scholarly journals Modulation of Phase Separation by RNA: A Glimpse on N6-Methyladenosine Modification

2021 ◽  
Vol 9 ◽  
Author(s):  
Yingfeng Su ◽  
Yasen Maimaitiyiming ◽  
Lingfang Wang ◽  
Xiaodong Cheng ◽  
Chih-Hung Hsu
Keyword(s):  
Phase Separation ◽  
Driving Force ◽  
Rna Binding ◽  
Rna Binding Protein ◽  
The Other ◽  
Biological Processes ◽  
Essential Component ◽  
Modulated Phase ◽  
New Perspective ◽  
Cellular Components

Phase separation is the driving force behind formation of various biomolecular condensates (BioMCs), which sub-compartmentalize certain cellular components in a membraneless manner to orchestrate numerous biological processes. Many BioMCs are composed of proteins and RNAs. While the features and functions of proteins are well studied, less attention was paid to the other essential component RNAs. Here, we describe how RNA contributes to the biogenesis, dissolution, and properties of BioMCs as a multivalence providing scaffold for proteins/RNA to undergo phase separation. Specifically, we focus on N6-methyladenosine (m6A), the most widely distributed dynamic post-transcriptional modification, which would change the charge, conformation, and RNA-binding protein (RBP) anchoring of modified RNA. m6A RNA-modulated phase separation is a new perspective to illustrate m6A-mediated various biological processes. We summarize m6A main functions as “beacon” to recruit reader proteins and “structural switcher” to alter RNA–protein and RNA–RNA interactions to modulate phase separation and regulate the related biological processes.

Phase separation of RNA-binding protein promotes polymerase binding and transcription

2021 ◽  
Author(s):  
Wen Shao ◽  
Xianju Bi ◽  
Yixuan Pan ◽  
Boyang Gao ◽  
Jun Wu ◽  
...  
Keyword(s):  
Phase Separation ◽  
Rna Binding ◽  
Binding Protein ◽  
Rna Binding Protein ◽  
Polymerase Binding

scholarly journals TIA1 potentiates tau phase separation and promotes generation of toxic oligomeric tau

2021 ◽  
Vol 118 (9) ◽  
pp. e2014188118
Author(s):  
Peter E. A. Ash ◽  
Shuwen Lei ◽  
Jenifer Shattuck ◽  
Samantha Boudeau ◽  
Yari Carlomagno ◽  
...  
Keyword(s):  
Phase Separation ◽  
Polyethylene Glycol ◽  
Stress Response ◽  
Tau Protein ◽  
Rna Binding ◽  
Biological Activities ◽  
Rna Binding Protein ◽  
Physiological Conditions ◽  
Tau Oligomers

Tau protein plays an important role in the biology of stress granules and in the stress response of neurons, but the nature of these biochemical interactions is not known. Here we show that the interaction of tau with RNA and the RNA binding protein TIA1 is sufficient to drive phase separation of tau at physiological concentrations, without the requirement for artificial crowding agents such as polyethylene glycol (PEG). We further show that phase separation of tau in the presence of RNA and TIA1 generates abundant tau oligomers. Prior studies indicate that recombinant tau readily forms oligomers and fibrils in vitro in the presence of polyanionic agents, including RNA, but the resulting tau aggregates are not particularly toxic. We discover that tau oligomers generated during copartitioning with TIA1 are significantly more toxic than tau aggregates generated by incubation with RNA alone or phase-separated tau complexes generated by incubation with artificial crowding agents. This pathway identifies a potentially important source for generation of toxic tau oligomers in tau-related neurodegenerative diseases. Our results also reveal a general principle that phase-separated RBP droplets provide a vehicle for coassortment of selected proteins. Tau selectively copartitions with TIA1 under physiological conditions, emphasizing the importance of TIA1 for tau biology. Other RBPs, such as G3BP1, are able to copartition with tau, but this happens only in the presence of crowding agents. This type of selective mixing might provide a basis through which membraneless organelles bring together functionally relevant proteins to promote particular biological activities.

scholarly journals TDP-43 and HSP70 phase separate into anisotropic, intranuclear liquid spherical annuli

2020 ◽  
Author(s):  
Haiyang Yu ◽  
Shan Lu ◽  
Kelsey Gasior ◽  
Digvijay Singh ◽  
Olga Tapia ◽  
...  
Keyword(s):  
Phase Separation ◽  
Rna Binding ◽  
Rna Binding Protein ◽  
Proteasome Inhibition ◽  
Chaperone Activity ◽  
Cell Nuclei ◽  
The Core ◽  
Hsp70 Family ◽  
Age Related ◽  
Annulus Formation

