Dataset Information

Profile the composition of reconstituted nuclear speckles

ABSTRACT: Nuclear extract from Hela Cells are incubated with recombinant SON IDR2 protein, resulting condensates are spun down and the compostion profiled by mass spectrometry.

INSTRUMENT(S):

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Permanent Cell Line Cell, Cell Culture

DISEASE(S): Cervix Carcinoma

SUBMITTER: Bokai Zhu

LAB HEAD: Bokai Zhu

PROVIDER: PXD050371 | Pride | 2024-11-25

REPOSITORIES: Pride

ACCESS DATA

Dataset's files

Source:

			Action	DRS
	Nuclear_extract_input_rep1.pepXML	Pepxml
	Nuclear_extract_input_rep1.raw	Raw
	Nuclear_extract_input_rep2.pepXML	Pepxml
	Nuclear_extract_input_rep2.raw	Raw
	SON_IDR2_seed-rep1.pepXML	Pepxml

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Publications

SON-dependent nuclear speckle rehabilitation alleviates proteinopathies.

Dion William W Tao Yuren Y Chambers Maci M Zhao Shanshan S Arbuckle Riley K RK Sun Michelle M Kubra Syeda S Schaich Matthew A MA Nie Yuhang Y Ye Megan M Jamal Imran I Larsen Mads B MB Camarco Daniel D Ickes Eleanor E Wang Haokun H HH DuPont C C Wang Bingjie B Liu Silvia S Pi Shaohua S Van Houten Bennet B Chen Bill B BB Chen Yuanyuan Y Chen Xu X Zhu Bokai B

bioRxiv : the preprint server for biology 20250428

Current treatments targeting individual protein quality control pathways have limited efficacy in alleviating proteinopathies, highlighting the prerequisite for a common druggable target capable of global proteostasis modulation. Building upon our prior research establishing nuclear speckles as pivotal membrane-less organelles for transcriptional control of proteostasis, we aim to alleviate proteinopathies through nuclear speckle rehabilitation. We identified pyrvinium pamoate as a nuclear speck ...[more]

PMID: 38659924

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Project description:The nucleus of higher eukaryotes is a highly compartmentalized and dynamic organelle consisting of several biological condensates that regulate gene expression at multiple levels. First reported more than 100 years ago by Ramón y Cajal, nuclear speckles (NS) are among the most prominent of such condensates, which were independently rediscovered multiple times and are also known as interchromatin granule clusters (ICGs), splicing speckles, or SC35 domains. Despite their prevalence, research on the function of NS is virtually restricted to colocalization analyses, since an organizing core, without which NS cannot form, remains unidentiﬁed. The monoclonal antibody SC35, which was raised against a spliceosomal extract, is a frequently used reagent to mark NS since its debut in 1990. Despite its proliﬁc use, and the consensus regarding its primary target as SRSF2, clear inconsistencies between observations made with mAb SC35, and transgenic SRSF2 constructs in the same experiment are noted. Intrigued by these disparities, we carried out a systematic re-characterization of this monoclonal antibody and its cellular targets. Unexpectedly, we found no evidence for SC35 mAb recognizing SRSF2 in human cells. In contrast, our results show that the 35 kDa namesake protein recognized by SC35 mAb is SRSF7, a related but distinct SR protein. More importantly, using mass-spectrometry, CRISPR-mediated knock-ins, immunoblotting and ﬂuorescence microscopy, we show that the main target of SC35 mAb is SRRM2, a large (300 kDa), spliceosome-associated protein with prominent intrinsically disordered regions (IDRs) that sharply localizes to NS. Analysis of protein length evolution among metazoa revealed a peculiar IDR extension speciﬁcally for SRRM2 and SON in vertebrates, a hallmark of condensate formation. Combining these results, we tested a long-standing question in the study of NS: whether NS are formed around a speciﬁc core, or various SR-proteins self-assemble into a phase-separated compartment without the need for a deﬁned core. Here we show that, the elusive core of NS is formed by SON and SRRM2, since depletion of SON leads only to a partial disassembly of NS, while combined depletion of SON together with SRRM2, but not other NS associated factors, or depletion of SON in a cell line where IDRs of SRRM2 are genetically deleted, leads to a near-complete dissolution of NS. This work, therefore, paves the way to study the role of NS under diverse physiological and stress conditions.