Genome wide mapping of effects of U6atac knockdown on pre-mRNA splicing
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE48263 | GEO | 2013/08/01
SECONDARY ACCESSION(S): PRJNA209423
REPOSITORIES: GEO
Similar Datasets
Project description:This project looks into experimentally identifying all minor introns by knocking down the minor spliceosome's catalytic snRNP, U6atac. Knockdown of U6atac by antisense morpholino followed by examining mRNA splicing by RNA-seq
2013-08-01 | E-GEOD-48263 | biostudies-arrayexpress
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.
2021-04-19 | GSE143392 | GEO
Project description: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.
2024-02-04 | GSE246283 | GEO
Project description:Minor intron-containing genes (MIGs) are crucial cell cycle regulators and are essential for cell survival. Thus, we explored whether MIGs play a role in cancer progression and cancer therapy resistance. Here we show that MIG-expression segregates sensitive and resistant prostate cancer cells from each other and them from benign tissue. Moreover, the rate of minor intron splicing, a crucial regulatory node for MIG-expression, was more efficient in patients with advanced prostate cancer. We show that the increased minor spliceosome (MiS) activity is downstream of the androgen receptor signaling axis in both therapy sensitive and insensitive prostate cancer types. The increased MiS activity was in line with the elevated expression of U6atac snRNA, a crucial MiS component. We propose the expression of U6atac as a potential point of vulnerability in cancer. MIGs are uniquely susceptible to the MiS, so we used siRNA against U6atac, which like protein-coding transcripts, is downregulated. Inhibition of MiS was sufficient to lower the tumor burden significantly in therapy-resistant (prostate) cancer cells and PCa patient-derived organoids. KD cells displayed elevated minor intron retention and alternative splicing across minor introns of MIGs which enriched for MAPK activity, DNA repair and cell cycle regulation. Importantly, these findings were confirmed by MassSpec analysis. Collectively our study nominates the MiS as a driver of highly proliferative, rapidly dividing (prostate) cancer cells that bears strong potential as a therapeutic target for many different cancer types.
2023-06-08 | PXD026949 | Pride
Project description:A Drosophila line (l(2)k01105) with a P-element insertion in U6atac snRNA, an essential component of the U12-type spliceosome, was used to investigate the impact of disrupted U12-dependent splicing during larval development. A custom-designed exon array was used to study the expression of genes containing U12-type introns and genes involved in downstream pathways affected by deficient U12-dependent splicing. Flies homozygous for the U6atac mutation (U6atac/U6atac) were compared to heterozygotes (U6atac/CyO-GFP) which have a normal phenotype.
2010-10-11 | GSE24148 | GEO
Project description:The minor spliceosome is evolutionarily conserved in higher eukaryotes, but its biological significance remains poorly understood. Here, by precise CRISPR/Cas9-mediated disruption of the U12 and U6atac snRNAs, we report that a defective minor spliceosome is responsible for spinal muscular atrophy (SMA) associated phenotypes in Drosophila. Using a newly developed bioinformatic approach, we identified a large set of minor spliceosome-sensitive splicing events and demonstrate that three sensitive intron-containing neural genes, Pcyt2, Zmynd10, and Fas3, directly contribute to disease development as evidenced by the ability of their cDNAs to rescue the SMA-associated phenotypes in muscle development, neuromuscular junctions, and locomotion. Interestingly, many splice sites in sensitive introns are recognizable by both minor and major spliceosomes, suggesting a new mechanism of splicing regulation through competition between minor and major spliceosomes. These findings reveal a vital contribution of the minor spliceosome to SMA and to regulated splicing in animals.
2020-04-01 | GSE138183 | GEO
Project description:A Drosophila line (l(2)k01105) with a P-element insertion in U6atac snRNA, an essential component of the U12-type spliceosome, was used to investigate the impact of disrupted U12-dependent splicing during larval development. A custom-designed exon array was used to study the expression of genes containing U12-type introns and genes involved in downstream pathways affected by deficient U12-dependent splicing. Flies homozygous for the U6atac mutation (U6atac/U6atac) were compared to heterozygotes (U6atac/CyO-GFP) which have a normal phenotype. Samples from homozygous and heterozygous U6atac mutant larvae at 1st, 2nd and 3rd larval instar were labelled with three colours and hybridized three samples per array. Three independently harvested biological replicates were made, with dye swap, except only two replicates for 3rd stage heterozygous control. This study presents exon-level data. Supplementary files: Raw data (.gpr) files. Series variables and repeats information.
2010-10-11 | E-GEOD-24148 | biostudies-arrayexpress
Project description:This project looks into how U1 snRNP inhibition causes a loss of telescripting through premature cleavage and polyadenylation based on the size and function of human genes.
2017-10-02 | GSE103252 | GEO
Project description:In this work, 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.
| MSV000092983 | MassIVE
Project description:Introns are removed by the spliceosome, a large complex composed of five ribonucleoprotein subcomplexes (U snRNP). In metazoans, the U1 snRNP, which binds to 5’ splice sites, also fulfills regulatory roles in splice site selection and possesses non-splicing related functions. Here, we show that an Arabidopsis U1 snRNP subunit, LUC7, affects constitutive and alternative splicing. Interestingly, LUC7 specifically promotes splicing of a subset of terminal introns. Splicing of LUC7-dependent terminal introns is a prerequisite for nuclear export and can be modulated by stress. Globally, intron retention under stress conditions occurs preferentially among first and terminal introns, uncovering an unknown bias for splicing regulation in Arabidopsis. Taken together, our study reveals that the Arabidopsis U1 snRNP is important for alternative splicing and removal of terminal introns and it suggests that Arabidopsis terminal introns fine-tune gene expression under stress conditions.
2017-05-24 | GSE98779 | GEO