Human introns contain conserved tissue-specific cryptic poison exons.

Area of Science:

• Genomics
• Molecular Biology
• Gene Expression

Background:

• Alternative splicing generates numerous transcripts from single genes, but exon catalogs are incomplete.
• Human introns harbor conserved elements with unknown functions, potentially including regulatory or coding sequences.
• Cryptic exons, expressed under specific conditions, may contribute to transcriptomic diversity.

Purpose of the Study:

• To investigate whether conserved intronic elements in human protein-coding genes represent cryptic exons.
• To determine the functional impact and expression patterns of identified cryptic exons.
• To validate the expression of cryptic exons using experimental methods.

Main Methods:

• Analysis of RNA-seq data from the Genotype-Tissue Expression (GTEx) dataset to identify potential cryptic exons.
• Comparison of cryptic exon characteristics (evolutionary conservation, read coverage) with annotated exons.
• RNA sequencing of A549 cells treated with cycloheximide to inhibit nonsense-mediated decay (NMD).
• Quantitative polymerase chain reaction (qPCR) to confirm the expression of selected cryptic exons.

Main Results:

• A set of cryptic exons was identified in human introns, exhibiting characteristics similar to annotated exons.
• Many identified cryptic exons function as poison exons, leading to NMD isoforms upon inclusion.
• Evidence suggests tissue-specific and cancer-specific expression and regulation of these cryptic exons.
• Experimental validation confirmed the expression of seven out of eight tested cryptic exons, particularly after NMD inhibition.

Conclusions:

• Human protein-coding gene introns contain cryptic poison exons within conserved regions.
• These cryptic exons are often unannotated due to limited representation in standard RNA-seq libraries.
• Their expression in specific conditions and potential role in NMD highlight their significance in gene regulation and disease.