Dataset Information

Casein kinase 1 is recruited to nuclear speckles by FAM83H and SON.

ABSTRACT: In some fibroblasts, casein kinase 1? (CK1?) is localized to nuclear speckles, which are sub-nuclear compartments supplying splicing factors, whereas it is recruited on keratin filaments in colorectal cancer cells such as DLD1 cells. In order to obtain a deeper understanding of why CK1? is localized to these different subcellular sites, we herein elucidated the mechanisms underlying its localization to nuclear speckles. CK1? and FAM83H were localized to nuclear speckles in RKO and WiDr colorectal cancer cells, which do not express simple epithelial keratins, and in DLD1 cells transfected with siRNAs for type I keratins. The localization of FAM83H to nuclear speckles was also detected in colorectal cancer cells with a poorly organized keratin cytoskeleton in colorectal cancer tissues. Using an interactome analysis of FAM83H, we identified SON, a protein present in nuclear speckles, as a scaffold protein to which FAM83H recruits CK1?. This result was supported by the knockdown of FAM83H or SON delocalizing CK1? from nuclear speckles. We also found that CK1? and ? are localized to nuclear speckles in a FAM83H-dependent manner. These results suggest that CK1 is recruited to nuclear speckles by FAM83H and SON in the absence of an intact keratin cytoskeleton.

SUBMITTER: Kuga T

PROVIDER: S-EPMC5041083 | biostudies-literature | 2016 Sep

REPOSITORIES: biostudies-literature

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Casein kinase 1 is recruited to nuclear speckles by FAM83H and SON.

Kuga Takahisa T Kume Hideaki H Adachi Jun J Kawasaki Naoko N Shimizu Maiko M Hoshino Isamu I Matsubara Hisahiro H Saito Youhei Y Nakayama Yuji Y Tomonaga Takeshi T

Scientific reports 20160929

In some fibroblasts, casein kinase 1α (CK1α) is localized to nuclear speckles, which are sub-nuclear compartments supplying splicing factors, whereas it is recruited on keratin filaments in colorectal cancer cells such as DLD1 cells. In order to obtain a deeper understanding of why CK1α is localized to these different subcellular sites, we herein elucidated the mechanisms underlying its localization to nuclear speckles. CK1α and FAM83H were localized to nuclear speckles in RKO and WiDr colorecta ...[more]

PMID: 27681590

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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

Casein kinase 1 is recruited to nuclear speckles by FAM83H and SON.

Publications

Casein kinase 1 is recruited to nuclear speckles by FAM83H and SON.

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