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

Crystal structure of the 25 kDa subunit of human cleavage factor Im

Cleavage factor Im is an essential component of the pre-messenger RNA 3′-end processing machinery in higher eukaryotes, participating in both the polyadenylation and cleavage steps. Cleavage factor Im is an oligomer composed of a small 25 kDa subunit (CF Im25) and a variable larger subunit of either 59, 68 or 72 kDa. The small subunit also interacts with RNA, poly(A) polymerase, and the nuclear poly(A)-binding protein. These protein–protein interactions are thought to be facilitated by the Nudix domain of CF Im25, a hydrolase motif with a characteristic α/β/α fold and a conserved catalytic sequence or Nudix box. We present here the crystal structures of human CF Im25 in its free and diadenosine tetraphosphate (Ap4A) bound forms at 1.85 and 1.80 Å, respectively. CF Im25 crystallizes as a dimer and presents the classical Nudix fold. Results from crystallographic and biochemical experiments suggest that CF Im25 makes use of its Nudix fold to bind but not hydrolyze ATP and Ap4A. The complex and apo protein structures provide insight into the active oligomeric state of CF Im and suggest a possible role of nucleotide binding in either the polyadenylation and/or cleavage steps of pre-messenger RNA 3′-end processing

A Small Covalent Allosteric Inhibitor of Human Cytomegalovirus DNA Polymerase Subunit Interactions

Human cytomegalovirus DNA polymerase comprises a catalytic subunit, UL54, and an accessory subunit, UL44, the interaction of which may serve as a target for the development of new antiviral drugs. Using a high-throughput screen, we identified a small molecule, (5-((dimethylamino)­methylene-3-(methylthio)-6,7-dihydrobenzo­[<i>c</i>]­thiophen-4­(5<i>H</i>)-one), that selectively inhibits the interaction of UL44 with a UL54-derived peptide in a time-dependent manner, full-length UL54, and UL44-dependent long-chain DNA synthesis. A crystal structure of the compound bound to UL44 revealed a covalent reaction with lysine residue 60 and additional noncovalent interactions that cause steric conflicts that would prevent the UL44 connector loop from interacting with UL54. Analyses of the reaction of the compound with model substrates supported a resonance-stabilized conjugation mechanism, and substitution of the lysine reduced the ability of the compound to inhibit UL44-UL54 peptide interactions. This novel covalent inhibitor of polymerase subunit interactions may serve as a starting point for new, needed drugs to treat human cytomegalovirus infections