Comprehensive proteomics analyses of spliceosomal complexes are currently limited to those in humans, and thus, it is unclear to what extent the spliceosome's highly complex composition and compositional dynamics are conserved among metazoans. Here we affinity purified Drosophila melanogaster spliceosomal B and C complexes formed in Kc cell nuclear extract. Mass spectrometry revealed that their composition is highly similar to that of human B and C complexes. Nonetheless, a number of Drosophila-specific proteins were identified, suggesting that there may be novel factors contributing specifically to splicing in flies. Protein recruitment and release events during the B-to-C transition were also very similar in both organisms. Electron microscopy of Drosophila B complexes revealed a high degree of structural similarity with human B complexes, indicating that higher-order interactions are also largely conserved. A comparison of Drosophila spliceosomes formed on a short versus long intron revealed only small differences in protein composition but, nonetheless, clear structural differences under the electron microscope. Finally, the characterization of affinity-purified Drosophila mRNPs indicated that exon junction complex proteins are recruited in a splicing-dependent manner during C complex formation. These studies provide insights into the evolutionarily conserved composition and structure of the metazoan spliceosome, as well as its compositional dynamics during catalytic activation.Pre-mRNA splicing is catalyzed by the spliceosome, a large RNP molecular machine consisting of the U1, U2, U4/U6, and U5 snRNPs plus a multitude of non-snRNP proteins (reviewed in reference 59). Spliceosome assembly is an ordered, highly dynamic process that leads to the stepwise formation of the catalytic site(s) responsible for catalyzing the excision of an intron of a pre-mRNA and ligation of its flanking exons (reviewed in reference 59). Initially, the U1 snRNP interacts with the conserved 5Ј splice site (5Ј ss) of the pre-mRNA, forming the spliceosomal E complex, and in the next step the U2 snRNP stably interacts with the pre-mRNA's branch site, leading to the A complex (i.e., prespliceosome). The preformed U4/U6.U5 tri-snRNP particle then interacts with the A complex to generate the precatalytic B complex. The latter is converted to the catalytically activated B* complex, a process involving major RNP rearrangements, including destabilization or loss of the U1 and U4 snRNPs. The first step of splicing subsequently ensues and involves a nucleophilic attack of the branch point adenosine at the 5Ј ss, generating a cleaved 5Ј exon and intron-3Ј exon lariat intermediate. The spliceosomal C complex is formed at this time and subsequently catalyzes the second step of splicing, which entails intron excision and concomitant ligation of the 5Ј and 3Ј exons to form mRNA. The mRNA, in the form of an RNP, is then exported to the cytoplasm for translation by the ribosome.During spliceosome assembly, a complex RNA-RNA network involving the snR...