Pre-mRNA splicing is an essential step in eukaryotic gene expression. The processes of intron excision and exon ligation are carried out by a mega-Dalton complex of RNAs and proteins called the spliceosome. This proposal seeks to develop and utilize new chemical and biophysical methods to study the mechanism of pre-mRNA splicing at the single molecule level. During the mentored phase, methods will be developed to site-specifically fluorescently label pre-mRNAs and yeast spliceosomal proteins in order to observe their dynamics during a complete splicing reaction by single molecule fluorescence microscopy. This work will focus on addressing questions relevent to the transition of the spliceosome between an enzyme capable of carrying out lariat formation and an enzyme competent for exon ligation, including direct observation of the interaction of DExD/H-box proteins/fidelity factors such as Prp16 and Prp22 with the spliceosome. During the independent phase, a transition will be made to the human spliceosome. This work will focus on questions specific to human splicing such as the influence of SR proteins on pre-mRNA dynamics during the chemical steps of splicing. In addition, chemical methods will be developed and employed to provide a high kinetic resolution analysis of 3'splice site recognition and exon ligation using photocaging groups and light to trigger these events in solution and at the single molecule level. I have received extensive training in mechanistic enzymology and nucleotide synthesis while studying de novo purine biosynthesis as a graduate student with JoAnne Stubbe at MIT. As a result, I wish to employ chemical and detailed kinetic methods to the study of complex cellular machines such as the spliceosome during my independent career. As a NIH NRSA postdoctoral research fellow with Melissa Moore and Jeff Gelles, I have expanded my training to include RNA biochemistry, yeast genetics, and single molecule fluorescence microscopy. This enivronment has allowed me to formulate a career path in which I will be able to use chemistry and phyiscs to provide unique and fundamental insight into human biology.
Pre-mRNA splicing is an essential step in human gene expression and is therefore of fundamental importance to the study of human health and biology. The vast majority of human genes are alternatively spliced and defects in this process may account for >15% of human genetic diseases. The research proposed here will have a significant impact on human health by elucidating this step in gene expression.
