Conservation, evolution, and regulation of splicing during prefrontal cortex development in humans, chimpanzees, and macaques

Abstract

Changes in splicing are known to affect the function and regulation of genes. We analyzed splicing events that take place during the postnatal development of the prefrontal cortex in humans, chimpanzees, and rhesus macaques based on data obtained from 168 individuals. Our study revealed that among the 38,822 quantified alternative exons, 15% are differentially spliced among species, and more than 6% splice differently at different ages. Mutations in splicing acceptor and/or donor sites might explain more than 14% of all splicing differences among species and up to 64% of high-amplitude differences. A reconstructed trans-regulatory network containing 21 RNA-binding proteins explains a further 4% of splicing variations within species. While most age-dependent splicing patterns are conserved among the three species, developmental changes in intron retention are substantially more pronounced in humans.

Keywords: RNA-seq; alternative splicing; brain development; transcriptomics.

FIGURE 1.

Splicing variability in the human, chimpanzee, and macaque brain. (A) Age distribution of individuals used in the study. Each symbol represents one individual (circle—female, triangle—male). The rectangles indicate individual samples pooled in DS3. The colors show species: humans—red, chimpanzees—blue, macaques—green. (B,C) Multidimensional scaling of samples based on the Pearson correlation of the PSI values. (D) The numbers and the types of gene segments showing: (i) significant splicing differences between species (H–C, H–M, C–M), (ii) species-specific splicing differences (H-spec, C-spec, M-spec), and (iii) age-dependent splicing changes within species (H-age, C-age, M-age). (E) Comparison of splicing variation between species detected based on DS1 and DS3. The colors show species’ comparisons. Only segments with significant splicing differences in DS1 are shown. (F) Assessment of splicing differences detected based on RNA-seq data using RT-PCR. (G) Example of large splicing difference between species in the SNHG11 gene. Shown are the average coverage plots for each species: (humans—red, chimpanzees—blue, macaques—green), human gene annotation (black), and PSI distributions for selected gene segments (boxplots). (H) Neighbor joining tree based on splicing divergence (one minus Person correlation coefficient). The numbers of divergent splicing events are shown in the legend.

FIGURE 2.

Age-dependent splicing changes in the human, chimpanzee, and macaque brain. (A) The numbers of gene segments showing age-dependent splicing in each species. Here and further the colors indicate the following: humans—red, chimpanzees—blue, and macaques—green. (B) The distributions of the Pearson correlation coefficients based on age-dependent splicing changes measured using DS1 and DS2. (C) The distribution of age-scale coefficients. The dashed lines show location of the distribution modes shown in the legend. (D) The distributions of the Pearson correlation coefficients based on age-dependent splicing changes detected in both compared species. (E,F) Six age-dependent splicing patterns identified for protein-coding segments and retained introns. Each panel represents one pattern; the dots show the mean z-score transformed PSI. The curves are drawn using cubic splines with four degrees of freedom. Numbers of segments in each cluster are shown in title. (G) Mean normalized expression profile for 24 human-specific genes showing age-related intron retention.

FIGURE 3.

Splicing regulation and function. (A) The distribution of the Pearson correlation coefficients based on the PSI of age-dependent retained introns and the expression level of the respective genes (solid lines) or randomly selected genes (dashed lines). The legend shows P-values of the Wilcoxon test comparing the observed and control distributions. (B) The proportions of the last (top) and the second-to-last (bottom) non-age-dependent introns or age-dependent introns in one of the species among all retained introns. The error bars show 95% confidence intervals. Only genes with at least five introns were included. (C) The relationship between PSI variation between species and splicing propensity differences at the alternative donor sites. (D) The proportions of splicing differences between species that could be explained by the sequence differences within the core splicing sites at a different PSI change amplitudes. (E,F) The distributions of PSI values at the alternative donor sites within the PARP2 and ULK3 genes. Reference and alternative alleles (red) are shown under the consensus donor site sequence. In the ULK3 sequence, SNP overlaps both ancestral and alternative donor sites. (G) The distributions of the average primate phastCons scores for protein-coding cassette exons and adjacent introns.