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

Area of Science:

• Molecular Biology
• Genetics
• Biochemistry

Background:

• Alternative splicing generates mRNA diversity in human cells.
• The spliceosome accurately pairs exons, but rare aberrant and non-canonical splicing events remain unquantified at the nucleotide level.
• Understanding these splicing events is crucial for comprehending gene expression regulation and disease mechanisms.

Purpose of the Study:

• To quantify the frequency and identity of aberrant and non-canonical pre-mRNA splicing events at single nucleotide resolution.
• To investigate the impact of mutations within SMN exon 7 on splicing fidelity.
• To determine the driving forces behind non-canonical splice site pairing.

Main Methods:

• Ultra-deep sequencing was employed to analyze splicing events across three splice junctions flanking SMN1 exon 7.
• Bioinformatic analysis was performed to correct for background noise from PCR amplification and sequencing.
• RNA secondary structure analysis and assessment of splice site strength were conducted.

Main Results:

• Pre-mRNA splicing was found to have a low overall rate of aberrant and non-canonical events after noise correction.
• Several previously unannotated splicing events at SMN1 exon|intron junctions were identified.
• Mutations in SMN exon 7 were shown to decrease splicing fidelity by altering RNA structure, regulatory protein binding, and 5' splice site strength, sometimes creating de novo splice sites.

Conclusions:

• Pre-mRNA splicing exhibits high fidelity in human cells.
• The immediate sequence context surrounding splice sites is the primary determinant of non-canonical splice site selection.
• Understanding splicing fidelity and the impact of mutations is vital for diagnosing and potentially treating genetic disorders like Spinal Muscular Atrophy.