Pre-mRNA splicing is catalyzed by the spliceosome, a nanometer-scale cellular machine composed of small nuclear RNAs (snRNAs) and dozens of protein components. Spliceosomes assemble on pre-mRNA transcripts at particular locations and must become “activated” in order to catalyze the splicing reaction. Activation involves removal of of the U1 and U4 snRNPs, loss of several tri-snRNP proteins, rearrangement of the snRNAs and intron, and recruitment of the Prp19 complex (NTC). Ultimately this creates an enzyme active site into which the intronic splice sites and branch site can dock. Despite recent elucidation of several spliceosome structures by cryo-EM, the mechanism of activation remains largely unknown. We have constructed a library of yeast strains for mapping the pathways of spliceosome activation using Colocalization Single Molecule Spectroscopy (CoSMoS). Using this library, we are elucidating when splicing factors are released from or recruited to spliceosomes undergoing activation. We show that release of the heteroheptameric Lsm ring from the U6 snRNP occurs after destabilization of the U4 snRNP and recruitment of the NTC. These results strongly suggest a conformational change in the NTC during activation. Since the spliceosome appears to be avoiding loss of the Lsm ring in the absence of the NTC, we wondered if both the Lsm and NTC could share a common function within the spliceosome. We speculate that this function involves annealing and stabilizing helix II formed from the U2 and U6 snRNAs. In agreement with this hypothesis, purified Lsm rings can anneal U2 and U6 snRNAs. These results in combination with structural conservation between eukaryotic Lsm proteins and bacterial Hfq suggest that the RNA annealing function of bacterial Hfq is evolutionarily conserved in the Lsm ring found in eukaryotic spliceosomes.