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Loss of the RNA trimethylguanosine cap is compatible with nuclear accumulation of spliceosomal snRNAs but not pre-mRNA splicing or snRNA processing during animal development

ABSTRACT: The 2,2,7-trimethylguanosine (TMG) cap is synthesized by the conserved Tgs1 enzyme, which is abundantly present on snRNAs essential for pre-mRNA splicing. In our study, we characterize new mutations in the Drosophila tgs1 gene and studied their effects on development and on splicing. Utilizing a hypomorphic tgs1 mutation, we uncovered a prominent role of Tgs1 in pre-mRNA splicing, particularly of introns with smaller sizes and weaker splice sites. Remarkably, stronger tgs1 mutations generated by CRISPR-Cas9 cause lethality without disrupting splicing, likely due to the preponderance of a maternal contribution and stable TMG capped snRNAs maintaining a near normal level of TMG modified snRNAs, which further proved that the TMG modification is important in splicing.

ORGANISM(S): Drosophila melanogaster

PROVIDER: GSE143488 | GEO | 2020/10/09

REPOSITORIES: GEO

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Project description:In this study we investigated snRNA-mediated modulation of pre-mRNA splicing across the human transcriptome. We first quantified the relative abundance of snRNAs across a comprehensive range of healthy adult and fetal tissues, revealing a surprising variation in the relative snRNA levels both inter- and intra-tissue. To study the role of snRNAs in cancer-relevant splicing, we used breast cancer as a model, since it exhibits a high rate of aberrant splicing48, but a low frequency of mutations in the splicing machinery52. We observed fluctuations in snRNA adundance across the majority of patients, across all breast cancer subtypes. To investigate the impact of snRNA levels using a controlled system, we knocked down snRNAs in vitro, to analyze splicing both transcriptome-wide and at the level of individual exons and introns. Depletion of each specific snRNA resulted in differential splicing across more than a thousand exon junctions within mRNA transcripts. Knock-down of U1 and U2 levels was primarily associated with changes in exon inclusion rates, whereas U4 and U6 depletion predominantly caused incomplete intron removal and a resulting retention of introns in the mature mRNA. Rather than being driven by a single factor, the observed splicing changes were associated with multiple other splicing-relevant features and mechanisms, including mRNA transcription, intron size, and nucleotide composition. The snRNA-mediated changes to the splicing program were enriched within genes encoding components of core cellular pathways and processes, including multiple aspects of RNA and protein metabolism, and thereby have the potential to impact cell growth and identity. The exons and introns that were sensitive to snRNA levels displayed a high variability in splicing in vivo across primary breast cancer samples, indicating that snRNA dysregulation may contribute to aberrant splicing in cancer. We suggest that the cellular composition of snRNAs constitutes a previously unrecognized layer of splicing regulation within the cell, and that variations or disruptions in the relative abundance of the snRNAs can affect the transcriptome of both healthy and malignant cells.

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Loss of the RNA trimethylguanosine cap is compatible with nuclear accumulation of spliceosomal snRNAs but not pre-mRNA splicing or snRNA processing during animal development

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