RNA processing has emerged as an important step in post-transcriptional gene regulation. This grant will detail experiments designed to understand a subset of these processes, pre-mRNA 3'end processing and siRNA production, and their potential link via an important scaffolding protein, Symplekin, in Drosophila melanogaster. All eukaryotic pre-mRNAs require processing of their 3'ends to form mature mRNAs. Replication-dependant histone pre-mRNAs are only cleaved at the 3'end, while all other eukaryotic mRNAs require cleavage followed by polyadenylation to form a mature message. Interestingly, even though this variation in 3'end processing necessitates differences in many cis and trans acting factors, recent data in Drosophila indicate that 3'end processing of all eukaryotic mRNAs is performed by the same protein complex. This complex, the core cleavage complex, contains three proteins: CPSF73, CPSF100 and Symplekin. While we understand the biological role of this complex, the molecular requirements for its assembly and activity have not been determined. Recently, a new class of biomolecules, small RNAs, has emerged as being integral for proper cellular function. These small interfering or micro RNAs (siRNAs or miRNAs, respectively) are derived from both genomic and extracellular sources and interact with larger RNA molecules to affect downstream cellular processes such as transcription, translation and heterochromatin formation. Our preliminary experiments suggest that Symplekin, the essential component of the core cleavage complex described in the previous paragraph, interacts with another protein, Dicer-2, to cut siRNAs from larger precursor RNAs. While my previous work with Symplekin detailed its importance in pre- mRNA 3'end processing, its involvement in the siRNA maturation pathway is a novel discovery. Specifically, we aim to characterize molecular interactions in the core cleavage factor, determine the role of Symplekin in Drosophila siRNA metabolism and investigate the potential link between pre-mRNA 3'end processing and siRNA biogenesis. We hypothesize that Symplekin participates in siRNA biogenesis and that the siRNA production pathway in Drosophila is intimately connected to pre-mRNA 3'end processing via Symplekin. Experiments detailed in this grant will test this hypothesis.
Aberent post-transcriptional gene regulation can cause disease, including cancer. The experiments described in this grant aim to dissect two aspects of post- transcriptional gene regulation at the molecular level. First, we hope to better understand interactions required to form a functional mRNA 3'end core cleavage complex. Second, we investigate production of a new class of small RNAs, endo- siRNAs. Finally, we try to understand how these two RNA pathways function in concert. Data gathered from these experiments will further our knowledge of post- transcriptional gene regulation, therefore, promoting our understanding of disease.