Violating the splicing rules: TG dinucleotides function as alternative 3' splice sites in U2-dependent introns

Area of Science:

• Molecular Biology
• Genetics
• RNA Splicing

Background:

• Eukaryotic pre-mRNA splicing typically relies on U2-dependent introns with a 3' AG dinucleotide.
• Anecdotal evidence suggested functional non-AG 3' splice sites exist, prompting further investigation.

Purpose of the Study:

• To conduct a human genome-wide screen for non-AG 3' splice sites.
• To investigate the functional relevance and characteristics of identified non-AG splice sites.

Main Methods:

• Genome-wide screening of human genes.
• Experimental validation of identified splice variants.
• Sequence conservation analysis between orthologous genes.
• Tissue-specificity analysis of alternative splicing ratios.

Main Results:

• Identified TG dinucleotides as functional alternative 3' splice sites in 36 human genes.
• Achieved a 92% experimental validation rate for TG-derived splice variants.
• Observed tissue-specific alternative splicing ratios and significant conservation of TG splice sites across vertebrates.
• Found TG splice sites exclusively as alternative sites, never the sole 3' splice site, with a distance constraint for TG-AG tandems.

Conclusions:

• The U2 spliceosome demonstrates high specificity for 3' AG sites early in splicing but can select 3' TG sites later.
• Cis-acting sequence elements play a crucial role in defining splice site selection.
• TG-AG 3' splice site tandems are valuable models for studying the mechanisms of 3' splice site selection.