Tyramide signal amplification mass spectrometry (TSA-MS) ratio identifies nuclear speckle proteins

We present a simple ratio method to infer protein composition within cellular structures using proximity labeling approaches but compensating for the diffusion of free radicals. We used tyramide signal amplification (TSA) and label-free mass spectrometry (MS) to compare proteins in nuclear speckles versus centromeres. Our “TSA-MS ratio” approach successfully identified known nuclear speckle proteins. For example, 96% and 67% of proteins in the top 30 and 100 sorted proteins, respectively, are known nuclear speckle proteins, including proteins that we validated here as enriched in nuclear speckles. We show that MFAP1, among the top 20 in our list, forms droplets under certain circumstances and that MFAP1 expression levels modulate the size, stability, and dynamics of nuclear speckles. Localization of MFAP1 and its binding partner, PRPF38A, in droplet-like nuclear bodies precedes formation of nuclear speckles during telophase. Our results update older proteomic studies of nuclear speckles and should provide a useful reference dataset to guide future experimental dissection of nuclear speckle structure and function.

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Revealing RCOR2 as a regulatory component of nuclear speckles

10.21203/rs.3.rs-978769/v1 ◽

2021 ◽

Author(s):

Carlos Rivera ◽

Daniel Verbel ◽

Duxan Arancibia ◽

Anna Lappala ◽

Marcela González ◽

...

Keyword(s):

System Development ◽

Cell Types ◽

Nervous System Development ◽

Nuclear Bodies ◽

Nuclear Speckles ◽

Nuclear Speckle ◽

Multiple Cell ◽

Rna Maturation ◽

Nuclear Processes ◽

The Brain

Abstract Background Nuclear processes such as transcription and RNA maturation can be impacted by subnuclear compartmentalization in condensates and nuclear bodies. Here we characterize the nature of nuclear granules formed by REST corepressor 2 (RCOR2), a nuclear protein essential for pluripotency maintenance and central nervous system development. Results Using biochemical approaches and high-resolution microscopy, we reveal that RCOR2 is localized in nuclear speckles across multiple cell types, including neurons in the brain. RCOR2 forms complexes with nuclear speckle components such as SON, SRSF7, and SRRM2. When cells are exposed to chemical stress, RCOR2 behaves as a core component of the nuclear speckle and is stabilized by RNA. In turn, nuclear speckle morphology appears to depend on RCOR2. Specifically, RCOR2 knockdown results larger nuclear speckles, whereas overexpressing RCOR2 leads to smaller and rounder nuclear speckles. Conclusion Our study suggests that RCOR2 is a regulatory component of the nuclear speckle bodies, setting this co-repressor protein as a factor that controls nuclear speckles behavior.

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Revealing RCOR2 as a regulatory component of nuclear speckles

Epigenetics & Chromatin ◽

10.1186/s13072-021-00425-4 ◽

2021 ◽

Vol 14 (1) ◽

Author(s):

Carlos Rivera ◽

Daniel Verbel-Vergara ◽

Duxan Arancibia ◽

Anna Lappala ◽

Marcela González ◽

...

Keyword(s):

System Development ◽

Cell Types ◽

Nervous System Development ◽

Nuclear Bodies ◽

Nuclear Speckles ◽

Nuclear Speckle ◽

Multiple Cell ◽

Rna Maturation ◽

Nuclear Processes ◽

The Brain

Abstract Background Nuclear processes such as transcription and RNA maturation can be impacted by subnuclear compartmentalization in condensates and nuclear bodies. Here, we characterize the nature of nuclear granules formed by REST corepressor 2 (RCOR2), a nuclear protein essential for pluripotency maintenance and central nervous system development. Results Using biochemical approaches and high-resolution microscopy, we reveal that RCOR2 is localized in nuclear speckles across multiple cell types, including neurons in the brain. RCOR2 forms complexes with nuclear speckle components such as SON, SRSF7, and SRRM2. When cells are exposed to chemical stress, RCOR2 behaves as a core component of the nuclear speckle and is stabilized by RNA. In turn, nuclear speckle morphology appears to depend on RCOR2. Specifically, RCOR2 knockdown results larger nuclear speckles, whereas overexpressing RCOR2 leads to smaller and rounder nuclear speckles. Conclusion Our study suggests that RCOR2 is a regulatory component of the nuclear speckle bodies, setting this co-repressor protein as a factor that controls nuclear speckles behavior.

