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Ultra-deep sequencing reveals pre-mRNA splicing as a sequence driven high-fidelity process

ABSTRACT: Alternative splicing diversifies mRNA transcripts in human cells. While the spliceosome pairs exons with a high degree of accuracy, the rates of rare aberrant and non-canonical pre-mRNA splicing have not been evaluated at the nucleotide level to determine the quantity and identity of these events across splice junctions. Using ultra-deep sequencing the frequency of aberrant and non-canonical splicing events for three splice junctions flanking exon 7 of SMN1 were determined at single nucleotide resolution. After correction for background noise introduced by PCR amplification and sequencing steps, pre-mRNA splicing was shown to maintain a low overall rate of aberrant and non-canonically spliced events. Several previously unannotated splicing events across 3 exon|intron junctions in SMN1 were identified. Mutations within SMN exon 7 were shown to affect splicing fidelity by modulating RNA secondary structures, by altering the binding site of regulatory proteins and by changing the 5’ splice site strength. Mutations also create a truncated SMN1 exon 7 through the introduction of a de novo non-canonical 5’ splice site. The results from the ultra-deep sequencing approach highlight the impressive fidelity of pre-mRNA splicing and demonstrate that the immediate sequence context around splice sites is the main driving force behind non-canonical splice site pairing.

ORGANISM(S): Homo sapiens

PROVIDER: GSE137883 | GEO | 2019/09/24

REPOSITORIES: GEO

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Project description:MSI (Microsatellite Instability) colorectal cancer (CRC) show improved survival, are less prone to metastasis and show poor response to chemotherapy (compared to MSS tumors). The underlying reasons for these characteristics are still not understood and no specific therapeutic approach for MSI colon tumours (15% of CRC overall) has yet been developed. The MSI process is oncogenic when it affects DNA repeat sequences that have a functional role, e.g. Small Coding Repeats (SCR). MSI also frequently affects Long Non-Coding Repeats (LNCR) in tumour DNA. In contrast to SCR, only a few LNCR are endowed with biological activity. Consequently, this area has received very little attention. Our group recently identified HSP110 mutant chaperone protein in MSI CRC that was generated by somatic deletion of a LNCR. Of interest, HSP110 mutant (due to exon skipping) have anti-oncogenic properties and the survival of MSI CRC patients receiving chemotherapy is positively associated with HSP110 mutations in tumour DNA. The aim of the current project is to identify additional clinically relevant MSI-associated splicing aberrations due to mutations in LNCR located in splice acceptor sites. The four main steps are as follows: 1. To identify exon/intron sites affected by aberrant splicing events due to MSI in CRC . All RNASeq data will be exploited to identify recurrent splicing aberrations (mostly exon skipping) that occur specifically in MSI colon tumours; 2. To investigate for possible functional links between MSI and any detected aberrant splicing events . All specific aberrant splicing events detected by RNAseq in MSI CRC samples will be first confirmed (quantitative RT-PCR) in order to eliminate false positive cases. For validated exon candidates, the allelic profiles of adjacent intronic LNCR will be analysed (PCR and fluorescence genotyping) in CRC cell lines and primary tumours (MSI and MSS), as well as in matching normal mucosa samples in order to assess their polymorphic status; 3. To identify splicing events and LNCR mutations with clinical relevance in MSI CRC patients . All LNCR with a confirmed role in gene splicing in MSI CRC will be analysed. The clinical relevance of candidate genes will be assessed using multivariate survival regression models for Relapse- Free Survival, with interaction terms (response to chemotherapy); 4. To initiate functional studies on a limited number of clinically relevant, cancer-related genes whose splicing is perturbed in MSI cancer cells, and to develop biological tools to simplify screening in future clinical assays Similar to HSP110, we will focus on 4 or 5 mutant proteins that are promising drug therapeutic targets. Functional assays will be developed to further elucidate their role in the pathophysiology of MSI tumours. We also aim to develop biological tools for these candidate genes, such as the detection of wild-type or mutant proteins by immunohistochemistry.

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Ultra-deep sequencing reveals pre-mRNA splicing as a sequence driven high-fidelity process

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