Structure, regulation, and evolution of the splicing machinery

PROJECT SUMMARY
The complexity of human splicing is daunting, yet intervention in splicing for treatment of diseases holds
huge potential. Based on strong preliminary results, we propose three areas of investigation that leverage our
group’s deep knowledge of splicing to address critical open questions, and to explore the potential for innovative
engineering. The first area addresses the mechanism by which U2 snRNP captures the intron branchpoint
early in spliceosome assembly, a step altered by recurrent cancer mutations and targeted in nature by
antibiotic-producing bacteria. Using new reporters in which two branchpoints compete for recognition, we have
identified a novel splicing fidelity mechanism we call “NO-BP decay,” in which U2 complexes that fail due to
aberrant branchpoint selection are destroyed. We will characterize this process, applying a battery of candidate
gene-based suppressor screens and biochemical tests in splicing extracts. The second area of investigation
addresses how splicing is integrated with transcription and cell growth at the individual gene and cellular
levels, an emerging area in need of innovation if splicing is to be successfully engineered. Preliminary results
indicate that yeast cells have a limited capacity for splicing that creates competition for pre-mRNAs that is critical
to cell function. We will measure both splicing capacity and the dynamics of competition, using RNA sequencing
to develop a predictive model that explains how splicing is coordinated at a systems level. To understand the
contribution of individual genes to this system we are applying synthetic biology approaches. We have
engineered site-specific pauses of RNA polymerase II and shown that they alter splicing efficiency and
alternative splicing, by unknown mechanism(s) that we will dissect. We will also explore in detail the role of
splicing noise (stochastic variations in splicing output over time) on the ability of splicing to control stable
homeostatic expression settings (as it does in many RNA binding protein genes) as well as to control a bistable
switch (as it does in the Drosophila Sex lethal gene). These experiments will define the operational principles of
simple splicing regulatory circuits. The third area of investigation is focused on the process of intron gain
and its roles in eukaryotic gene creation and gene diversification. Our recent discovery that the spliceosome
can convert the lariat intron to a true intron circle after splicing indicates that it can carry out reverse splicing
reactions in vivo, raising questions about whether and how it might promote formation of new introns. We
propose to test biochemical steps predicted to be necessary for spliceosome-mediated intron gain, and have
already set up experiments to document intron gain in vivo. Given the fundamental conservation of the splicing
machinery, this work promises to translate directly into new understanding of the mechanisms of gene regulation
in eukaryotes, including humans. Defects in splicing are frequently recognized as contributors to disease, and
interventions that address splicing defects are increasingly successful pathways to treatment.

Grant Number: 5R35GM145266-05
NIH Institute/Center: NIH
Principal Investigator: Manuel Ares