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Limited Proteolysis-Coupled Mass Spectrometry Identifies Phosphatidylinositol 4,5-Bisphosphate Effectors in Human Nuclear Proteome

Cells ◽

10.3390/cells10010068 ◽

2021 ◽

Vol 10 (1) ◽

pp. 68

Author(s):

Martin Sztacho ◽

Barbora Šalovská ◽

Jakub Červenka ◽

Can Balaban ◽

Peter Hoboth ◽

...

Keyword(s):

Gene Expression ◽

Mass Spectrometry ◽

Cell Cycle ◽

Rna Splicing ◽

Binding Capacity ◽

Limited Proteolysis ◽

Bioinformatic Analysis ◽

Nuclear Pore ◽

Label Free ◽

Nuclear Speckles

Specific nuclear sub-compartments that are regions of fundamental processes such as gene expression or DNA repair, contain phosphoinositides (PIPs). PIPs thus potentially represent signals for the localization of specific proteins into different nuclear functional domains. We performed limited proteolysis followed by label-free quantitative mass spectrometry and identified nuclear protein effectors of the most abundant PIP—phosphatidylinositol 4,5-bisphosphate (PIP2). We identified 515 proteins with PIP2-binding capacity of which 191 ‘exposed’ proteins represent a direct PIP2 interactors and 324 ‘hidden’ proteins, where PIP2 binding was increased upon trypsin treatment. Gene ontology analysis revealed that ‘exposed’ proteins are involved in the gene expression as regulators of Pol II, mRNA splicing, and cell cycle. They localize mainly to non-membrane bound organelles—nuclear speckles and nucleolus and are connected to the actin nucleoskeleton. ‘Hidden’ proteins are linked to the gene expression, RNA splicing and transport, cell cycle regulation, and response to heat or viral infection. These proteins localize to the nuclear envelope, nuclear pore complex, or chromatin. Bioinformatic analysis of peptides bound in both groups revealed that PIP2-binding motifs are in general hydrophilic. Our data provide an insight into the molecular mechanism of nuclear PIP2 protein interaction and advance the methodology applicable for further studies of PIPs or other protein ligands.

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Hsp70 gene association with nuclear speckles is Hsp70 promoter specific

The Journal of Cell Biology ◽

10.1083/jcb.201004041 ◽

2010 ◽

Vol 191 (4) ◽

pp. 711-719 ◽

Cited By ~ 30

Author(s):

Yan Hu ◽

Matt Plutz ◽

Andrew S. Belmont

Keyword(s):

Heat Shock ◽

Integration Site ◽

Promoter Sequence ◽

Artificial Chromosome ◽

Nuclear Bodies ◽

Nuclear Speckles ◽

Transcript Accumulation ◽

Nuclear Speckle ◽

Hsp70 Promoter ◽

Acid Processing

Many mammalian genes localize near nuclear speckles, nuclear bodies enriched in ribonucleic acid–processing factors. In this paper, we dissect cis-elements required for nuclear speckle association of the heat shock protein 70 (Hsp70) locus. We show that speckle association is a general property of Hsp70 bacterial artificial chromosome transgenes, independent of the chromosome integration site, and can be recapitulated using a 2.8-kilobase HSPA1A gene fragment. Association of Hsp70 transgenes and their transcripts with nuclear speckles is transcription dependent, independent of the transcribed sequence identity, but dependent on the Hsp70 promoter sequence. Transgene speckle association does not correlate with the amount of transcript accumulation, with large transgene arrays driven by different promoters showing no speckle association, but smaller Hsp70 transgene arrays with lower transcript accumulation showing high speckle association. Moreover, despite similar levels of transcript accumulation, Hsp70 transgene speckle association is observed after heat shock but not cadmium treatment. We suggest that certain promoters may direct specific chromatin and/or transcript ribonucleoprotein modifications, leading to nuclear speckle association.

