The Development of Methods for the Site-Specific Fluorescent Labeling of Spliceosomal Proteins for use in Single-Molecule Studies

Abstract

Spliceosomes are multi-megadalton ribonucleoprotein (RNP) complexes responsible for catalyzing the removal of noncoding introns from eukaryotic precursor messenger RNA (pre-mRNA) transcripts and ligating the flanking coding exon sequences to produce a mature messenger RNA (mRNA). Spliceosomal assembly and catalysis require a highly dynamic coordination of proteins and RNAs with the purpose of producing mature mRNA that can be properly transcribed by the ribosome. Due to the complexity of the process and a lack of suitable tools, there is still much unknown about the assembly of the spliceosome. Single molecule fluorescence microscopy tools have recently been developed for studies of spliceosome assembly and dynamics of the pre-mRNA substrate throughout splicing. In an effort to advance the study of splicing and delineate the exact timing and dynamics required to achieve splicing, we are currently investigating methods for the site-specific labeling of proteins, one of these methods being protein trans-splicing (PTS) using a split intein. Protein splicing is a naturally occurring process in which a protein editor, called an intein, excises itself out of a host protein in which it is embedded creating a new peptide bond between its two flanking regions, the exteins. PTS uses an artificially or naturally split intein to create a new peptide bond between flanking exteins similar to protein splicing. Upon incubation of the two components, protein splicing will take place, resulting in the ligation of the two extein regions. By fluorescently labeling one of the extein components, we hope to use PTS to site-specifically label several spliceosomal proteins with the ultimate goal of correlating the association and dissociation of spliceosomal components with pre-mRNA dynamics in order to gain a better understanding of the mechanism of pre-mRNA splicing.