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PXD021814 is an original dataset announced via ProteomeXchange.

Dataset Summary
TitleSON and SRRM2 form nuclear speckles in human cells
DescriptionThe 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.
ReviewLevelPeer-reviewed dataset
DatasetOriginOriginal dataset
RepositorySupportUnsupported dataset by repository
PrimarySubmitterDavid Meierhofer
SpeciesList scientific name: Homo sapiens (Human); NCBI TaxID: 9606;
ModificationListiodoacetamide derivatized residue
InstrumentQ Exactive HF
Dataset History
RevisionDatetimeStatusChangeLog Entry
02020-10-05 03:50:05ID requested
12020-10-26 01:09:52announced
Publication List
Ilik İ A, Malszycki M, L, ü, bke AK, Schade C, Meierhofer D, Akta, ş T, SON and SRRM2 are essential for nuclear speckle formation. Elife, 9():(2020) [pubmed]
Keyword List
submitter keyword: SRRM2
nuclear speckles
Contact List
Tugce, Aktas
contact affiliationMax Planck Research Group Leader, Quantitative RNA Biology OWL, MPIMG Berlin, Germany
contact emailaktas@molgen.mpg.de
lab head
David Meierhofer
contact affiliationMass Spectrometry Facility MPIMG
contact emailmeierhof@molgen.mpg.de
dataset submitter
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