AbstractThe RNA binding protein TDP-43 naturally phase separates within cell nuclei and forms cytoplasmic aggregates in age-related neurodegenerative diseases. Here we show that acetylation-mediated inhibition of TDP-43 binding to RNA produces co-de-mixing of acetylated and unmodified TDP-43 into symmetrical, intranuclear spherical annuli whose shells and cores have liquid properties. Shells are anisotropic, like liquid crystals. Consistent with our modelling predictions that annulus formation is driven by components with strong self-interactions but weak interaction with TDP-43, the major components of annuli cores are identified to be HSP70 family proteins, whose chaperone activity is required to maintain liquidity of the core. Proteasome inhibition, mimicking reduction in proteasome activity during aging, induces TDP-43-containing annuli in neurons in rodents. Thus, we identify that TDP-43 phase separation is regulated by acetylation, proteolysis, and ATPase-dependent chaperone activity of HSP70.One Sentence SummaryAcetylation of TDP-43 drives its phase separation into spherical annuli that form a liquid-inside-a-liquid-inside-a-liquid.

scholarly journals RNA Binding Protein HuR Promotes Autophagosome Formation by Regulating Expression of Autophagy-Related Proteins 5, 12, and 16 in Human Hepatocellular Carcinoma Cells

2019 ◽  
Vol 39 (6) ◽  
Author(s):  
Eunbyul Ji ◽  
Chongtae Kim ◽  
Hoin Kang ◽  
Sojin Ahn ◽  
Myeongwoo Jung ◽  
...  
Keyword(s):  
Hepatocellular Carcinoma ◽  
Rna Binding ◽  
Binding Protein ◽  
Rna Binding Protein ◽  
Autophagic Flux ◽  
Autophagosome Formation ◽  
Cellular Autophagy ◽  
Related Proteins ◽  
Cellular Components ◽  
Hcc Cells

ABSTRACT Autophagy is a process of lysosomal self-degradation of cellular components by forming autophagosomes. Autophagosome formation is an essential process in autophagy and is fine-tuned by various autophagy-related gene (ATG) products, including ATG5, ATG12, and ATG16. Although several reports have shown that numerous factors affect multiple levels of gene regulation to orchestrate cellular autophagy, the detailed mechanism of autophagosome formation still needs further investigation. In this study, we demonstrate that the RNA binding protein HuR (human antigen R) performs an essential function in autophagosome formation. We observe that HuR silencing leads to inhibition of autophagosome formation and autophagic flux in liver cells. Ribonucleoprotein immunoprecipitation (RIP) assay allows the identification of ATG5, ATG12, and ATG16 mRNAs as the direct targets of HuR. We further show that HuR mediates the translation of ATG5, ATG12, and ATG16 mRNAs by binding to their 3′ untranslated regions (UTRs). In addition, we show that HuR expression positively correlates with the levels of ATG5 and ATG12 in hepatocellular carcinoma (HCC) cells. Collectively, our results suggest that HuR functions as a pivotal regulator of autophagosome formation by enhancing the translation of ATG5, ATG12, and ATG16 mRNAs and that augmented expression of HuR and ATGs may participate in the malfunction of autophagy in HCC cells.

scholarly journals The SARS-CoV-2 nucleocapsid protein is dynamic, disordered, and phase separates with RNA

2020 ◽  
Author(s):  
Jasmine Cubuk ◽  
Jhullian J. Alston ◽  
J. Jeremías Incicco ◽  
Sukrit Singh ◽  
Melissa D. Stuchell-Brereton ◽  
...  
Keyword(s):  
Phase Separation ◽  
Single Molecule ◽  
Protein Function ◽  
Rna Binding ◽  
Binding Protein ◽  
Rna Binding Protein ◽  
Genome Packaging ◽  
Binding Motifs ◽  
N Protein ◽  
Single Genome

AbstractThe SARS-CoV-2 nucleocapsid (N) protein is an abundant RNA binding protein critical for viral genome packaging, yet the molecular details that underlie this process are poorly understood. Here we combine single-molecule spectroscopy with all-atom simulations to uncover the molecular details that contribute to N protein function. N protein contains three dynamic disordered regions that house putative transiently-helical binding motifs. The two folded domains interact minimally such that full-length N protein is a flexible and multivalent RNA binding protein. N protein also undergoes liquid-liquid phase separation when mixed with RNA, and polymer theory predicts that the same multivalent interactions that drive phase separation also engender RNA compaction. We offer a simple symmetry-breaking model that provides a plausible route through which single-genome condensation preferentially occurs over phase separation, suggesting that phase separation offers a convenient macroscopic readout of a key nanoscopic interaction.

scholarly journals Fusion protein EWS-FLI1 is incorporated into a protein granule in cells

2020 ◽  
Author(s):  
Nasiha S. Ahmed ◽  
Lucas M. Harrell ◽  
Jacob C. Schwartz
Keyword(s):  
Phase Separation ◽  
Fusion Protein ◽  
Ewing Sarcoma ◽  
Rna Binding ◽  
Binding Protein ◽  
Rna Binding Protein ◽  
Rna Pol Ii ◽  
Pol Ii ◽  
Protein Granules ◽  
In Cells

