Different mutants impact on the transcriptome response to nutrients depletion
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ABSTRACT:
ORGANISM(S): Saccharomyces cerevisiae
PROVIDER: GSE198451 | GEO | 2025/05/27
REPOSITORIES: GEO
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Project description:Cross-linking of isotope-labelled RNA coupled with mass spectrometry (CLIR-MS) was used to study the interaction between the ubiquitin-like domain (UBL) of the protein SF3A1, a component of the U2 snRNP, with stem-loop 4 RNA (SL4) from the U1 snRNP. The complex was cross-linked and analysed both in isolation, and in broader context with components of the U1 snRNP. Cross-linking was performed using irradiation under 254 nm light, relying on the inherent reactivity of ribonucleotides.
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Project description:Transcription of the mammalian genome is pervasive, but productive transcription outside of protein-coding genes is limited by unknown mechanisms. In particular, although RNA polymerase II (RNAPII) initiates divergently from most active gene promoters, productive elongation occurs primarily in the sense-coding direction. Here we show in mouse embryonic stem cells that asymmetric sequence determinants flanking gene transcription start sites control promoter directionality by regulating promoter-proximal cleavage and polyadenylation. We find that upstream antisense RNAs are cleaved and polyadenylated at poly(A) sites (PASs) shortly after initiation. De novo motif analysis shows PAS signals and U1 small nuclear ribonucleoprotein (snRNP) recognition sites to be the most depleted and enriched sequences, respectively, in the sense direction relative to the upstream antisense direction. These U1 snRNP sites and PAS sites are progressively gained and lost, respectively, at the 5' end of coding genes during vertebrate evolution. Functional disruption of U1 snRNP activity results in a dramatic increase in promoter-proximal cleavage events in the sense direction with slight increases in the antisense direction. These data suggest that a U1-PAS axis characterized by low U1 snRNP recognition and a high density of PASs in the upstream antisense region reinforces promoter directionality by promoting early termination in upstream antisense regions, whereas proximal sense PAS signals are suppressed by U1 snRNP. We propose that the U1-PAS axis limits pervasive transcription throughout the genome. 3' end sequencing of poly (A) + RNAs in mouse ES cells with and without U1 snRNP inhibition using antisense morpholino oligonucleotides (AMO). Each with two biological replicates.
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Project description:Transcription and splicing are intricately linked processes, with emerging evidence highlighting the involvement of splicing factors mediating their coupling. U1 small nuclear ribonucleoprotein particle (U1 snRNP), a key splicing factor, not only serves as the initial component of the spliceosome but also plays a crucial role in preventing premature cleavage and polyadenylation, facilitating long-distance transcription elongation. Here, we show that U1 snRNP regulates alternative promoter usage through inhibition of premature polyadenylation. Inhibiting U1 snRNP with antisense oligonucleotides or introducing a premature polyadenylation leads to a significant decrease in downstream promoter activity in genes with premature polyadenylation sites in between two promoters. Conversely, restoring U1 snRNP activity or inhibiting premature polyadenylation sites using a gene-specific U1 snRNP rescues downstream promoter activity. Mechanistically, U1 snRNP inhibition correlates with reduced chromatin accessibility and serine 5 phosphorylation levels of RNA polymerase II at downstream promoters. Our findings propose a model where U1 snRNP facilitates productive transcription from upstream promoters, triggering downstream promoter activation by destabilizing nucleosomes and promoting promoter escape.
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