Polyadenylation-related isoform switching in human evolution revealed by full-length transcript structure

Rhesus macaque is a unique nonhuman primate model for human evolutionary and translational study, but the error-prone gene models critically limit its applications. Here, we de novo defined full-length macaque gene models based on single molecule, long-read transcriptome sequencing in four macaque tissues (frontal cortex, cerebellum, heart and testis). Overall, 8588227 poly(A)-bearing complementary DNA reads with a mean length of 14106 nt were generated to compile the backbone of macaque transcripts, with the fine-scale structures further refined by RNA sequencing and cap analysis gene expression sequencing data. In total, 51605 macaque gene models were accurately defined, covering 89.7% of macaque or 75.7% of human orthologous genes. Based on the full-length gene models, we performed a human-macaque comparative analysis on polyadenylation (PA) regulation. Using macaque and mouse as outgroup species, we identified 79 distal PA events newly originated in humans and found that the strengthening of the distal PA sites, rather than the weakening of the proximal sites, predominantly contributes to the origination of these human-specific isoforms. Notably, these isoforms are selectively constrained in general and contribute to the temporospatially specific reduction of gene expression, through the tinkering of previously existed mechanisms of nuclear retention and microRNA (miRNA) regulation. Overall, the protocol and resource highlight the application of bioinformatics in integrating multilayer genomics data to provide an intact reference for model animal studies, and the isoform switching detected may constitute a hitherto underestimated regulatory layer in shaping the human-specific transcriptome and phenotypic changes.

Automated summary

The study utilized single molecule, long-read transcriptome sequencing to define full-length gene models in four tissues of rhesus macaques, covering a majority of rhesus macaque and human orthologous genes. Through a human-macaque comparative analysis, it was found that the formation of human-specific isoforms is mainly due to the strengthening of distal polyadenylation sites, potentially contributing to temporospatial-specific reduction in gene expression.