scholarly journals Transcription organizes euchromatin via microphase separation

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Lennart Hilbert ◽  
Yuko Sato ◽  
Ksenia Kuznetsova ◽  
Tommaso Bianucci ◽  
Hiroshi Kimura ◽  
...  
Keyword(s):  
Phase Separation ◽  
Cell Nucleus ◽  
Transcriptional Activity ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Microphase Separation ◽  
Rna Polymerases ◽  
Typical Pattern ◽  
Inactive Chromatin ◽  
Theory And Experiment

AbstractIn eukaryotes, DNA is packed inside the cell nucleus in the form of chromatin, which consists of DNA, proteins such as histones, and RNA. Euchromatin, which is permissive for transcription, is spatially organized into transcriptionally inactive domains interspersed with pockets of transcriptional activity. While transcription and RNA have been implicated in euchromatin organization, it remains unclear how their interplay forms and maintains transcription pockets. Here we combine theory and experiment to analyze the dynamics of euchromatin organization as pluripotent zebrafish cells exit mitosis and begin transcription. We show that accumulation of RNA induces formation of transcription pockets which displace transcriptionally inactive chromatin. We propose that the accumulating RNA recruits RNA-binding proteins that together tend to separate from transcriptionally inactive euchromatin. Full phase separation is prevented because RNA remains tethered to transcribed euchromatin through RNA polymerases. Instead, smaller scale microphases emerge that do not grow further and form the typical pattern of euchromatin organization.

scholarly journals The SRSF4-GAS5-Glucocorticoid Receptor Axis Regulates Ventricular Hypertrophy

2021 ◽  
Author(s):  
Javier Larrasa-Alonso ◽  
María Villalba ◽  
Carlos Martí-Gómez ◽  
Paula Ortiz-Sánchez ◽  
Marina López-Olañeta ◽  
...  
Keyword(s):  
Glucocorticoid Receptor ◽  
Cardiac Hypertrophy ◽  
Diastolic Dysfunction ◽  
Ventricular Hypertrophy ◽  
Transcriptional Activity ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Left Ventricular ◽  
Therapeutic Tools ◽  
Nucleotide Resolution

Rationale: RNA-binding proteins (RBPs) play critical roles in human biology and disease. Aberrant RBP expression affects various steps in RNA processing, altering the function of the target RNAs. The RBP serine/arginine-rich splicing factor 4 (SRSF4) has been linked to neuropathies and cancer. However, its role in the heart is completely unknown. Objective: To investigate the role of SRSF4 in the heart. Methods and Results: Echocardiography of mice specifically lacking SRSF4 in the heart (SRSF4 KO) revealed left ventricular hypertrophy and increased cardiomyocyte area, which led to progressive diastolic dysfunction with age. SRSF4 KO mice showed altered electrophysiological activity under isoproterenol-induced cardiac stress, with a post-QRS depression and a longer QT interval, indicating an elevated risk of sudden cardiac death. RNA-Seq analysis revealed expression changes in several long non-coding RNAs (lncRNAs), including GAS5 (growth arrest specific 5), which we identified as a direct SRSF4 target in cardiomyocytes by individual-nucleotide-resolution cross-linking and immuno-precipitation (iCLIP). GAS5 is a repressor of the glucocorticoid receptor (GR) and was downregulated in SRSF4 KO hearts. This corresponded with elevated GR transcriptional activity in cardiomyocytes, leading to increases in hypertrophy markers and cell size. Furthermore, hypertrophy in SRSF4 KO cardiomyocytes was reduced by overexpressing GAS5. Conclusions: Loss of SRSF4 expression results in cardiac hypertrophy, diastolic dysfunction, and abnormal repolarization. The molecular mechanism underlying this effect involves GAS5 downregulation and consequent elevation of GR transcriptional activity. Our findings may help to develop new therapeutic tools for the treatment of cardiac hypertrophy and myocardial pathology in Cushing's syndrome patients.

scholarly journals Nonclassical nuclear localization signals mediate nuclear import of CIRBP

2020 ◽  
Vol 117 (15) ◽  
pp. 8503-8514 ◽  
Author(s):  
Benjamin Bourgeois ◽  
Saskia Hutten ◽  
Benjamin Gottschalk ◽  
Mario Hofweber ◽  
Gesa Richter ◽  
...  
Keyword(s):  
Phase Separation ◽  
Nuclear Localization ◽  
Nuclear Import ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Specific Interaction ◽  
Stress Granule ◽  
Rich Region ◽  
Nuclear Localization Signals ◽  
Cargo Proteins

