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Distinct functions for the paralogous RBM41 and U11/U12-65K proteins in the minor spliceosome

ABSTRACT: 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 the U11/U12-65K protein, a known unique component of the minor spliceosome that functions during the early steps of minor intron recognition as a component of the U11/U12 di-snRNP. We show that 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 the U11/U12-65K protein, which uses the N-terminal region to interact with U11 snRNP during the intron recognition step. Finally, we show that while RBM41 knockout cells are viable, they show alterations in the splicing of U12-type introns, particularly differential U12-type 3' splice site usage. Together, our results highlight the role 3’-terminal stem-loop of U12 snRNA as a dynamic binding platform for the paralogous U11/U12-65K and RBM41 proteins, which function at distinct stages of minor spliceosome assembly/disassembly cycle.

ORGANISM(S): Homo sapiens

PROVIDER: GSE246283 | GEO | 2024/02/04

REPOSITORIES: GEO

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Project description:U12-type introns were originally recognized based on their highly conserved non-consensus AT-AC termini1,2, which are spliced by a separate minor spliceosome3,4. Padgett and Krainer groups later showed that terminal dinucleotides do not differentiate U12-type from U2-type introns, as there are U12-type introns with GT-AG termini and U2-type introns with AT-AC termini5,6. Rather, U12-type introns are recognized by their divergent and highly conserved 5’ splice site (5’ss) and branch point sequences, which both differ from the consensus sequences found in U2-type introns. To date, no functional differences have been ascribed to AT-AC or GT-AG subtypes of U12-type introns, nor have RNAseq analyses of minor spliceosome diseases reported any subtype specificity. Here, we describe a novel protein component of the minor spliceosome, encoded by the CENATAC locus, that is required for accurate splicing of AT-AC but not GT-AG type minor introns. CENATAC was initially identified in a subset of Mosaic Variegated Aneuploidy (MVA) patients with mutations in CENATAC, which lead to chromosome congression defects during mitosis. Earlier large-scale proteomic analyses tentatively classified CENATAC as a spliceosome component and phylogenetic analyses showed co-segregation of CENATAC with minor spliceosome components. Targeted depletion of CENATAC in HeLa cells, followed by RNAseq revealed global retention of AT-AC minor subtype introns with more than 60% showing statistically significant (up to 90%) intron retention (IR). Additionally, U12-type introns with 5’-AT, but divergent 3’-terminal dinucleotides also showed significant IR. We also detected cryptic U2-type splice site activation near affected AT-AC introns. In contrast, about 10% of GT-AG subtype introns responded to CENATAC depletion. Co-IP experiments revealed that CENATAC is not a U11/U12 di-snRNP component as expected for a specificity factor, but rather associates with the U4atac/U6atac.U5 tri-snRNP via interaction with PRPF3/4, suggesting a role for minor tri-snRNP in initial 5’ss recognition. CENATAC also interacts with TXNL4B, a paralog of TXNL4A in the major tri-snRNP. Finally, several genes encoding chromosome congression factors harbor U12 AT-AC-type introns that were highly retained in CENATAC depleted cells, potentially explaining the aneuploidy phenotype observed in MVA patients.

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Distinct functions for the paralogous RBM41 and U11/U12-65K proteins in the minor spliceosome

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