Quantitative modeling of 5' splice site subclass regulation and evolution

Abstract

Pre-mRNA splicing is an essential molecular process required for eukaryotic gene expression. In this thesis, I present a previously unknown mechanism of splicing regulation where a family of splicing factors, the LUC7 family, compete to differentially impact 5→ splice site (5→ SS) selection in a sequence-dependent manner. I quantitatively characterize two major subclasses of 5→ SS in eukaryotes and outline distinctive features of 5→ SS in exons affected by the three human LUC7 paralogs: LUC7L2 and LUC7L enhance splicing of “right-handed” 5→ SS that exhibit stronger consensus matching on the intron side of the nearly-invariant / GU, while LUC7L3 boosts splicing of “left-handed” 5→ SS with stronger consensus matching upstream of the /GU. Using a range of experimental systems, from human cells to mutant plants, I show that LUC7 paralogs have opposing effects on these two 5→ SS subclasses and that this regulatory mechanism likely originated in the last common ancestor of animals and plants over 1.5 billion years ago. I further evaluate a competing model of 5→ SS subclass regulation involving METTL16- mediated U6 snRNA modification and reconcile both models by devising computational tools that identify sequence features predictive splicing dysregulation in transcriptome-wide datasets. Finally, I examine the evolutionary dynamics of left- and right-handed 5→ SS and propose a model of intron evolution in which codon and intron phase constraints in protein-coding genes shape both minor-to-major intron conversion and transitions between left- and right- 5→ SS subclasses.