The specific interaction of importins with nuclear localization signals (NLSs) of cargo proteins not only mediates nuclear import but also, prevents their aberrant phase separation and stress granule recruitment in the cytoplasm. The importin Transportin-1 (TNPO1) plays a key role in the (patho-)physiology of both processes. Here, we report that both TNPO1 and Transportin-3 (TNPO3) recognize two nonclassical NLSs within the cold-inducible RNA-binding protein (CIRBP). Our biophysical investigations show that TNPO1 recognizes an arginine-glycine(-glycine) (RG/RGG)–rich region, whereas TNPO3 recognizes a region rich in arginine-serine-tyrosine (RSY) residues. These interactions regulate nuclear localization, phase separation, and stress granule recruitment of CIRBP in cells. The presence of both RG/RGG and RSY regions in numerous other RNA-binding proteins suggests that the interaction of TNPO1 and TNPO3 with these nonclassical NLSs may regulate the formation of membraneless organelles and subcellular localization of numerous proteins.

scholarly journals Charged sequence motifs increase affinity towards liquid-liquid phase separation

2021 ◽  
Author(s):  
András L. Szabó ◽  
Anna Sánta ◽  
Zoltán Gáspári
Keyword(s):  
Phase Separation ◽  
Charge Density ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Structural Characteristics ◽  
Protein Sequences ◽  
Sequence Motifs ◽  
High Charge ◽  
Charged Residue ◽  
High Charge Density

AbstractProtein phase separation has been shown to be a major governing factor in multiple cellular processes, especially ones concerning RNA and RNA-binding proteins. Despite many key observations, the exact structural characteristics of proteins involved in the process are still not fully deciphered. In this work we show that proteins harbouring sequences with specific regions of charged residues are significantly associated with phase separation phenomena. In particular, regions with repetitive arrays of alternating charges (termed charged residue repeats, CRRs) show the strongest association, whereas segments with generally high charge density (charge-dense regions, CDRs) and single alpha-helices (SAHs) show also detectable but weaker connections.It is known to contribute to the formation of membrane-less organelles (MLOs) and to an extent the aggregation of proteins. The causes and consequences of phase separation has been a rigorously researched topic in the last few years, as the condensation of specific phase-separating proteins is known to promote several diseases.In this work we carried out a computational analysis to examine the presence of repetitive segments with high charge density in proteins prone to phase separation. Free resources such as the Charged Single α-Helix (CSAH) web server and the PhaSepDB online database were used to examine possible links between the charged side-chain content of protein sequences and their partition into membrane-less condensates. Furthermore, we carried out the development of a novel algorithm aimed to detect a larger variety of charged protein segments, in order to examine their relationship to the phenomenon. Fisher’s exact test of independence was implemented on several generated data sets to confirm correlation between charged residue repeats (CRRs) and charge-dense regions (CDRs) within human protein sequences and their affinity for phase separation.

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 SARS-CoV-2 nucleocapsid protein undergoes liquid-liquid phase separation stimulated by RNA and partitions into phases of human ribonucleoproteins

2020 ◽  
Author(s):  
Theodora Myrto Perdikari ◽  
Anastasia C. Murthy ◽  
Veronica H. Ryan ◽  
Scott Watters ◽  
Mandar T. Naik ◽  
...  
Keyword(s):  
Phase Separation ◽  
Liquid Phase ◽  
Nucleocapsid Protein ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Liquid Phase Separation ◽  
Host Protein ◽  
Host Cells ◽  
Liquid Liquid Phase Separation

AbstractTightly packed complexes of nucleocapsid protein and genomic RNA form the core of viruses and may assemble within viral factories, dynamic compartments formed within the host cells. Here, we examine the possibility that the multivalent RNA-binding nucleocapsid protein (N) from the severe acute respiratory syndrome coronavirus (SARS-CoV-2) compacts RNA via protein-RNA liquid-liquid phase separation (LLPS) and that N interactions with host RNA-binding proteins are mediated by phase separation. To this end, we created a construct expressing recombinant N fused to a N-terminal maltose binding protein tag which helps keep the oligomeric N soluble for purification. Using in vitro phase separation assays, we find that N is assembly-prone and phase separates avidly. Phase separation is modulated by addition of RNA and changes in pH and is disfavored at high concentrations of salt. Furthermore, N enters into in vitro phase separated condensates of full-length human hnRNPs (TDP-43, FUS, and hnRNPA2) and their low complexity domains (LCs). However, N partitioning into the LC of FUS, but not TDP-43 or hnRNPA2, requires cleavage of the solubilizing MBP fusion. Hence, LLPS may be an essential mechanism used for SARS-CoV-2 and other RNA viral genome packing and host protein co-opting, functions necessary for viral replication and hence infectivity.

