4sU metabolic labeled transcripts in mESCs

We carried out an extensive functional genomics analysis to elucidate the roles, if any, of splicing at enhancer-associated lincRNA loci (elincRNA). While we found little evidence supporting conservation of elincRNA exons, their splice sites and other splicing associated motifs accumulated significantly fewer mutations than neutrally evolving regions, suggesting that selection has acted to preserve their splicing. We showed that in contrast to other lincRNAs, not only were multi-exonic elincRNAs efficiently and rapidly spliced, they were also specifically associated with enhanced expression of proximal genes and increased local chromosomal interactions. Enrichment of chromatin signatures associated with high enhancer activity at these loci further supports the contribution of elincRNA splicing to enhancer function. Our results elucidate the previously observed evolutionary constraint at elincRNA splice sites, but not their exons, and corroborate the impact of elincRNA splicing modulation on neighboring gene expression regulation, highlighting an unexpected contribution of elincRNA splicing to enhancer function. Overall design: Mouse embryonic stem cells (mESCs) were labeled with 4-thiouridine for 15, 30 and 60 minutes in duplicates. Labeled and total RNA were sequenced on Illumina HiSeq 2500.