A Single-Molecule FRET Strategy that Enables the Ordering the Proximities of Yeast Specific U1 Proteins to the 5 Splice Site

Abstract

In the first step of formation of spliceosome toward catalyzing mRNA formation, U1 particle would come to recognize the 5 splice site on the pre-mRNA. Later, a ATPase/helicase named Prp28 comes in to disrupt the U1/pre-mRNA base-pairing to prepare the pre-mRNA to engage with the U6 particle, which is a ribozyme that will perform the catalysis. In yeast, however, it was discovered that mutations on several yeast-specific U1 proteins including Snu71p would allow the system to bypass the control by Prp28. In order to understand the bypass mechanism, we set out to study the communication between those yeast-specific proteins and the 5 splice site. To facilitate such, we have created a single-molecule strategy to measure the proximity between 5 splice site and a yeast-specific protein. In brief, the fluorescence resonance energy transfer experiment is designed for a donor labeled on the yeast-specific protein and an acceptor on the 5 splice site. The donor is carried by an off-resistant donor-labeled calmodulin that is also immobilized on the microscope slide surface while the 5 splice site labeled by an acceptor, of which the presence is monitored by a modified alternating laser excitation that can reduce the photo-damage of the dye.