scholarly journals Phase separation of RNA-binding protein promotes polymerase engagement and transcription

2021 ◽  
Author(s):  
Wen Shao ◽  
Xianju Bi ◽  
Boyang Gao ◽  
Jun Wu ◽  
Yixuan Pan ◽  
...  
Keyword(s):  
Phase Separation ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Embryonic Stem ◽  
Transcription Control ◽  
Pol Ii ◽  
Genome Wide ◽  
Condensate Formation ◽  
Active Transcription ◽  
Biochemical Analyses

An RNA-involved phase-separation model has been proposed for transcription control. Yet, the molecular links that connect RNA binding to the transcription machinery remain missing. Here we find RNA-binding proteins (RBPs) constitute half of the chromatin proteome in embryonic stem cells (ESCs), and some are colocalized with RNA polymerase (Pol) II at promoters and enhancers. Biochemical analyses of representative RBPs--such as PSPC1 and PTBP1--show that the paraspeckle protein PSPC1 not only prevents the RNA-induced premature release of Pol II, and also makes use of RNA as multivalent molecules to promote Pol II engagement and activity, by enhancing the phase separation and subsequent phosphorylation and release of polymerase condensates. In ESCs, auxin-induced acute degradation of PSPC1 leads to genome-wide defects in Pol II phosphorylation and chromatin-binding and nascent transcription. We propose that the synergistic interplay of RBPs and RNA aids in the rate-limiting step of polymerase condensate formation to promote active transcription.

scholarly journals RNA Droplets

2020 ◽  
Vol 49 (1) ◽  
pp. 247-265 ◽  
Author(s):  
Kevin Rhine ◽  
Velinda Vidaurre ◽  
Sua Myong
Keyword(s):  
Phase Separation ◽  
Intrinsically Disordered Proteins ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Disordered Proteins ◽  
In Vivo Studies ◽  
Anionic Polymer ◽  
Temporal And Spatial Distribution ◽  
Intrinsically Disordered ◽  
Rnp Granules

Liquid–liquid phase separation is emerging as the universal mechanism by which membraneless cellular granules form. Despite many previous studies on condensation of intrinsically disordered proteins and low complexity domains, we lack understanding about the role of RNA, which is the essential component of all ribonucleoprotein (RNP) granules. RNA, as an anionic polymer, is inherently an excellent platform for achieving multivalency and can accommodate many RNA binding proteins. Recent findings have highlighted the diverse function of RNA in tuning phase-separation propensity up or down, altering viscoelastic properties and thereby driving immiscibility between different condensates. In addition to contributing to the biophysical properties of droplets, RNA is a functionally critical constituent that defines the identity of cellular condensates and controls the temporal and spatial distribution of specific RNP granules. In this review, we summarize what we have learned so far about such roles of RNA in the context of in vitro and in vivo studies.

scholarly journals The Sam68 nuclear body is composed of two RNase-sensitive substructures joined by the adaptor HNRNPL

2016 ◽  
Vol 214 (1) ◽  
pp. 45-59 ◽  
Author(s):  
Taro Mannen ◽  
Seisuke Yamashita ◽  
Kozo Tomita ◽  
Naoki Goshima ◽  
Tetsuro Hirose
Keyword(s):  
Cell Nucleus ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Nuclear Body ◽  
Nuclear Bodies ◽  
Cdna Clones ◽  
Rna Recognition ◽  
Rna Recognition Motifs ◽  
Rnase Treatment ◽  
Recognition Motifs

The mammalian cell nucleus contains membraneless suborganelles referred to as nuclear bodies (NBs). Some NBs are formed with an architectural RNA (arcRNA) as the structural core. Here, we searched for new NBs that are built on unidentified arcRNAs by screening for ribonuclease (RNase)-sensitive NBs using 32,651 fluorescently tagged human cDNA clones. We identified 32 tagged proteins that required RNA for their localization in distinct nuclear foci. Among them, seven RNA-binding proteins commonly localized in the Sam68 nuclear body (SNB), which was disrupted by RNase treatment. Knockdown of each SNB protein revealed that SNBs are composed of two distinct RNase-sensitive substructures. One substructure is present as a distinct NB, termed the DBC1 body, in certain conditions, and the more dynamic substructure including Sam68 joins to form the intact SNB. HNRNPL acts as the adaptor to combine the two substructures and form the intact SNB through the interaction of two sets of RNA recognition motifs with the putative arcRNAs in the respective substructures.

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