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Arginine dimethylation and liquid-liquid phase separation_Supplementary

ABSTRACT: Recently, arginine methylation was found to regulate the protein LLPS which drives the formation of MLOs. Here, we developed a chemical enrichment method coupled with LC-MS/MS to analyze dynamic change of arginine di-methylation upon SG formation.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Qi Wang

LAB HEAD: Mingliang Ye

PROVIDER: PXD035137 | Pride | 2022-10-20

REPOSITORIES: Pride

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			Action	DRS
	LZX_QE_hmJurkat_trypsin_aDMA_antibody_rep1.raw	Raw
	LZX_QE_hmJurkat_trypsin_aDMA_antibody_rep2.raw	Raw
	LZX_QE_hmJurkat_trypsin_aDMA_antibody_rep3.raw	Raw
	LZX_QE_hmJurkat_trypsin_sDMA_antibody_rep1.raw	Raw
	LZX_QE_hmJurkat_trypsin_sDMA_antibody_rep2.raw	Raw

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Arginine dimethylation and liquid-liquid phase separation

Project description:Recently, arginine methylation was found to regulate the protein LLPS which drives the formation of MLOs. Here, we developed a chemical enrichment method coupled with LC-MS/MS to analyze dynamic change of arginine di-methylation upon SG formation.

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Protein methylation analysis of Jurkat cells

Project description:Protein methylation, especially in arginine and lysine, is one of most important post-translational modifications (PTMs). In this project, we developed a novel chemical strategy which enabled almost complete depletion of histidine-conatining peptides in protein digestion and thereby resulted in the identification of more low-abundance arginine/lysine methylpeptides.

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First direct observation of elongated conformational states in alpha-synuclein upon liquid-liquid phase separation

Project description:α-Synuclein (α-syn) is an intrinsically disordered protein (IDP) that undergoes liquid-liquid phase separation (LLPS), fibrillation, and forms insoluble intracellular Lewy’s bodies in neurons, which are the hallmark of Parkinson’s Disease (PD). Neurotoxicity precedes the formation of aggregates and is probably related to LLPS of α-syn in the cell. The molecular mechanisms underlying the early stages of LLPS are still elusive. To obtain structural insights into α-syn upon LLPS, we take advantage of cross-linking/mass spectrometry (XL-MS) and introduced an innovative new approach, termed COMPASS (COMPetitive PAiring StatisticS). COMPASS unravels transient interactions between α-syn molecules in liquid droplets to derive structural information of α-syn upon LLPS. In this work, we show that the conformational ensemble of α-syn shifts towards more elongated conformational states upon LLPS. We obtain insights into the critical initial stages of PD and establish a novel mass spectrometry-based approach that will aid to solve open questions in LLPS structural biology.

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The phase separation-dependent FUS interactome reveals nuclear and cytoplasmic function of liquid-liquid phase separation

Project description:Liquid-liquid phase separation (LLPS) of proteins and RNAs has emerged as the driving force underlying the formation of membrane-less organelles. Such biomolecular condensates have various biological functions and have been linked to disease. One of the best studied proteins undergoing LLPS is Fused in Sarcoma (FUS), a predominantly nuclear RNA-binding protein. Mutations in FUS have been causally linked to Amyotrophic Lateral Sclerosis (ALS), an adult-onset motor neuron disease, and LLPS followed by aggregation of cytoplasmic FUS has been proposed to be a crucial disease mechanism. In spite of this, it is currently unclear how LLPS impacts the behaviour of FUS in cells, e.g. its interactome. In order to study the consequences of LLPS on FUS and its interaction partners, we developed a method that allows for the purification of phase separated FUS-containing droplets from cell lysates. We observe substantial alterations in the interactome of FUS, depending on its biophysical state. While non-phase separated FUS interacts mainly with its well-known interaction partners involved in pre-mRNA processing, phase-separated FUS predominantly binds to proteins involved in chromatin remodelling and DNA damage repair. Interestingly, factors with function in mitochondria are strongly enriched with phase-separated FUS, providing a potential explanation for early changes in mitochondrial gene expression observed in mouse models of ALS-FUS. In summary, we present a methodology that allows to investigate the interactome of phase-separating proteins and provide evidence that LLPS strongly shapes the FUS interactome with important implications for function and disease.

