The majority of messenger RNA sequences in eukaryotes are in fragments that need to be correctly spliced in order to be translated into functional proteins. Defects in pre-mRNA splicing have been linked to many human diseases including neurological dysfunction and cancer. The broad, long-term objectives of this grant are to understand the structure and function of the large ribonucleoprotein complex, called the spliceosome, that carries out the pre-mRNA splicing reaction. The spliceosome field has made tremendous progress since its conception almost 20 years ago; however, it is still not clear what factors exist in the core of the spliceosome and how the core is activated or remodeled for catalysis. This proposal will investigate the role of the U6 small nuclear RNA in spliceosome catalysis as well as the function of extrinsic splicing protein factors at the spliceosome core. Specific aim 1 is to study the 3' intramolecular stem-loop (ISL) of yeast U6 snRNA, which binds a magnesium ion required for the first transesterification reaction. We will test whether the magnesium at the U6/3'ISL is at the 5' splice site of the pre-mRNA by using double sulfur substitutions and rescuing with a thiophilic metal ion. We will use crosslinking strategies to test whether the U6/3'ISL is held in the spliceosome core by RNA or by protein. Specific aim 2 is to study how an RNA helicase called Prp2 remodels the core of the spliceosome. We will investigate how a prp2-suppressor protein or an intermolecular U2/U6 helix RNA contributes to the spliceosome recognition by Prp2. We will also use crosslinking and yeast genetics approaches to test the interaction between Prp2 and the spliceosomal RNAs. The specific aim 3 is to investigate whether the activation of the spliceosome by Prp2 is conserved in human. We will inhibit DBP2 by expressing a small interference RNA (siRNA) or a dominant-negative mutant targeting DBP2 in human cells. We will analyze the splicing machinery when DBP2 function is impaired in vivo or in vitro. The proposed study shall reveal the dynamic features and the remodeling of the spliceosome core in yeast and human.