4-12-05: Interaction between microtubule-associated protein tau and RNA binding proteins stimulates tau misfolding and stress granule formation

2015 ◽  
Vol 11 (7S_Part_6) ◽  
pp. P300-P301
Author(s):  
Tara Vanderweyde ◽  
Kathrerine Youmans-Kidder ◽  
Daniel J. Apicco ◽  
Peter E.A. Ash ◽  
Casey Cook ◽  
...  
Keyword(s):  
Binding Proteins ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Stress Granule ◽  
Granule Formation ◽  
Protein Tau ◽  
Microtubule Associated Protein Tau

scholarly journals Osmotic stress-induced, rapid clustering of IGF2BP proteins nucleates stress granule assembly

2019 ◽  
Author(s):  
Wei-jie Zeng ◽  
Chuxin Lu ◽  
Yuanyuan Shi ◽  
Xinxin Chen ◽  
Jie Yao
Keyword(s):  
Osmotic Stress ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Optical Resolution ◽  
Super Resolution ◽  
Dense Granule ◽  
Atp Depletion ◽  
Intrinsically Disordered ◽  
Resolution Imaging ◽  
Multiple Clusters

AbstractStress granules (SGs) are formed in the cytoplasm by liquid-liquid phase separation (LLPS) of translationally-stalled mRNA and RNA-binding proteins during stress response. Understanding the mechanisms governing SG assembly requires imaging SG formation in real time. Here we used live cell imaging and super-resolution imaging to visualize SG assembly in human cells. We found that IGF2BP proteins formed microscopically visible clusters almost instantaneously upon osmotic stress, prior to the recruitment of G3BP1 and TIA1. The rapid clustering of IGF2BP1 was ATP-independent and was mediated by its KH3/4 di-domains and an intrinsically disordered region (IDR), whereas ATP depletion inhibited the recruitment of G3BP1 and TIA1. Moreover, we detected cytoplasmic clusters of IGF2BP1 below the optical resolution in normal cells and found IGF2BP1 forming a dense granule associated with multiple clusters of poly(A) mRNA in mature SGs. Thus, ATP-independent, rapid clustering of IGF2BP nucleates SG assembly during osmotic stress.

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