Splicing of internal large exons is defined by novel cis-acting sequence elements.

Area of Science:

• Genomics
• Molecular Biology
• Gene Regulation

Background:

• Human internal exons are typically small (<300 nt), facilitating exon definition during splicing.
• Previously, large internal exons (>1000 nt) were primarily associated with alternative splicing.
• The functional significance and regulatory mechanisms of large internal exons remain incompletely understood.

Purpose of the Study:

• To identify and characterize large internal exons in the human genome.
• To investigate the splicing patterns and regulatory elements associated with these large exons.
• To identify novel sequence motifs that promote the splicing of large exons.

Main Methods:

• Bioinformatic identification of human internal exons exceeding 1000 nt.
• Analysis of gene expression levels and conservation across mammals.
• Comparative sequence analysis to identify enriched motifs in large exons.
• Functional validation of identified motifs through mutational analysis.

Main Results:

• 1115 large internal exons were identified in 5% of human protein-coding genes, with most being expressed.
• 40% of these large exons exhibited constitutive splicing patterns, similar to flanking exons.
• Constitutively spliced large exons showed higher enhancer/silencer ratios and greater mammalian conservation.
• 38 novel C-rich sequences with a central CA dinucleotide, termed large exon splicing enhancers (LESEs), were identified.
• Mutational analysis confirmed the importance of LESEs for large exon recognition by the splicing machinery.

Conclusions:

• The human genome contains a significant number of large internal exons, challenging previous assumptions.
• A subset of large exons are constitutively spliced, regulated by specific sequence elements (LESEs).
• LESEs play a crucial role in the efficient recognition and splicing of large exons, contributing to gene expression regulation.