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Label-free microRNA detection through analyzing the length distribution pattern of the residual fragments of probe DNA produced during exonuclease III assisted signal amplification by mass spectrometry

Talanta ◽

10.1016/j.talanta.2021.122414 ◽

2021 ◽

Vol 231 ◽

pp. 122414

Author(s):

Cai-Xia Shi ◽

Yan-Zi Li ◽

Qing Liu ◽

Zeng-Ping Chen ◽

Sha-Sha Li ◽

...

Keyword(s):

Mass Spectrometry ◽

Distribution Pattern ◽

Signal Amplification ◽

Length Distribution ◽

Label Free ◽

Exonuclease Iii ◽

Microrna Detection ◽

Residual Fragments

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Label Free Quantitative Mass Spectrometry Identifies Processes Linked to Platelet Degranulation As Early Events during Platelet Storage

Blood ◽

10.1182/blood.v128.22.2638.2638 ◽

2016 ◽

Vol 128 (22) ◽

pp. 2638-2638 ◽

Cited By ~ 1

Author(s):

Maaike Rijkers ◽

Floris P. van Alphen ◽

Pieter F. van der Meer ◽

Dirk de Korte ◽

Frank W.G. Leebeek ◽

...

Keyword(s):

Mass Spectrometry ◽

Board Of Directors ◽

Protein Composition ◽

Enrichment Analysis ◽

Prolonged Storage ◽

Biological Processes ◽

Label Free ◽

Quantitative Mass Spectrometry ◽

Advisory Committees ◽

Platelet Storage

Abstract Background Platelet concentrates (PCs) are stored at room temperature to preserve their biological activity. To minimize the risk of bacterial outgrowth, storage time is limited to 7 days. It is well-established that prolonged storage of PCs results in modifications that result in a decreased hemostatic efficacy. This loss of platelet functionality during storage is commonly referred to as the platelet storage lesion (PSL). Typical events linked to development of the PSL are shape changes, platelet activation and loss of receptors crucial for platelet functionality. Two-dimensional (2D) differential gel electrophoresis (DIGE), isotope tagging and isotope-coded affinity tagging (ICAT) have been previously used to monitor changes in protein composition during storage. These studies have provided valuable insights into the changes associated with the PSL, however, these studies generally focused on a limited set of proteins. Aim We aimed to generate an overview of changes in the platelet proteome during storage using label free quantitative mass spectrometry. Furthermore, we employed Gene ontology (GO) enrichment analysis to identify pathways and biological processes that were linked to development of the PSL. Methods Three independently pooled PCs were stored in plasma under standard blood bank conditions for 16 days. Tryptic peptides were separated by nanoscale C18 reverse phase chromatography coupled on line to an Orbitrap Fusion Tribrid mass spectrometer. The RAW mass spectrometry files were processed with the MaxQuant computational platform. The global changes in protein level during platelet storage were assessed employing the analysis-of-variance functions of PERSEUS. Gene ontology enrichment analysis of biological processes, molecular functions and cellular compartments of the significantly different proteins was performed using the Cytoscape plug-in BiNGO. Results A total number of 2501 proteins was detected in all 3 biological replicates in at least one of the time points analyzed. The analysis showed that 18 proteins were down-regulated over time, whereas the level of 3 proteins was found to increase. CytoScape BinGo analysis of these significantly downregulated proteins revealed that the majority of this set was linked to GO-terms platelet degranulation, secretion and regulated exocytosis. This set of proteins included von Willebrand factor (VWF), serglycin (SRGN), SPARC, amyloid beta A4 protein (APP), multimerin-1 (MMRN1) and platelet factor 4 (PF4). A significant decline in these protein levels was observed at day 5 of storage, suggesting that release of α-granules is a relatively early event during platelet storage. At day 5 also a marked decline in S100A9 was observed. S100A9 has been implicated in degranulation in neutrophils, and may therefore also be linked to platelet granule release. Levels of membrane surface platelet glycoproteins such as glycoprotein Ibα did not significantly change at day 5. Only one single protein, histone H2A, was found to be consistently decreased already at two days of storage, but the significance of this finding is not clear. Upon prolonged storage (13 and 16 days) an increase in the level of α-2-macroglobulin (A2M), immunoglobulin M (IGM) and glycogenin-1 (GYG1) was observed suggesting that platelets acquire an (increased) potential to bind and/or internalize proteins from their environment. Consistent with this notion we also detected significant levels of several serine protease inhibitors, although levels of these proteins did not change upon storage. Conclusions Overall, our findings highlight dynamic changes in protein composition of platelets during storage. Our data provide evidence for sustained release of α-granules over time which becomes significant at day 5. Our data also suggest that during storage, platelets can bind or ingest proteins from their environment which may have impact on the hemostatic properties of stored platelets. Disclosures Leebeek: CSL Behring: Membership on an entity's Board of Directors or advisory committees, Research Funding; Baxalta: Consultancy, Membership on an entity's Board of Directors or advisory committees; Dutch Hemphilia Foundation: Research Funding.

