Deep sequencing of subcellular RNA fractions shows splicing to be predominantly co-transcriptional in the human genome but inefficient for lncRNAs

Abstract

Splicing remains an incompletely understood process. Recent findings suggest that chromatin structure participates in its regulation. Here, we analyze the RNA from subcellular fractions obtained through RNA-seq in the cell line K562. We show that in the human genome, splicing occurs predominantly during transcription. We introduce the coSI measure, based on RNA-seq reads mapping to exon junctions and borders, to assess the degree of splicing completion around internal exons. We show that, as expected, splicing is almost fully completed in cytosolic polyA+ RNA. In chromatinassociated RNA (which includes the RNA that is being transcribed), for 5.6% of exons, the removal of the surrounding introns is fully completed, compared with 0.3% of exons for which no intron-removal has occurred. The remaining exons exist as a mixture of spliced and fewer unspliced molecules, with a median coSI of 0.75. Thus, most RNAs undergo splicing while being transcribed: ''co-transcriptional splicing.'' Consistent with co-transcriptional spliceosome assembly and splicing, we have found significant enrichment of spliceosomal snRNAs in chromatin-associated RNA compared with other cellular RNA fractions and other nonspliceosomal snRNAs. CoSI scores decrease along the gene, pointing to a ''first transcribed, first spliced'' rule, yet more downstream exons carry other characteristics, favoring rapid, co-transcriptional intron removal. Exons with low coSI values, that is, in the process of being spliced, are enriched with chromatin marks, consistent with a role for chromatin in splicing during transcription. For alternative exons and long noncoding RNAs, splicing tends to occur later, and the latter might remain unspliced in some cases.[Supplemental material is available for this article.]Central in the pathway leading from primary transcripts to mature functional RNAs is splicing, the process by which intervening sequences in the primary transcript (introns) are excised and the remaining sequences (exons) are concatenated together to form the mature eukaryotic RNAs. Conserved sequence motifs, the splice sites, mark exon-intron boundaries and are recognized by elements of the splicing machinery. Splice site sequences, however, do not carry enough information to unequivocally specify exon-intron boundaries, and a plethora of other sequence motifs, recognized by a variety of RNA binding proteins, contribute to define and regulate splice site selection (Graveley 2000;Smith and Valcárcel 2000;Wang and Burge 2008). While there have been considerable advances in modeling splicing from features in the primary transcript sequence (Wang et al. 2004;Barash et al. 2010), it is currently close to impossible to predict from the analysis of mammalian primary RNA sequence alone neither the entire exonintron structure of transcripts nor their tissue specific expression pattern (i.e., the abundance of given transcript in a given cell type).It appears thus that other factors, not necessarily encoded in the sequence of the primary transcript, may play a role in...

The landscape of human mutually exclusive splicing

“…To validate the predicted MXE candidates, we made use of over 15 billion publically available RNA‐Seq reads, selecting 515 samples comprising 31 tissues and organs, 12 cell lines and seven developmental stages (Barbosa‐Morais et al , 2012; Djebali et al , 2012; Tilgner et al , 2012; Xue et al , 2013; Yan et al , 2013; Fagerberg et al , 2014; Dataset EV1). The data were chosen to encompass common and rare potential splice events in a broad range of tissues, cell types and embryonic stages.…”

Co-expressed genes prepositioned in spatial neighborhoods stochastically associate with SC35 speckles and RNA polymerase II factories

“…In earlier differentiation experiments we have seen that higher passage number hMaDS cells did not respond to hormonal stimulation to the same extent as cells with low passage number. Splicing often occurs co-transcriptionally [47], so we asked if dispersal of SC35 splicing speckles might also be affecting gene expression. We performed quantitative Rt-PCR with hMaDS-CLK2GFP +DOX/−DOX and found that expression of the seven tested genes was decreased in the doxycycline-treated cells (supplemental information Fig.…”

“…It is known that most splicing is occurring co-transcriptionally [47] and that the splicing factor SC35 has an important role in transcriptional elongation [53]. association of highly active genes with the periphery of speckles brings them to an environment with highly increased concentrations of splicing factors and therefore enables more efficient transcription and processing.…”

The role of long non-coding RNAs in neurodevelopment, brain function and neurological disease

“…Interestingly, a recent study by Tilgner et al [29] showed that the efficiency of lncRNA splicing is significantly lower than for mRNAs, and that many lncRNA transcripts (including well-studied functional examples such as Airn and KCNQ1OT1) remain unspliced.…”