Pre-mRNA splicing is a fundamental process governing gene expression in eukaryotes and is orchestrated by the spliceosome, a dynamic ribonucleoprotein complex. Spliceosomes catalyze splicing in two steps and are composed of both small nuclear RNAs (snRNAs; U1, U2, U4, U5, U6) and numerous proteins. Dynamic interactions between snRNAs, proteins, and the pre-mRNA substrate occur during splicing which are central to regulating splice site recognition, catalysis, and fidelity. This thesis examines multiple facets of RNA-protein interactions within spliceosome complexes beginning with snRNA biogenesis, through formation of the spliceosome active site, and during the second step of chemistry, exon ligation. Functional mechanisms into splicing, such as selection of intron recognition sites, are examined. Function of the main catalytic component of the spliceosome, the U6 snRNA, is first explored. Transcription and post-transcriptional processing of U6 is unique among snRNAs and therefore may be required to generate a functional U6. Transcription by RNAP II instead of RNAP III produces a functional U6 molecule. Defects in stability, likely a result of incorrect post-transcriptional processing and binding of stabilizing proteins, result in changes to in vivo distributions of spliceosome sub particles called snRNPs. Transcription of U6 by RNAP II is useful for the incorporation of genetic tags for endogenous fluorescent labeling or purification. Additionally, new applications of endogenous fluorescent labeling techniques within the U4 snRNA are presented, paving the way for single molecule studies of snRNA dynamics and Brr2 helicase function during activation. Mango and MS2 tags are well tolerated in the yeast U4 snRNA and with several of the tagged U4 constructs also minimally impacting splicing activities. Future single molecule experiments will examine the timing of the U4 snRNA release from the spliceosome compared to the release of Prp3, a protein associated with U6 and U4 snRNAs. Finally, validation of a proposed novel splicing factor, Fyv6, and study of its influence on 3′ SS represent major contributions to the field of splicing. Utilizing a new high resolution P complex structure of the spliceosome containing Fyv6 solved by Max Wilkinson, I examined multiple contacts of Fyv6 with other splicing factors, notably Prp22. Genetic studies in yeast show that interactions with the protein Syf1 and Prp22 are important for Fyv6 function. The absence of Fyv6 from spliceosomes results in transcriptome-wide splicing defects, largely due to changes in 3′ SS usage. From these studies, Fyv6 can be added to the list of splicing factors that impact the second step of splicing and affect fidelity of 3′ SS selection.