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Paraspeckles interact with SWI/SNF subunit ARID1B to regulate transcription and splicing.

ABSTRACT: Paraspeckles are subnuclear RNA-protein structures that are implicated in important processes including cellular stress response, differentiation, and cancer progression. However, it is unclear how paraspeckles impart their physiological effect at the molecular level. Through biochemical analyses, we show that paraspeckles interact with the SWI/SNF chromatin-remodeling complex. This is specifically mediated by the direct interaction of the long-non-coding RNA NEAT1 of the paraspeckles with ARID1B of the cBAF-type SWI/SNF complex. Strikingly, ARID1B depletion, in addition to resulting in loss of interaction with the SWI/SNF complex, decreases the binding of paraspeckle proteins to chromatin modifiers, transcription factors, and histones. Functionally, the loss of ARID1B and NEAT1 influences the transcription and the alternative splicing of a common set of genes. Our findings reveal that dynamic granules such as the paraspeckles may leverage the specificity of epigenetic modifiers to impart their regulatory effect, thus providing a molecular basis for their function.

SUBMITTER: Reddy D

PROVIDER: S-EPMC9827562 | biostudies-literature | 2023 Jan

REPOSITORIES: biostudies-literature

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Paraspeckles interact with SWI/SNF subunit ARID1B to regulate transcription and splicing.

Reddy Divya D Bhattacharya Saikat S Levy Michaella M Zhang Ying Y Gogol Madelaine M Li Hua H Florens Laurence L Workman Jerry L JL

EMBO reports 20221110 1

Paraspeckles are subnuclear RNA-protein structures that are implicated in important processes including cellular stress response, differentiation, and cancer progression. However, it is unclear how paraspeckles impart their physiological effect at the molecular level. Through biochemical analyses, we show that paraspeckles interact with the SWI/SNF chromatin-remodeling complex. This is specifically mediated by the direct interaction of the long-non-coding RNA NEAT1 of the paraspeckles with ARID1 ...[more]

PMID: 36354291

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Project description:Protein samples were analyzed by Multidimensional Protein Identification Technology (MudPIT), as described previously. Briefly, precipitated proteins were resuspended in 30uL of 100mM Tris pH 8.5 with 8M urea to denature proteins. Cysteines were reduced and alkylated prior to digestion with recombinant LysC and modified trypsin. Reactions were quenched by the addition of formic acid to the final concentration of 5%. After digestion, peptide samples were pressure-loaded onto 100 um fused silica microcapillary columns packed first with 9 cm of reverse phase material, followed by 3 cm of 5um Strong Cation Exchange material, followed by 1 cm of 5um C18 RP. The loaded microcapillary columns were placed in-line with a 1260 Quartenary HPLC. The application of a 2.5 kV distal voltage electrosprayed the eluting peptides directly into LTQ linear ion trap mass spectrometers equipped with a custom-made nano-LC electrospray ionization source. Full MS spectra were recorded on the eluting peptides over a 400 to 1600 m/z range, followed by fragmentation in the ion trap on the first to fifth most intense ions selected from the full MS spectrum. Dynamic exclusion was enabled for 120 s. Mass spectrometer scan functions and HPLC solvent gradients were controlled by the XCalibur data system. RAW files were extracted into .ms2 file format using RawDistiller v. 1.0, in-house developed software. RawDistiller D(g, 6) settings were used to abstract MS1 scan profiles by Gaussian fitting and to implement dynamic offline lock mass using six background polydimethylcyclosiloxane ions as internal calibrants. MS/MS spectra were first searched using ProLuCID with a mass tolerance of 500 ppm for peptide and fragment ions. Trypsin specificity was imposed on both ends of candidate peptides during the search against a protein database containing 44, 080 human proteins (NCBI 2019-11-03 release), as well as 426 common contaminants such as human keratins, IgGs and proteolytic enzymes. To estimate false discovery rates (FDR), each protein sequence was randomized (keeping the same amino acid composition and length) and the resulting "shuffled" sequences were added to the database, for a total search space of 89, 038 amino acid sequences. Masses of 57 Da was differentially added to cysteine residues to account for alkylation by CAM and 16 Da were differentially added to methionine residues. DTASelect v.1.9 was used to select and sort peptide/spectrum matches (PSMs) passing the following criteria set: PSMs were only retained if they had a DeltCn of at least 0.08; minimum XCorr values of 1.8 for singly-, 2.1 for doubly-, and 2.5 for triply-charged spectra; peptides had to be at least 7 amino acids long. Results from each sample were merged and compared using CONTRAST. Combining all replicate runs, proteins had to be detected by at least 2 peptides and/or 2 spectral counts. Proteins that were subsets of others were removed using the parsimony option in DTASelect on the proteins detected after merging all runs. Proteins that were identified by the same set of peptides (including at least one peptide unique to such protein group to distinguish between isoforms) were grouped together, and one accession number was arbitrarily considered as representative of each protein group. NSAF7 was used to create the final reports on all detected peptides and non-redundant proteins identified across the different run

Dataset Information

Paraspeckles interact with SWI/SNF subunit ARID1B to regulate transcription and splicing.

Publications

Paraspeckles interact with SWI/SNF subunit ARID1B to regulate transcription and splicing.

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