Ilik, I. A. | Search bioRxiv

bioRxiv2020

SON and SRRM2 form nuclear speckles in human cells

The nucleus of higher eukaryotes is a highly compartmentalized and dynamic organelle consisting of several biomolecular condensates that regulate gene expression at multiple levels (1, 2). First reported more than 100 years ago by Ramon y Cajal, nuclear speckles (NS) are among the most prominent of such condensates (3). 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 (4, 5). The monoclonal antibody SC35, which was raised against a spliceosomal extract, is a frequently used reagent to mark NS since its debut in 1990 (6). Unexpectedly, we found that this antibody has been misidentified and the main target of SC35 mAb is SRRM2, a large ([~]300 kDa), spliceosomeassociated (7) protein with prominent intrinsically disordered regions (IDRs) that sharply localizes to NS (8). 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.

molecular biology↗

bioRxiv2017

uvCLAP: a fast, non-radioactive method to identify in vivo targets of RNA-binding proteins

RNA-binding proteins (RBPs) play important and essential roles in eukaryotic gene expression regulating splicing, localization, translation and stability of mRNAs. Understanding the exact contribution of RBPs to gene regulation is crucial as many RBPs are frequently mis-regulated in several neurological diseases and certain cancers. While recently developed techniques provide binding sites of RBPs, they are labor-intensive and generally rely on radioactive labeling of RNA. With more than 1,000 RBPs in a human cell, it is imperative to develop easy, robust, reproducible and high-throughput methods to determine in vivo targets of RBPs. To address these issues we developed uvCLAP (UV crosslinking and affinity purification) as a robust, reproducible method to measure RNA-protein interactions in vivo. To test its performance and applicability we investigated binding of 15 RBPs from fly, mouse and human cells. We show that uvCLAP generates reliable and comparable data to other methods. Unexpectedly, our results show that despite their different subcellular localizations, STAR proteins (KHDRBS1-3, QKI) bind to a similar RNA motif in vivo. Consistently a point mutation (KHDRBS1Y440F) or a natural splice isoform (QKI-6) that changes the respective RBP subcellular localization, dramatically alters target selection without changing the targeted RNA motif. Combined with the knowledge that RBPs can compete and cooperate for binding sites, our data shows that compartmentalization of RBPs can be used as an elegant means to generate RNA target specificity.

molecular biology↗