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More than 200 proteins associate with human spliceosomes, but little is known about their relative abundances in a given spliceosomal complex. Here we describe a novel two-dimensional (2D) electrophoresis method that allows separation of high-molecular-mass proteins without in-gel precipitation and thus without loss of protein. Using this system coupled with mass spectrometry, we identified 171 proteins altogether on 2D maps of stage-specific spliceosomal complexes. By staining with a fluorescent dye with a wide linear intensity range, we could quantitate and categorize proteins as present in high, moderate, or low abundance. Affinity-purified human B, B(act), and C complexes contained 69, 63, and 72 highly/moderately abundant proteins, respectively. The recruitment and release of spliceosomal proteins were followed based on their abundances in A, B, B(act), and C spliceosomal complexes. Staining with a phospho-specific dye revealed that approximately one-third of the proteins detected in human spliceosomal complexes by 2D gel analyses are phosphorylated. The 2D gel electrophoresis system described here allows for the first time an objective view of the relative abundances of proteins present in a particular spliceosomal complex and also sheds additional light on the spliceosome's compositional dynamics and the phosphorylation status of spliceosomal proteins at specific stages of splicing.

The 17S U2 snRNP plays an essential role in branch point selection and catalysis during pre-mRNA splicing. Much remains to be learned about the molecular architecture of the U2 snRNP, including which proteins contact the functionally important 5′ end of the U2 snRNA. Here, RNA-protein interactions within immunoaffinity-purified human 17S U2 snRNPs were analyzed by lead(II)-induced RNA cleavage and UV cross-linking. Contacts between the U2 snRNA and SF3a60, SF3b49, SF3b14a/p14 and SmG and SmB were detected. SF3b49 appears to make multiple contacts, interacting with the 5′ end of U2 and nucleotides in loops I and IIb. SF3a60 also contacted different regions of the U2 snRNA, including the base of stem-loop I and a bulge in stem-loop III. Consistent with it contacting the pre-mRNA branch point adenosine, SF3b14a/p14 interacted with the U2 snRNA near the region that base pairs with the branch point sequence. A comparison of U2 cross-linking patterns obtained with 17S U2 snRNP versus purified spliceosomal A and B complexes revealed that RNA-protein interactions with stem-loop I and the branch site-interacting region of U2 are dynamic. These studies provide important insights into the molecular architecture of 17S U2 snRNPs and reveal U2 snRNP remodeling events during spliceosome assembly.