Elucidating biochemical mechanisms in eukaryotic RNA metabolism with chemistry, biology, and single molecule analysis
In the cell, RNA is built, processed, and degraded by a number of macromolecular machines. Many of these machines are composed of dozens, or even hundreds, of protein components. Studying the underlying biochemistry of these enzymes is particularly challenging given these huge numbers of parts. Work in my laboratory utilizes a number of assays, including multi-wavelength single molecule fluorescence microscopy, to elucidate these processes.
We are particularly focused on studies of pre-mRNA splicing--the process of removing introns from nascent transcripts. This reaction is carried out by the spliceosome. For every intron that is removed, the spliceosome is built, activated for catalysis, and disassembled on the pre-mRNA transcript. This is a remarkable feat of cellular engineering involving the coordinated actions of ~100 proteins and 5 snRNAs
In order to study this process, we simplify our system by studying one spliceosome at a time using CoSMoS: Co-localization Single Molecule Spectroscopy. In this technique, biomolecules (such as pre-mRNAs) are fluorescently labeled and tethered to a glass or silica surface. We then study association of other fluorescent bio-molecules (such as spliceosomes) with the tethered biomolecules. By analyzing the comings and goings of many molecules, we are able to provide mechanistic insight into complex systems even in a whole cell extract!
Recognition of Splice Sites during Spliceosome Assembly and Activation
Arguably, the most important function of the spliceosome is not the chemistry per se but determining the correct location of the chemistry. The spliceosome is not just a machine--it is an intelligent machine! This project focuses on using kinetic measurements to determine how the spliceosome chooses the 5' splice site and branchsite in a pre-mRNA sequence. Work on this project makes use of single molecule fluorescence and ensemble experiments to study these events. In addition to carrying out these reactions in cell extracts, efforts in this area will also include work on purified systems, the use of yeast genetic engineering, and many types of biochemical assays.
Coupling between Eukaryotic RNA Processing Events
The spliceosome does not function in isolation within the cell's nucleus. Many experiments have suggested a tight coupling of nuclear RNA processing events including chromatin remodeling, transcription, splicing, capping, poly-A tail formation, RNA decay, and transport into the cytosol. The work in this area focuses on understanding the biochemical mechanisms behind coupling of splicing with two processes: transcription and RNA capping. These projects will make use of both single molecule and many molecule experiments to uncover the biochemistry behind these phenomena.
Chemical Tools for Modifying RNPs in vitro and in vivo
While currently there exist many methods for studying proteins in cells by fluorescence, analysis of in vivo RNAs is limited and often confined to analysis of mRNAs rather than structured ribonucleoproteins (RNPs). The goal of this research is to develop new chemical tools for studying RNPs in vitro and in vivo in both wild type and disease-related backgrounds. This project will combine organic synthesis, biochemical assays, and cell biology to create "RNA Tags" for cellular imaging.