The Investigation of Cleavage Factor IM by Crystallographic and Biochemical Techniques

RNA maturation involves several steps prior to export of the mRNA out of the nucleus and translation in the cytoplasm. PremRNA 3’end processing is one of such steps, and comprises the endonucleolytic cleavage and polyadenylation of the 3’end of the premRNA. These two steps involve more than 14 processing factors that coordinate multiple proteinprotein and proteinRNA interactions necessary to coordinate efficient cleavage and polyadenylation. To date, many of these interactions have been investigated biochemically and require additional structural characterization both to confirm and highlight key residues involved in substrate contacts. Further structural characterization will also open investigation into the mechanism of 3’end processing by providing structural insight into the coordination of multiple binding components. The cleavage factor Im, CF Im, is a component of the 3’end processing machinery and plays an important role early, during endonucleolytic cleavage, and additionally to increase polyadenylation efficiency and regulate poly(A) site recognition. CF Im is composed of a small 25 kDa subunit, CF Im25, and a large, either 58 kDa, 68 kDa, or 72 kDa subunit. The 25 kDa subunit of CF Im interacts with both the RNA and other processing factors such as the poly(A) polymerase, Clp1, and the larger subunit of CF Im. It is our goal to crystallize CF Im25 alone and in complex with one of its interacting partners to better understand CF Im25 contributions to premRNA 3’end processing. The structural investigation of CF Im25 and its binding partners has accomplished four major objectives: 1) Characterized the crystal structure of CF Im25 alone and bound to diadenosine tetraphosphate, 2) Provided insight into the oligomeric state of the CF Im complex, 3) Determined the binding properties of the Nudix domain of CF Im25 and its function in 3’end processing, 4) Further characterize the interactions between CF Im25 and PAP, CF Im68, and Clp1. These results demonstrate CF Im25 is a dimer both in solution and in the crystal suggesting that it is likely to be a dimer in the CF Im complex. The nucleotide binging capability of CF Im25 has no apparent role in 3’end processing in vitro but may provide a function outside of 3’end processing or may directly be involved in RNA recognition. The additional investigation of complex interactions with the 25 kDa subunit of CF Im25 suggests that although these factors interact during the 3’end processing event additional mechanisms may play a role in stabilizing those interactions