ABSTRACTEwing sarcoma is driven by fusion proteins containing a low complexity (LC) domain that is intrinsically disordered and a powerful transcriptional regulator. The most common fusion protein found in Ewing sarcoma, EWS-FLI1, takes its LC domain from the RNA-binding protein EWSR1 (Ewing Sarcoma RNA-binding protein 1) and a DNA-binding domain from the transcription factor FLI1 (Friend Leukemia Virus Integration 1). EWS-FLI1 binds RNA polymerase II (RNA Pol II) and can self-assemble through a process known as phase separation. The ability of self-oligomerizing RNA-binding proteins like EWSR1 to assemble into ribonucleoprotein granules in cells has received significant attention but the role of phase separation in EWS-FLI1 activity is less understood. We investigated the intersecting roles of EWSR1 and EWS-FLI1 to control gene expression and tumorigenic cell growth in Ewing sarcoma. We also studied interactions among EWS-FLI1, EWSR1, and RNA Pol II. We applied a crosslinking approach to demonstrate the incorporation of EWSR1 and RNA Pol II into protein granules in cells. We also identified protein granules in cells associated with the fusion protein, EWS-FLI1. Interactions through the LC domain, which allow EWS-FLI1 to bind EWSR1 and RNA Pol II, were found to be required for inclusion into the cellular granules observed by TEM. The physical characterization of EWS-FLI1 assemblies reported here offers insight into a large protein assembly that may allow EWS-FLI1 to engage its wide network of protein partners while driving tumorigenesis.

scholarly journals The low-complexity domain of the FUS RNA binding protein self-assembles via the mutually exclusive use of two distinct cross-β cores

2021 ◽  
Vol 118 (42) ◽  
pp. e2114412118
Author(s):  
Masato Kato ◽  
Steven L. McKnight
Keyword(s):  
Phase Separation ◽  
Rna Binding ◽  
Binding Protein ◽  
Rna Binding Protein ◽  
Low Complexity ◽  
Terminal Region ◽  
Aqueous Environment ◽  
Fused In Sarcoma ◽  
Biochemical Assays ◽  
Biologic Function

The low-complexity (LC) domain of the fused in sarcoma (FUS) RNA binding protein self-associates in a manner causing phase separation from an aqueous environment. Incubation of the FUS LC domain under physiologically normal conditions of salt and pH leads to rapid formation of liquid-like droplets that mature into a gel-like state. Both examples of phase separation have enabled reductionist biochemical assays allowing discovery of an N-terminal region of 57 residues that assembles into a labile, cross-β structure. Here we provide evidence of a nonoverlapping, C-terminal region of the FUS LC domain that also forms specific cross-β interactions. We propose that biologic function of the FUS LC domain may operate via the mutually exclusive use of these N- and C-terminal cross-β cores. Neurodegenerative disease–causing mutations in the FUS LC domain are shown to imbalance the two cross-β cores, offering an unanticipated concept of LC domain function and dysfunction.

scholarly journals Expression of phage P4 integrase is regulated negatively by both Int and Vis

2006 ◽  
Vol 87 (8) ◽  
pp. 2423-2431 ◽  
Author(s):  
D. Piazzolla ◽  
S. Calì ◽  
E. Spoldi ◽  
F. Forti ◽  
C. Sala ◽  
...  
Keyword(s):  
Rna Binding ◽  
Transcription Termination ◽  
Rna Binding Protein ◽  
Mrna Degradation ◽  
Negative Regulation ◽  
The Other ◽  
Terminal Portion ◽  
N Terminus ◽  
Different Levels ◽  
Complex Manner

Phage P4 int gene encodes the integrase responsible for phage integration into and excision from the Escherichia coli chromosome. Here, the data showing that P4 int expression is regulated in a complex manner at different levels are presented. First of all, the Pint promoter is regulated negatively by both Int and Vis, the P4 excisionase. The N-terminal portion of Int appears to be sufficient for such a negative autoregulation, suggesting that the Int N terminus is implicated in DNA binding. Second, full-length transcripts covering the entire int gene could be detected only upon P4 infection, whereas in P4 lysogens only short 5′-end covering transcripts were detectable. On the other hand, transcripts covering the 5′-end of int were also very abundant upon infection. It thus appears that premature transcription termination and/or mRNA degradation play a role in Int-negative regulation both on the basal prophage transcription and upon infection. Finally, comparison between Pint–lacZ transcriptional and translational fusions suggests that Vis regulates Int expression post-transcriptionally. The findings that Vis is also an RNA-binding protein and that Int may be translated from two different start codons have implications on possible regulation models of Int expression.

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