Study of the RNA splicing kinetics via in vivo 5-EU labelling

Splicing, a process of intron removal from eukaryotic RNA transcripts, is an important step of gene expression in all eukaryotes. Splice sites might be used with different efficiency giving rise to alternative splicing products. At the same time, splice sites might be utilised with a variable rate. In this work we aimed to determine splicing rates for each donor and acceptor splice sites used in HeLa cells. Nascent RNA transcripts undergoing splicing comprise a very small share of total cellular RNA. In order to observe a higher share of intermediates we performed 10 and 20 minutes pulse labelling of nascent RNA in HeLa cell culture by 5-ethynyl uridine (EU) followed by nascent RNA purification and high throughput sequencing. Each experiment was done in 3 biological replicates. Additionally, we performed standard polyA+ RNA-seq to determine the steady state level of unexcised introns to account for intron retention events.