Universal alternative splicing of noncoding exons

The human transcriptome is so large, diverse and dynamic that, even after a decade of investigation by RNA sequencing (RNA-Seq), we are yet to resolve its true dimensions. RNA-Seq suffers from an expression-dependent bias that impedes discovery of low-abundance transcripts and has prevented a complete census of gene expression. Here we performed targeted single-molecule and short-read RNA-Seq to survey the transcriptional landscape of a single human chromosome (Hsa21) at unprecedented resolution. Our analysis reaches the lower limits of the transcriptome and identifies a fundamental distinction between the architecture of protein-coding and noncoding gene content. Unlike their coding counterparts, noncoding exons undergo universal alternative splicing to produce a seemingly limitless variety of isoforms. Targeted RNA-Seq analysis of syntenic regions of the mouse genome shows that few noncoding exons are shared between human and mouse. Despite this divergence, human alternative splicing profiles are recapitulated on Hsa21 in mouse cells, indicative of regulation by a local splicing code that is more strongly conserved than the noncoding isoforms themselves. We propose that noncoding exons are functionally modular, with combinatorial alternative splicing generating an enormous repertoire of potential regulatory RNAs and a rich transcriptional reservoir for gene evolution. Duplicate or triplcate tissue samples from adult human, mouse or Tc1 mouse (carries human chromosome 21), analyzed by RNA CaptureSeq targeted to Chr21 and syntentic regions of the mouse genome