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

We generate high-precision measurements of the in vivo rates of both chemical steps of pre-mRNA splicing across the genome-wide complement of substrates in yeast by coupling metabolic labeling, multiplexed primer-extension sequencing, and kinetic modeling. We demonstrate that the rates of intron removal vary widely, splice-site sequences are primary determinants of 1 step but have little apparent impact on 2 step rates, and the 2 step is generally faster than the 1 step. Ribosomal protein genes (RPGs) are spliced faster than non-RPGs at each step, and RPGs share evolutionarily conserved properties that may contribute to their faster splicing. A genetic variant defective in the 1 step of the pathway reveals a genome-wide defect in the 1 step but an unexpected, transcript-specific change in the 2 step. Our work demonstrates that extended co-transcriptional association is an important determinant of splicing rate, a conclusion at odds with recent claims of ultra-fast splicing.