Intron looping is mediated during transcription by U1 snRNP and RNA polymerase II [RIP-seq]

In the earliest step of spliceosome assembly, the two splice sites flanking an intron are brought into proximity by U1 snRNP and U2AF. The mechanism that facilitates this intron looping is poorly understood. Using a CRISPR interference-based approach to halt RNA polymerase II transcription in the middle of introns, we discovered that the 5 splice site base pairs with a U1 snRNA that is tethered to RNA polymerase II during intron synthesis. Correlation with splicing outcomes demonstrate that these associations are functional. The interactions between 5 splice sites, U1 snRNP, and elongating RNA polymerase II occurs genome-wide. Our findings reveal that during intron synthesis the upstream 5 splice site remains attached to the transcriptional machinery and is thus brought into proximity of the 3 splice site to enable rapid splicing. Overall design: RNA-ChIP-Seq of Pol2-ser2 and U1 and double RNA-ChIP-Seq of Pol2-ser2-U1 with and without AMT treatment Please note that the processed data for the *_AMT samples is generated from a merged version of both replicates and is linked to the corresponding *rep2 sample records.