The patterns and mechanisms by which eukaryotic cells regulate the expression of their genetic information are highly complex and intricate. The transmittance of this information from nuclear repository to cytoplasmic translation contains within it several steps, including the selective removal and concomitant joining of pieces of information in a process called alternative splicing. The projects detailed within this document describe the regulation of alternative splicing through the interaction of specific proteins with specific pre-mRNA transcripts.

The Rio lab has studied PSI, a protein involved in the regulation of the P element transposase transcript, for many years. It has since been shown to regulate the splicing of hundreds of other transcripts. The experiments described here look at the organization of PSI and other proteins on the P element transcript by site-specific labeling of the transcript using radioactive 32P. We also investigate two phosphorylation events of PSI, identifying the kinases responsible and demonstrate that these events may change the protein-protein interaction partners of PSI.

It has become increasingly apparent that alternative splicing may not only be regulated by protein/RNA interactions, but also by RNA/RNA interactions. To probe this, we designed experiments to test if some well-known small RNA-associated proteins are regulating alternative splicing. Using splice junction microarrays, we determined that Argonaute-2 (Ago-2) regulated the splicing of over 100 splice junctions, and further experiments using ChIP-seq and mRNA-seq of Ago-2 mutants revealed that Ago-2 also has a role in transcriptional repression, possibly through being incorporating in complexes composed of polycomb-group genes. We also used CLIP-seq to determine the RNA binding profile and preferences of Ago-2 in Drosophila tissue culture cells.

Finally, we characterized the functions of a Drosophila specific splicing factor called LS2. LS2 is orthologous to the highly conserved splicing factor dU2AF50, but its origin through retroduplication and subsequent divergence to acquire distinct sequence specificity, expression pattern, and function show it to be an interesting case in the evolution of alternative splicing regulation. This may be a mechanism that underlies the existence of some members of the large families of splicing factors, including hnRNP proteins and SR proteins. That is, by duplicating functional copies of genes, cellular systems create new proteins to tinker with and acquire new functions while keeping the former functionality and stability of the parent protein.

While these projects are essentially independent of each other, they all fall under the umbrella of protein regulation of RNA metabolism and hopefully contribute to a more complete understanding of the regulation of gene expression.