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

Abstract

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.

Structural basis of UGUA recognition by the Nudix protein CFI m 25 and implications for a regulatory role in mRNA 3′ processing

“…CFI m 25 forms a homodimer in solution (16,22), and the same dimer conformation is retained upon RNA binding, as shown in our crystal structure. CFI m 25 therefore has the potential to specifically bind two UGUA elements simultaneously.…”

“…The CFI m 25-RNA complex retains the same homodimer conformation previously observed in the apo structure (16,22). Multimeric organization is a common feature of single-stranded RNA binding proteins and has been demonstrated to facilitate both higher affinity and specificity (39)(40)(41)(42)(43).…”

“…The SO 2− 4 molecule was found to occupy the same location as the γ-phosphate of Ap 4 A (16,22). Each of these small molecules binds CFI m 25 in a manner that excludes the possibility of RNA binding.…”

“…Found throughout all three kingdoms, Nudix proteins participate in a wide range of crucial housekeeping functions, including the hydrolysis of mutagenic nucleotides, the modulation of the levels of signaling molecules, and the monitoring of metabolic intermediates (15). CFI m 25 possesses the characteristic α/β/ α Nudix fold and is able to bind Ap 4 A (diadenosine tetraphosphate), but it has no hydrolase activity, due to the absence of two of the four glutamate residues that coordinate the divalent cations important for substrate hydrolysis (16).…”

Structural basis of pre-mRNA recognition by the human cleavage factor Im complex

“…Crystals obtained from the full-length CF I m 25 in a complex with CF I m-68RRM were not of an adequate quality to allow data collection. In the structure of apo-CF I m 25, residues 1-20 were not observed in the electron density map and residues 21-32 formed an extended loop structure [16]. To obtain crystals for high-resolution studies, a truncated CF I m 25 protein, with residues 1-33 removed, was constructed.…”