Isoform-specific m6A deposition and coordinated splicing shape mammalian transcriptome evolution

Area of Science:

• Genomics
• Evolutionary Biology
• Molecular Biology

Background:

• Alternative isoform usage and RNA modifications are key drivers of transcriptome evolution.
• N6-methyladenosine (m6A) is the most abundant internal mRNA modification, crucial for gene regulation.
• Short-read sequencing limits understanding of evolutionary conservation and phenotypic impact of transcript isoforms and m6A.

Purpose of the Study:

• To conduct a comparative evolutionary analysis of transcript isoforms and m6A modifications using direct-RNA long-read sequencing.
• To investigate the conservation and functional significance of these elements across mammalian clades and an avian outgroup.

Main Methods:

• Direct-RNA long-read sequencing of six tissues from multiple mammalian species and an avian outgroup.
• Comparative analysis of transcript isoform diversity and N6-methyladenosine (m6A) modification sites.
• Identification of conserved and species-specific isoforms and m6A modification patterns.

Main Results:

• 71% of transcript isoforms are species-specific but contribute minimally to gene expression; 18% of conserved isoforms dominate expression.
• 14.2% of m6A sites are conserved across mammals, enriched in 3'-UTRs and near stop codons.
• Conserved m6A sites show isoform-specific deposition, and coordinate splicing acts as a buffering mechanism for isoform regulation.

Conclusions:

• Post-transcriptional regulation, including alternative splicing and m6A modifications, significantly shapes phenotypic diversity and evolutionary adaptation in mammals.
• Conserved transcript isoforms and m6A modifications are critical for maintaining gene expression and functional transcript diversity.
• Direct-RNA long-read sequencing provides novel insights into the evolutionary dynamics of the transcriptome.