Abstract
RNA splicing is an essential part of eukaryotic gene expression. Although the mechanism of splicing has been extensively studied in vitro, in vivo kinetics for the two-step splicing reaction remain poorly understood. Here, we combine transient transcriptome sequencing (TT-seq) and mathematical modeling to quantify RNA metabolic rates at donor and acceptor splice sites across the human genome. Splicing occurs in the range of minutes and is limited by the speed of RNA polymerase elongation. Splicing kinetics strongly depends on the position and nature of nucleotides flanking splice sites, and on structural interactions between unspliced RNA and small nuclear RNAs in spliceosomal intermediates. Finally, we introduce the 'yield' of splicing as the efficiency of converting unspliced to spliced RNA and show that it is highest for mRNAs and independent of splicing kinetics. These results lead to quantitative models describing how splicing rates and yield are encoded in the human genome.
Highlights
Transcription of eukaryotic genes produces precursor RNA molecules that are processed by splicing
These observations were consistent with capture of newly synthesized precursor RNA because spliced introns are more rapidly degraded than exons that are maintained in mature, stable RNA
Even though the contributions of several mechanistic processes to the observed kinetics cannot be disentangled, our results reveal which nucleotide positions around splice sites are critical for fast splicing kinetics
Summary
Transcription of eukaryotic genes produces precursor RNA molecules that are processed by splicing. Previous studies have measured how fast introns are removed, but it remained unclear how long it takes to cut individual splice sites genome-wide To address this question, Wachutka, Caizzi et al combined a mathematical approach with a biochemical method that purifies newly made RNA in human cells. The combination of single molecule intron tracking (SMIT) and long read sequencing in yeast shows that splicing is 50% complete when Pol II is 45 nt downstream the acceptor spice site (Oesterreich et al, 2016) Despite these advances, the in vivo kinetics of splicing remain poorly understood. Our analysis provides genome-wide metabolic rates for donor and acceptor splice sites and identifies RNA-RNA interactions in the spliceosome that could contribute to in vivo splicing kinetics. From this work emerges a comprehensive global view of splicing kinetics and yield in human cells
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