Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

Dataset Information

ICLIP of Tra2b, Son-GFP and Srrm2-GFP in mESCs

ABSTRACT: In order to investigate the role of SR protein assemblies on GA-multivalent mRNA regions and their relationship to the nuclear speckle. We performed iCLIP of Tra2b, Son-GFP and Srrm2-GFP in mESCs. The GFP-tagged proteins are endogenously tagged and immunoprecipitated using an anti-GFP antibody, while Tra2b was immunoprecipitated using an antibody against the endogenous protein. Srrm2 and Son are very large proteins, but signal was observed at a variety of molecular weights along the membrane, suggesting indirect interactions. We isolated two fractions for each Srrm2 or Son sample: one 'high MW', cut at ~300kDa upwards, and one 'low MW' cut from 40 kDa to 300 kDa. These samples were prepared and sequenced separately. The data provided here has been demultiplexed and trimmed, and therefore should not contain adapter sequences or barcodes. UMI sequences have been moved to the fastq header. Also included are transcriptomic coordinates of deduplicated crosslinks for each sample, using one transcript per gene, as used to plot transcriptomic metaprofiles around multivalent sites.

INSTRUMENT(S): Illumina HiSeq 4000

ORGANISM(S): Mus musculus

SUBMITTER: Jernej Ule

PROVIDER: E-MTAB-13331 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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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.

Dataset Information

ICLIP of Tra2b, Son-GFP and Srrm2-GFP in mESCs

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