Functional inhibition of core spliceosomal machinery activates intronic premature cleavage and polyadenylation of pre-mRNAs
Ontology highlight
ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE283688 | GEO | 2025/01/29
REPOSITORIES: GEO
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Project description:In eukaryotes, U1 small nuclear ribonucleoprotein (snRNP) forms spliceosomes in equal stoichiometry with U2, U4, U5 and U6 snRNPs; however, its abundance in human far exceeds that of the other snRNPs. Here we used antisense morpholino oligonucleotide to U1 snRNA to achieve functional U1 snRNP knockdown in HeLa cells, and identified accumulated unspliced pre-mRNAs by genomic tiling microarrays. In addition to inhibiting splicing, U1 snRNP knockdown caused premature cleavage and polyadenylation in numerous pre-mRNAs at cryptic polyadenylation signals, frequently in introns near (<5 kilobases) the start of the transcript. This did not occur when splicing was inhibited with U2 snRNA antisense morpholino oligonucleotide or the U2-snRNP-inactivating drug spliceostatin A unless U1 antisense morpholino oligonucleotide was also included. We further show that U1 snRNA–pre-mRNA base pairing was required to suppress premature cleavage and polyadenylation from nearby cryptic polyadenylation signals located in introns. These findings reveal a critical splicing-independent function for U1 snRNP in protecting the transcriptome, which we propose explains its overabundance.
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Project description:To investigate the extent to which U6 inhibits premature cleavage and polyadenylation at the transcriptome level. we performed 3’-seq in control, U1 and U6-AMO treated sample using lexogen 3’-seq kit (Cat. 016.24) that could accurately quantify the global PAS usage. Indeed, we found both U1 and U6 could globally suppress intronic PCPA.
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Project description:U4/U6 di-snRNPs were disrupted and singular U4 and U6 snRNPs accumulated in egy mutant embryos, establishing the recycling function of p110 in vivo. Based on microarray analyses, a subset of spliceosome components and splicing-related factors was coordinately upregulated in the egy mutant. This revealed an extensive network of coregulated components of the spliceosome cycle, compensating – albeit inefficiently – for the recycling defect. In contrast, another set of genes, many of them eye- and pancreas-specific, was downregulated in the egy mutant embryos. Keywords: mut / wt comparison
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Project description:Alternative splicing (AS) can produce multiple transcripts with different exon-intron structures from a single pre-mRNA. Pre-mRNA splicing is catalyzed by a dynamic macromolecular ribonucleoprotein (RNP) complex termed the spliceosome. The spliceosome consists of several small nuclear ribonucleoproteins (snRNPs) that bind uridine-rich small nuclear RNA (snRNA). In U1, U2, U4 and U5 snRNPs, snRNA interacts with the conserved Smith antigen (Sm) proteins via a bipartite Sm sequence motif. In eukaryotes, seven Sm proteins (B, D1/2/3, E, F and G) form a heptameric ring-shaped complex surrounding the snRNA. Here, we performed a tandem affinity purification using SMEB as a bait in Arabidopsis cell suspension cultures. At least 45 known/hypothesized and potential novel spliceosome components were identified in Arabidopsis.
2023-03-03 | PXD016607 | Pride
Project description:U4/U6 di-snRNPs were disrupted and singular U4 and U6 snRNPs accumulated in egy mutant embryos, establishing the recycling function of p110 in vivo. Based on microarray analyses, a subset of spliceosome components and splicing-related factors was coordinately upregulated in the egy mutant. This revealed an extensive network of coregulated components of the spliceosome cycle, compensating â albeit inefficiently â for the recycling defect. In contrast, another set of genes, many of them eye- and pancreas-specific, was downregulated in the egy mutant embryos. Experiment Overall Design: Zebrafish earl grey (egy) mutant embryos carry an autosomal recessive defect in the p110-orthologous gene which leads to microcephaly, microphthalmia, underdevelopment of the pharyngeal arches, and thymus hypoplasia by day 8 of development. To characterize the defect on the transcriptional level, egy whole embryos (n>100) were collected and morphologically separated into pools of mutant (mut) and wildtype (wt) sibling embryos. Pools of embryos were collected at 2 time points (3 and 4 days post fertilization) with 2 and 3 biological replicates, resp.. After RNA extraction, labelled cRNA was hybridized onto Affymetrix microarrays.
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Project description:Brr2 is a DExD/H-box helicase responsible for U4/U6 unwinding, a critical step in spliceosomal activation. Brr2 contains an N-terminal domain and two tandem sets of a helicase-like domain followed by a Sec63 domain with unknown function. We determined the crystal structure of the second Sec63 domain, which unexpectedly resembles domains 4 and 5 of DNA helicase Hel308. The helicase-like domain upstream of Sec63 has clear sequence similarity with domains 1-3 of Hel308. In addition modeling indicates that Brr2 is composed of an N-terminal domain and two consecutive Hel308-like modules (Hel308-I and II). Together this provides our first glimpse of the overall structure of this large and unique spliceosomal ATPase and helicase. Our structural model and mutagenesis data suggest that Brr2 shares a similar helicase mechanism to Hel308, that differs from many DEAD-box proteins. We demonstrate that Hel308-II interacts with Prp8 and Snu114 in vitro and in vivo, potentially serving as a mediator for the regulation of Brr2’s activity by Prp8. We further find that the C-terminal region of Prp8 (Prp8-CTR) facilitates the binding of the Brr2/Prp8-CTR complex to U4/U6, suggesting a potential role of Prp8-CTR as an auxiliary substrate binding and specificity domain for Brr2.
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