Nested small open reading frames are translated from alternative transcripts.

Area of Science:

• Genomics and Transcriptomics
• Molecular Biology
• Human Gene Expression

Background:

• Internal open reading frames (iORFs) were previously thought to be rare in the human genome.
• The functional significance and expression of human iORFs remain largely uncharacterized.
• Annotated coding sequences (CDSs) were considered the primary functional units of human genes.

Purpose of the Study:

• To investigate the existence and expression of functional human iORFs.
• To develop methods for identifying and validating iORF expression from alternative transcripts.
• To explore the functional impact of iORF expression on gene activity.

Main Methods:

• Development of a novel bioinformatics pipeline for assigning translated iORFs to alternative transcripts.
• Utilized long-read sequencing for transcriptomic analysis and validation.
• Performed molecular validation experiments to confirm iORF expression in human genes.

Main Results:

• Identified hundreds of cases where alternative transcripts reprogram human genes to express iORF-encoded microproteins.
• Demonstrated that many non-coding alternative transcripts are poorly annotated but can lead to iORF translation.
• Validated the expression of numerous human iORFs using sequencing and molecular techniques.
• Showcased a conserved DEDD2 iORF that alters gene function from pro-apoptotic to anti-apoptotic.

Conclusions:

• Alternative transcript variants significantly expand the coding potential of the human genome by enabling iORF expression.
• This mechanism reveals a previously unrecognized layer of complexity in the human transcriptome and proteome.
• iORF expression can functionally reprogram genes, as exemplified by the DEDD2 gene.