Project 1: Alternative Polyadenylation in Cancer. In non-proliferating or non-transformed cells, the position of cleavage and polyadenylation is located as distal as possible relative to the stop codon. The use of this distal poly(A) site (dPAS) ensures maximal negative regulation by microRNAs as well as AU-rich destabilizing elements. The translational output of these longer mRNAs is therefore under more exquisite control by the cell. However, during the processes of transformation, tumor progression, or enhanced proliferation; the decision of where to put the poly(A) tail is altered and a more proximal poly(A) site (pPAS) is chosen. This typically does not change sequences within the open reading frame, the reduced 3’UTR length removes negative regulatory elements increasing protein expression. In the Wagner laboratory, we are focused on the mechanism of this change and the critical target mRNAs that undergo this process. We use a combination of cellular and in vivo models to investigate APA as it relates to tumor biology.
Alternative Polyadenylation (APA). In nontransformed cells a distal poly(A) site is used. After transformation, a proximal site is used and the mRNA is translated more efficiently.
Project 2: The Role of the Integrator Complex in Gene Expression Regulation. The Integrator Complex was discovered in 2005 as a 12 membered group of proteins that are required for the 3’ end formation of small nuclear RNA (snRNA). The Integrator Complex is thought to interact with machinery at the snRNA promoter including members of the SNAP complex, which leads Integrator association with the CTD of RNAPII. The molecular details of these interactions have not been elucidated to this date. Once Integrator is loaded onto RNAPII, it will cleave the pre-snRNA using its catalytic RNA endonuclease subunit: IntS11. More recently, Integrator has been shown to function in the pause/release of RNAPII at immediate early genes. It plays a key role in the recruitment of pTEF-b and ultimate transcriptional activation through enhanced elongation competence. The Wagner laboratory is interested in determining the mechanism of Integrator action at these various gene types. To that end, we have developed model systems in both human and Drosophila cells in address its various unknowns.
Schematic of Integrator function at snRNA genes. Both the SNAPc and Oct1 proteins recognize unique elements within the snRNA promoter recruiting Integrator and RPAP2. Following unique CTD phosphorylations, Integrator associates with RNAPII and cleaves the 3’ end of the snRNA recognizing the terminal stem loop of the snRNA and the 3’ box located downstream.
Model of Integrator function in pause-release. Upon activation by EGF, phosphorylated ELK1 and SRF mediate the enhanced recruitment of Integrator to the pause RNAPII. Integrator, in turn recruits pTEFb and stimulates the release of negative elongation factors (DSIF and NELF). This leads to the release of theelongation competent RNAPII.
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