Transcript-specific determinants of pre-mRNA splicing revealed through in vivo kinetic analyses of the 1st and 2nd chemical steps

Abstract

Understanding how the spliceosome processes its composite of substrates through the two chemical steps required for mRNA production will be essential to deciphering splicing regulation and mis-regulation. Here we measure the in vivo rates of these steps across the genome by coupling metabolic RNA labeling, targeted sequencing, and first order kinetic modeling. We reveal a wide variety of rates by which introns are removed, that splice site sequences are primary determinants of 1st step rates, and that the 2nd step is generally faster than the 1st step. We show that ribosomal protein genes (RPGs) are spliced faster than non-RPGs at each step, and that RPGs share evolutionarily conserved cis-features which facilitate their splicing. A genetic variant defective at the 1st step shows the expected defect in the 1st step, but an unexpected change in the 2nd step which suggests how co-transcriptional splicing functions as an important determinant of splicing rates.