Conservation of the Protein Composition and Electron Microscopy Structure of Drosophila melanogaster and Human Spliceosomal Complexes

Abstract

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.

SUPPLEMENTAL MATERIAL

Supplemental material for this article may be found at http://mcb.asm.org/ .

ACKNOWLEDGMENTS

We are grateful to Gabi Heyne, Thomas Conrad, and Hossein Kohansal for excellent technical assistance. We also thank Monika Raabe and Uwe Plessmann for their excellent help in MS analysis, Reinhard Rauhut for help in homology searches, and Christian Merz for help in preparing MS2-MBP and also for providing PCR template for transcription of the Ftz-M3 pre-mRNA.

This work was supported by grants from the DFG, the European Commission (EURASNET-518238), Fonds der Chemischen Industrie, and the Ernst Jung Stiftung to R.L. and a YIP grant from EURASNET to H.U.