Pre-mRNA splicing by the spliceosome requires the precise and regulated efforts of the five snRNAs (U1, U2, U4, U5, and U6) and numerous associated proteins. Following assembly and activation of the spliceosome, two consecutive reactions result in intron removal and exon ligation from pre-mRNA substrates. It has been established that several members of the DExH/D-box family of helicases act transiently on the spliceosome prior to the chemical steps to authorize the successive reactions by hydrolyzing ATP and consequently inducing structural rearrangements. While it has been suggested that these changes produced in the structure of the spliceosome result in optimal positioning of the reactive species, the mechanisms and products of these reorganizations remain uncharacterized. The work presented here describes the genetic strategy for accumulating and purifying spliceosomes arrested in vivo, during the catalytic steps of the splicing cycle. Using these complexes, we have defined the components required to proceed through the first and second steps of splicing, in addition to the factors required for the release of the spliced message. Analysis of these functional, synchronized particles has also allowed us to define a function for Prp2p in initiating the first step of pre-mRNA splicing. Our data suggest that Prp2p may act in an ATP-independent manner to remodel the spliceosome prior to using its ATPase function to displace the SF3 complex. We propose that the SF3 complex, in addition to its role in identification of the branchpoint, also acts to sequester the reactive 2’OH of the branchpoint adenosine to prevent premature reactivity. Following the two catalytic steps of the splicing cycle, the spliceosome must disassemble and recycle its snRNPs for further rounds of splicing. The essential U6 snRNP component Prp24p, mediates one of the early assembly events - the annealing between the U4 and U6 snRNAs. We have discovered that although Prp24p is essential for viability, its function(s) can be bypassed by overexpressing the U6 snRNA. Additionally, biochemical characterizations of various forms of the U4/U6 snRNP provide evidence that Prp24p must be released before other components of the U4/U6 snRNP are permitted to interact and facilitate tri-snRNP formation. / text