Most human introns are recognized via multiple and tissue-specific branchpoints

Although branchpoint recognition is an essential component of intron excision during the RNA splicing process, the branchpoint itself is frequently assumed to be a basal, rather than regulatory, sequence feature. However, this assumption has not been systematically tested due to the technical difficulty of identifying branchpoints and quantifying their usage. Here, we analyzed ∼1.31 trillion reads from 17,164 RNA sequencing data sets to demonstrate that almost all human introns contain multiple branchpoints. This complexity holds even for constitutive introns, 95% of which contain multiple branchpoints, with an estimated five to six branchpoints per intron. Introns upstream of the highly regulated ultraconserved poison exons of SR genes contain twice as many branchpoints as the genomic average. Approximately three-quarters of constitutive introns exhibit tissue-specific branchpoint usage. In an extreme example, we observed a complete switch in branchpoint usage in the well-studied first intron of HBB (β-globin) in normal bone marrow versus metastatic prostate cancer samples. Our results indicate that the recognition of most introns is unexpectedly complex and tissue-specific and suggest that alternative splicing catalysis typifies the majority of introns even in the absence of differences in the mature mRNA.