Fig. 1

Schematic overview of the approach to characterize the interdependencies between mRNA transcription initiation and processing events. a Identified full-length reads (reads with RNA inserts between 5′ and 3′ primers) are clustered into unique transcript structures using the ICE algorithm and further polished using the partial reads, where one of the primer sequences is missing. b Based on available transcripts per locus, available sequence (union of all exonic sequences that are observed at each locus) and unique set of features and splice sites are identified. Feature sets comprise unique transcriptional start sites (TSS), exons, and polyadenylation sites (PAS). The unique set of splice sites consists of unique donor and acceptor splice sites as well as all alternative TSSs and PASs. c The survey of coupling events is done by performing all possible pairwise tests between unique features in genes. The sum of the coverage of all transcripts that support the inclusion or exclusion of each pair is used in a contingency table to perform a Fisher’s exact test for statistical significance. The odds ratio (OR) is used to differentiate between mutually inclusive and exclusive coupling. d Set of interdependent coupling events were identified based on networks of coupling between features in each gene. Nodes represent features and links depict the mutual inclusivity (black edges) or mutual exclusivity (red edges) of each feature pair. Unique network components can thereby be filtered based on the type of interaction: mutual inclusive or mutual exclusive coupling events. e For all alternative exons that show significant coupling, a motif search is performed to assess the enrichment of specific RNA-binding protein motifs. For all alternative exons, 35-bp intronic sequences upstream of the acceptor site are defined as R1 domain (depicted in orange), 32-bp exonic sequences downstream of the acceptor site and upstream of the donor site are defined as R2 domain (depicted in dark gray), and 40-bp intronic sequences downstream of the donor site are defined as R3 domain (depicted in purple); 35-bp sequence upstream of each PAS (depicted in red) is searched for the presence of canonical and non-canonical poly(A) signals