Summary Cleavage Factor Im (CFIm) is a highly conserved component of the eukaryotic mRNA 3′ processing machinery that functions in sequence-specific poly(A) site recognition through the collaboration of two protein subunits, a 25 kDa subunit containing a nudix domain and a larger subunit of 59, 68, or 72 kDa containing an RNA recognition motif (RRM). Our previous work demonstrated that CFIm25 is both necessary and sufficient for sequence-specific binding of the poly(A) site upstream element UGUA. Here we report the crystal structure of CFIm25 complexed with the RRM domain of CFIm68 and RNA. The CFIm25 dimer is clasped on opposite sides by two CFIm68 RRM domains. Each CFIm25 subunit binds one UGUA element specifically. Biochemical analysis indicates that the CFIm68 RRMs serve to enhance RNA binding and facilitate RNA looping. The intrinsic ability of CFIm to direct RNA looping may provide a mechanism for its function in the regulation of alternative poly(A) site selection.

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.

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[c]thiophen-4(5H)-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.

Poly(A) polymerase (PAP) synthesizes the polyadenine tail at the 3′‐end of messenger RNA. A disulfide cross‐linking strategy was implemented to obtain a complex between bovine PAP (bPAP) and a 15‐mer oligo(A). All seven endogenous cysteines were mutated to eliminate nonspecific cross‐linked complexes. A cysteine residue was introduced at several different positions and A152C was found to achieve maximum specific cross‐linking efficiency. The resulting bPAP construct was active and, when mixed with a chemically modified RNA, yielded crystals of a bPAP–RNA complex. The crystals, which belonged to space group P2 and harbored two protein–RNA complexes per asymmetric unit, diffracted X‐rays to 2.25 Å resolution.

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