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Suppression of liquid-liquid phase separation by 1,6-hexanediol partially compromises the 3D genome organization in living cells

Project description:Liquid-liquid phase separation (LLPS) contributes to the spatial and functional segregation of molecular processes within the cell nucleus. However, the role played by LLPS in chromatin folding in living cells remains unclear. Here, using stochastic optical reconstruction microscopy (STORM) and Hi-C techniques, we studied the effects of 1,6-hexanediol (1,6-HD)- mediated LLPS disruption/modulation on higher-order chromatin organization in living cells. We found that 1,6-HD treatment caused the enlargement of nucleosome clutches and their more uniform distribution in the nuclear space. At a megabase-scale, chromatin underwent moderate but irreversible perturbations that resulted in the partial mixing of A and B compartments. The removal of 1,6-HD from the culture medium did not allow chromatin to acquire initial configurations, and resulted in more compact repressed chromatin than in untreated cells. 1,6-HD treatment also weakened enhancer-promoter interactions and TAD insulation but did not considerably affect CTCF-dependent loops. Our results suggest that 1,6-HD-sensitive LLPS plays a limited role in chromatin spatial organization by constraining its folding patterns and facilitating compartmentalization at different levels.

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Project description:Previous experiments have demonstrated that c-Maf can undergo liquid-liquid phase separation (LLPS) in MM. In order to search for the downstream target genes of c-Maf based on its LLPS regulation, we successfully constructed three kinds of cells with EV, c-Maf and already c-Maf-IDR mutation, and simultaneously subjected the three kinds of cells to ChIP sequencing (selecting the FLAG-tagged antibody), and then analysed them for the differential genes, thus screening the downstream targets of c-Maf based on its LLPS regulation.

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Arginine Methylation Regulates SARS-CoV-2 Nucleocapsid Protein Function and Viral Replication

Project description:Viral proteins are known to be methylated by host protein arginine methyltransferases (PRMTs) playing critical roles during viral infections. Herein, we show that PRMT1 methylates SARS-CoV-2 nucleocapsid (N) protein at residues R95 and R177 within RGG/RG sequences. Arginine methylation of N protein was confirmed by immunoblotting viral proteins extracted from SARS-CoV-2 virions isolated by cell culture. We demonstrate that arginine methylation of N protein is required for its RNA binding capacity, since treatment with a type I PRMT inhibitor (MS023) or substitution of R95K or R177K inhibited interaction with the 5’-UTR of the SARS-CoV-2 genomic RNA. We defined the N interactome in HEK293 cells with or without MS023 treatment and identified PRMT1 and many of its RGG/RG substrates including the known interactor, G3BP1, and other components of stress granules (SG). Methylation of N protein at R95 regulates another function namely its property to suppress the formation of SGs. MS023 treatment or R95K substitution blocked N-mediated suppression of SGs. Also, the co-expression of methylarginine reader TDRD3 quenched N-mediated suppression of SGs in a dose-dependent manner. Finally, pre-treatment of VeroE6 cells with MS023 significantly reduced SARS-CoV-2 replication. With type I PRMT inhibitors being in clinical trials for cancer treatment, inhibiting arginine methylation to target the later stages of the viral life cycle such as viral genome packaging and assembly of virions may be an additional therapeutic application of these drugs.

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CPSF role on APA regulation

Project description:We found that CPSF6, a component of the CFIm, can form liquid-liquid phase separation (LLPS) and the elevated LLPS induces the preferential usage of the distal poly(A) sites. CLK2, a kinase upregulated in cancer cells, destructs LLPS of CPSF6 by phosphorylating its arginine/serine-like domain. Albeit higher expression of CPSF6 in cancer, the reduction of LLPS leads to shortening 3’ UTR of cell cycle related genes and then promotes cell proliferation. These results reveal that LLPS regulation of CPSF6 is a fine tuning way of APA in cancer cells and provide a new mechanism for APA regulation by regulating LLPS of 3’ end processing factors through post-translational modification.

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