Reconstitution of the spliceosomal U1 snRNP from all recombinant subunits and its characterisation by ionspray Q-tof mass-spectrometry.
Muto Y., Krummel DA., Kambach C., Hernandez H., Robinson CV., Nagai K.
The first important step in pre-mRNA splicing is the recognition of the 5' splice site by the U1 snRNP. It consists of U1 snRNA and 10 protein subunits. We have reconstituted the U1 snRNP from all its ten proteins produced in E. coli and U1 snRNA transcribed in vitro. We have used nano spray time-of-flight (TOF) mass spectrometer in order to characterise the reconstituted U1 snRNP and its sub-assemblies which lack one of more subunits. The reconstituted U1 snRNP and its variants remained intact as multiply charged ions within the mass spectrometer and their mass was determined. By increasing collision energy subparticles are also observed. This method provides information not only about the stoichiometry of subunits within the complex but also about subsets of interacting proteins.