GSE108059
Spliceosome profiling visualizes the operations of a dynamic RNP in vivo at nucleotide resolution
Tools to understand how the spliceosome functions in vivo have lagged behind advances in its structural biology. We describe methods to globally profile spliceosome-bound precursor, intermediates and products at nucleotide resolution. We apply these tools to three divergent yeast species that span 600 million years of evolution. The sensitivity of the approach enables detection of novel cases of non- canonical catalysis including interrupted, recursive and nested splicing. Employing statistical modeling to understand the quantitative relationships between RNA features and the data, we uncover independent roles for intron size, position and number in substrate progression through the two catalytic stages. These include species-specific inputs suggestive of spliceosome-transcriptome coevolution. Further investigations reveal ATP-dependent discard of numerous endogenous substrates at both the precursor and lariat-intermediate stages and connect discard to intron retention, a form of splicing regulation. Spliceosome profiling is a quantitative, generalizable global technology to investigate an RNP central to eukaryotic gene expression. Overall design: Spliceosome profiling of samples from S. cerevisiae, S. pombe and C. neoformans. Detailed description of the method can be found in the supplementary PDF file. 2 replicates of each sample type are analyzed (3'' end profiling, spliceosome-bound RNA-seq, polyA RNA-seq, junction profiling and branch profiling.