SON and SRRM2 are essential for nuclear speckle formation

Abstract

The nucleus of higher eukaryotes is a highly compartmentalized and dynamic organelle consisting of several biomolecular condensates that regulate gene expression at multiple levels (Banani et al., 2017; Shin and Brangwynne, 2017). First reported more than 100 years ago by Ramón y Cajal, nuclear speckles (NS) are among the most prominent of such condensates (Spector and Lamond, 2011). 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 (Chen and Belmont, 2019; Galganski et al., 2017). The monoclonal antibody SC35, which was raised against a spliceosomal extract, is a frequently used reagent to mark NS since its debut in 1990 (Fu and Maniatis, 1990). Unexpectedly, we found that this antibody has been misidentified and the main target of SC35 mAb is SRRM2, a large (~300 kDa), spliceosome-associated (Jia and Sun, 2018) protein with prominent intrinsically disordered regions (IDRs) that sharply localizes to NS (Blencowe et al., 1994). Here we show that, the core of NS is likely formed by SON and SRRM2, since depletion of SON leads only to a partial disassembly of NS as reported previously (Ahn et al., 2011; Fei et al., 2017; Sharma et al., 2010), in contrast, 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.

The SC-35 Splicing Factor Interacts with RNA Pol II and A-Type Lamin Depletion Weakens This Interaction

“…The above-mentioned processes and molecular regulations inspired us to study cellular localization and dynamic properties of the main splicing factor, SC-35. Due to the fact that Ilik et al [ 21 ] showed that the antibody against SC-35 instead determines the SRRM2 protein, we verified, in a subset of experiments, the data by other antibodies, e.g., the antibody against SRRM2, and the SRSF7 protein as well. Primarily, by immunofluorescence, we studied the properties of the SC-35 protein by the use of anti-SC-35.…”

“…Here, we found that while the SC-35 protein is associated with the plasma membrane, A-type lamins are mostly decorated metaphase rosettes ( Figure 8 b). Due to the fact that Ilik et al [ 21 ] showed that the monoclonal antibody against SC-35 preferentially recognizes the SRRM2 protein, but not SRSF2, we additionally studied distribution properties by the use of additional antibodies against SRRM2, and also SRSF7.…”

“…At this point, we must point out that Ilik et al [ 21 ] showed that monoclonal antibody against nuclear speckles, specified here as anti-SC-35, preferentially recognizes the SRRM2 protein, but not SRSF2 (syn. SC-35), as it was previously published [ 52 ].…”

Tau aggregates are RNA-protein assemblies that mis-localize multiple nuclear speckle components

“…This would be consistent with the trend that intrinsically disordered regions of proteins can promote their recruitment to membraneless organelles. To test this idea, we used the CRISPaint system to create two HEK293 tau biosensor cell lines that contained a halo tag inserted into endogenous SRRM2 (Ilik et al, 2020;Schmid-Burgk et al, 2016). These two cell lines were 1) a full length SRRM2 cell line referred to as SRRM2_FL-halo (insert at aa 2708), and 2) a cell line lacking the C-terminal IDR of SRRM2 referred to as SRRM2_dIDR-halo (insert at aa 430) ( Fig.…”

The SON RNA splicing factor is required for intracellular trafficking that promotes centriole assembly

“…The multi-domained nuclear speckle and splicing factor protein, SON, is required for centriole assembly (Balestra et al, 2013;Ilik et al, 2020). SON is required for progression through the cell cycle and affects the efficient splicing of a number of genes important for SON is required for centriole assembly.…”