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A novel nuclear speckle factor, USP42, promotes homologous recombination repair by resolving DNA double-strand break induced R-loop

10.1101/776823 ◽

2019 ◽

Author(s):

Misaki Matsui ◽

Ryo Sakasai ◽

Masako Abe ◽

Yusuke Kimura ◽

Shoki Kajita ◽

...

Keyword(s):

Homologous Recombination ◽

Strand Break ◽

Double Strand Break ◽

Nuclear Bodies ◽

Nuclear Speckles ◽

Dsb Repair ◽

Nuclear Speckle ◽

Spatial Regulation ◽

Intrinsically Disordered ◽

Dna Double Strand Break

AbstractThe nucleus of mammalian cells is highly compartmentalized by nuclear bodies, including nuclear speckles. While nuclear bodies are known to function in regulating gene expression, their involvement in DNA repair has not been actively investigated. Here, our focused screen for nuclear speckle factors involved in homologous recombination (HR), which is a faithful DNA double-strand break (DSB) repair mechanism, revealed that nuclear speckle factors regulating transcription are potentially involved in the regulation of HR. Among the top hits, we provide evidence showing that USP42, which is a deubiquitylating enzyme and a hitherto unidentified nuclear speckles factor, promotes HR by facilitating BRCA1 recruitment to DSB sites and DNA-end resection. We further showed that USP42 localizes to nuclear speckles via an intrinsically disordered region, which is required for efficient HR. Furthermore, we established that USP42 interacts with DHX9, which possesses DNA-RNA helicase activity, and is required for efficient resolution of DSB-induced R-loop. Mechanistically, USP42 antagonizes mono-ubiquitylation of DHX9 that is evoked after DSB induction. In conclusion, our data propose a model in which a novel nuclear speckle factor, USP42, facilitates DSB-induced R-loop resolution, BRCA1 loading to DSB sites and preferential DSB repair by HR, indicating the importance of spatial regulation of DSB repair choice mediated by nuclear bodies.Significant statementDefects in the repair of DNA double-strand break (DSB), which is one of the most harmful DNA insults, cause human diseases including cancers. It has been suggested that DSBs generated in the coding region tend to be repaired by homologous recombination (HR) that is error-free DSB repair pathway. To reveal the spatial regulation of HR, in this study, we investigated the potential contribution of nuclear bodies, especially nuclear speckles, to HR, identifying a deubiquitylating enzyme USP42 as a HR promoting factor. We found that USP42 deubiquitylates DHX9, facilitates resolution of DNA-RNA hybrid structure and enhances HR through BRCA1 loading to DSB sites.ClassificationBiological Sciences, Cell Biology

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