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P element transposase catalyzes the mobility of P element DNA transposons within the Drosophila genome. P element transposase exhibits several unique properties, including the requirement for a guanosine triphosphate cofactor and the generation of long staggered DNA breaks during transposition. To gain insights into these features, we determined the atomic structure of the Drosophila P element transposase strand transfer complex using cryo-EM. The structure of this post-transposition nucleoprotein complex reveals that the terminal single-stranded transposon DNA adopts unusual A-form and distorted B-form helical geometries that are stabilized by extensive protein-DNA interactions. Additionally, we infer that the bound guanosine triphosphate cofactor interacts with the terminal base of the transposon DNA, apparently to position the P element DNA for catalysis. Our structure provides the first view of the P element transposase superfamily, offers new insights into P element transposition and implies a transposition pathway fundamentally distinct from other cut-and-paste DNA transposases.

The polypyrimidine tract binding protein (PTB) binds pre-mRNAs to alter splice-site choice. We characterized a series of spliceosomal complexes that assemble on a pre-mRNA under conditions of either PTB-mediated splicing repression or its absence. In the absence of repression, exon definition complexes that were assembled downstream of the regulated exon could progress to pre-spliceosomal A complexes and functional spliceosomes. Under PTB-mediated repression, assembly was arrested at an A-like complex that was unable to transition to spliceosomal complexes. Trans-splicing experiments indicated that, even when the U1 and U2 small nuclear ribonucleoprotein particles (snRNPs) are properly bound to the upstream and downstream exons, the presence of PTB prevents the interaction of the two exon complexes. Proteomic analyses of these complexes provide a new description of exon definition complexes, and indicate that splicing regulators can act on the transition between the exon definition complex and an intron-defined spliceosome.

The Down syndrome cell adhesion molecule (Dscam) gene has essential roles in neural wiring and pathogen recognition in Drosophila melanogaster. Dscam encodes 38,016 distinct isoforms via extensive alternative splicing. The 95 alternative exons in Dscam are organized into clusters that are spliced in a mutually exclusive manner. The exon 6 cluster contains 48 variable exons and uses a complex system of competing RNA structures to ensure that only one variable exon is included. Here we show that the heterogeneous nuclear ribonucleoprotein hrp36 acts specifically within, and throughout, the exon 6 cluster to prevent the inclusion of multiple exons. Moreover, hrp36 prevents serine/arginine-rich proteins from promoting the ectopic inclusion of multiple exon 6 variants. Thus, the fidelity of mutually exclusive splicing in the exon 6 cluster is governed by an intricate combination of alternative RNA structures and a globally acting splicing repressor.