ABSTRACT
Here, we identify RBM41 as a novel unique protein component of the minor spliceosome. RBM41 has no previously recognized cellular function but has been identified as a paralog of U11/U12-65K, a known unique component of the U11/U12 di-snRNP. Both proteins use their highly similar C-terminal RRMs to bind to 3'-terminal stem-loops in U12 and U6atac snRNAs with comparable affinity. Our BioID data indicate that the unique N-terminal domain of RBM41 is necessary for its association with complexes containing DHX8, an RNA helicase, which in the major spliceosome drives the release of mature mRNA from the spliceosome. Consistently, we show that RBM41 associates with excised U12-type intron lariats, is present in the U12 mono-snRNP, and is enriched in Cajal bodies, together suggesting that RBM41 functions in the post-splicing steps of the minor spliceosome assembly/disassembly cycle. This contrasts with U11/U12-65K, which uses its N-terminal region to interact with U11 snRNP during intron recognition. Finally, while RBM41 knockout cells are viable, they show alterations in U12-type 3' splice site usage. Together, our results highlight the role of the 3'-terminal stem-loop of U12 snRNA as a dynamic binding platform for the U11/U12-65K and RBM41 proteins, which function at distinct stages of the assembly/disassembly cycle.
Subject(s)
DEAD-box RNA Helicases , RNA Splicing Factors , RNA, Small Nuclear , RNA-Binding Proteins , Ribonucleoproteins, Small Nuclear , Spliceosomes , Spliceosomes/metabolism , Spliceosomes/genetics , Ribonucleoproteins, Small Nuclear/metabolism , Ribonucleoproteins, Small Nuclear/genetics , Ribonucleoproteins, Small Nuclear/chemistry , RNA-Binding Proteins/metabolism , RNA-Binding Proteins/genetics , RNA-Binding Proteins/chemistry , Humans , RNA, Small Nuclear/metabolism , RNA, Small Nuclear/genetics , RNA, Small Nuclear/chemistry , DEAD-box RNA Helicases/metabolism , DEAD-box RNA Helicases/genetics , RNA Splicing , Introns/genetics , HeLa Cells , Protein Binding , Coiled Bodies/metabolism , HEK293 CellsABSTRACT
Kinetochores connect chromosomes to spindle microtubules to ensure their correct segregation during cell division. Kinetochores of human and yeasts are largely homologous, their ability to track depolymerizing microtubules, however, is carried out by the nonhomologous complexes Ska1-C and Dam1-C, respectively. We previously reported the unique anti-correlating phylogenetic profiles of Dam1-C and Ska-C found among a wide variety of eukaryotes. Based on these profiles and the limited presence of Dam1-C, we speculated that horizontal gene transfer could have played a role in the evolutionary history of Dam1-C. Here, we present an expanded analysis of Dam1-C evolution, using additional genome as well as transcriptome sequences and recently published 3D structures. This analysis revealed a wider and more complete presence of Dam1-C in Cryptista, Rhizaria, Ichthyosporea, CRuMs, and Colponemidia. The fungal Dam1-C cryo-EM structure supports earlier hypothesized intracomplex homologies, which enables the reconstruction of rooted and unrooted phylogenies. The rooted tree of concatenated Dam1-C subunits is statistically consistent with the species tree of eukaryotes, suggesting that Dam1-C is ancient, and that the present-day phylogenetic distribution is best explained by multiple, independent losses and no horizontal gene transfer was involved. Furthermore, we investigated the ancient origin of Dam1-C via profile-versus-profile searches. Homology among 8 out of the 10 Dam1-C subunits suggests that the complex largely evolved from a single multimerizing subunit that diversified into a hetero-octameric core via stepwise subunit duplication and subfunctionalization of the subunits before the origin of the last eukaryotic common ancestor.
Subject(s)
Kinetochores , Saccharomyces cerevisiae Proteins , Humans , Microtubule-Associated Proteins/genetics , Phylogeny , Microtubules , Cell Division , Cell Cycle Proteins/genetics , Saccharomyces cerevisiae Proteins/genetics , Chromosomal Proteins, Non-Histone/geneticsABSTRACT
Aneuploidy is the leading cause of miscarriage and congenital birth defects, and a hallmark of cancer. Despite this strong association with human disease, the genetic causes of aneuploidy remain largely unknown. Through exome sequencing of patients with constitutional mosaic aneuploidy, we identified biallelic truncating mutations in CENATAC (CCDC84). We show that CENATAC is a novel component of the minor (U12-dependent) spliceosome that promotes splicing of a specific, rare minor intron subtype. This subtype is characterized by AT-AN splice sites and relatively high basal levels of intron retention. CENATAC depletion or expression of disease mutants resulted in excessive retention of AT-AN minor introns in Ë 100 genes enriched for nucleocytoplasmic transport and cell cycle regulators, and caused chromosome segregation errors. Our findings reveal selectivity in minor intron splicing and suggest a link between minor spliceosome defects and constitutional aneuploidy in humans.
