Spliceosome profiling visualizes the operations of a dynamic RNP in vivo at nucleotide resolution

摘要: Tools to understand how the spliceosome functions in vivo have lagged behind advances its structural biology. We describe methods globally profile spliceosome-bound precursor, intermediates and products at nucleotide resolution. apply these tools three divergent yeast species that span 600 million years of evolution. The sensitivity approach enables detection novel cases non-canonical catalysis including interrupted, recursive nested splicing. Employing statistical modeling quantitative relationships between RNA features data, we uncover independent roles for intron size, position number substrate progression through two catalytic stages. These include species-specific inputs suggestive spliceosome-transcriptome coevolution. Further investigations reveal ATP-dependent discard numerous endogenous substrates both precursor lariat-intermediate stages connect retention, a form splicing regulation. Spliceosome profiling is quantitative, generalizable global technology investigate an RNP central eukaryotic gene expression.