Crystal structure of the 25 kDa subunit of human cleavage factor I m

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Abstract
Cleavage factor I m 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 I m is an oligomer composed of a small 25 kDa subunit (CF I m 25) 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 I m 25, 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 I m 25 in its free and diadenosine tetraphosphate (Ap 4 A) bound forms at 1.85 and 1.80 Å, respectively. CF I m 25 crystallizes as a dimer and presents the classical Nudix fold. Results from crystallographic and biochemical experiments suggest that CF I m 25 makes use of its Nudix fold to bind but not hydrolyze ATP and Ap 4 A. The complex and apo protein structures provide insight into the active oligomeric state of CF I m and suggest a possible role of nucleotide binding in either the polyadenylation and/or cleavage steps of pre-messenger RNA 3′-end processing.