Background

Differences in gene expression drive phenotypic differences between species, yet major organs and tissues generally have conserved gene expression programs. Several comparative transcriptomic studies have observed greater similarity in gene expression between homologous tissues from different vertebrate species than between diverse tissues of the same species. However, a recent study by Lin and colleagues reached the opposite conclusion. These studies differed in the species and tissues analyzed, and in technical details of library preparation, sequencing, read mapping, normalization, gene sets, and clustering methods.

Results

To better understand gene expression evolution we reanalyzed data from four studies, including that of Lin, encompassing 6-13 tissues each from 11 vertebrate species using standardized mapping, normalization, and clustering methods. An analysis of independent data showed that the set of tissues chosen by Lin et al. were more similar to each other than those analyzed by previous studies. Comparing expression in five common tissues from the four studies, we observed that samples clustered exclusively by tissue rather than by species or study, supporting conservation of organ physiology in mammals. Furthermore, inter-study distances between homologous tissues were generally less than intra-study distances among different tissues, enabling informative meta-analyses. Notably, when comparing expression divergence of tissues over time to expression variation across 51 human GTEx tissues, we could accurately predict the clustering of expression for arbitrary pairs of tissues and species.

Conclusions

These results provide a framework for the design of future evolutionary studies of gene expression and demonstrate the utility of comparing RNA-seq data across studies.

Many RNA binding proteins (RBPs) bind specific RNA sequence motifs, but only a small fraction (∼15%-40%) of RBP motif occurrences are occupied in vivo. To determine which contextual features discriminate between bound and unbound motifs, we performed an in vitro binding assay using 12,000 mouse RNA sequences with the RBPs MBNL1 and RBFOX2. Surprisingly, the strength of binding to motif occurrences in vitro was significantly correlated with in vivo binding, developmental regulation, and evolutionary age of alternative splicing. Multiple lines of evidence indicate that the primary context effect that affects binding in vitro and in vivo is RNA secondary structure. Large-scale combinatorial mutagenesis of unfavorable sequence contexts revealed a consistent pattern whereby mutations that increased motif accessibility improved protein binding and regulatory activity. Our results indicate widespread inhibition of motif binding by local RNA secondary structure and suggest that mutations that alter sequence context commonly affect RBP binding and regulation.