A kinetic mechanism for U6 snRNP biogenesis obtained by single molecule fluorescence colocalization microscopy.

Abstract

Pre-mRNA splicing is the process of removing introns and forming a mature mRNA product by the spliceosome. The U6 small nuclear RNA ribonucleoprotein (snRNP) is an essential component of the splicing machinery and is composed of the U6 snRNA, Prp24, and Lsm2-8 heteroheptameric ring. In the complex, there are both RNA-protein and protein-protein interactions that play critical roles in snRNP assembly. Here, we are studying how the Lsm2-8 ring is recruited to the U6 snRNA in real-time using single-molecule fluorescence colocalization microscopy (CoSMoS). By using fluorescently labeled RNAs and proteins, we have analyzed single molecule reaction trajectories to determine the initial kinetic parameters for snRNP formation and how these change due to post-transcriptional modification of the RNA. We have also elucidated the function of highly-conserved protein domains in Prp24 in both splicing factor recruitment and stabilization of the RNP. Overall, our single-molecule results allow us to propose a kinetic mechanism for snRNP assembly and elucidate how assembly can proceed efficiently in both the presence and absence of RNA modifications.