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. 2010 Jun 1;107(22):10062-7.
doi: 10.1073/pnas.1000848107. Epub 2010 May 17.

Structural Basis of UGUA Recognition by the Nudix Protein CFI(m)25 and Implications for a Regulatory Role in mRNA 3' Processing

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Free PMC article

Structural Basis of UGUA Recognition by the Nudix Protein CFI(m)25 and Implications for a Regulatory Role in mRNA 3' Processing

Qin Yang et al. Proc Natl Acad Sci U S A. .
Free PMC article

Abstract

Human Cleavage Factor Im (CFI(m)) is an essential component of the pre-mRNA 3' processing complex that functions in the regulation of poly(A) site selection through the recognition of UGUA sequences upstream of the poly(A) site. Although the highly conserved 25 kDa subunit (CFI(m)25) of the CFI(m) complex possesses a characteristic alpha/beta/alpha Nudix fold, CFI(m)25 has no detectable hydrolase activity. Here we report the crystal structures of the human CFI(m)25 homodimer in complex with UGUAAA and UUGUAU RNA sequences. CFI(m)25 is the first Nudix protein to be reported to bind RNA in a sequence-specific manner. The UGUA sequence contributes to binding specificity through an intramolecular G:A Watson-Crick/sugar-edge base interaction, an unusual pairing previously found to be involved in the binding specificity of the SAM-III riboswitch. The structures, together with mutational data, suggest a novel mechanism for the simultaneous sequence-specific recognition of two UGUA elements within the pre-mRNA. Furthermore, the mutually exclusive binding of RNA and the signaling molecule Ap(4)A (diadenosine tetraphosphate) by CFI(m)25 suggests a potential role for small molecules in the regulation of mRNA 3' processing.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Overall structure of the CFIm25-RNA complex. (A) View of the crystal packing interactions of the CFIm25-UUGUAU complex. One asymmetric unit contains one CFIm25 homodimer (Molecule A in teal and Molecule B in dark blue) and one UUGUAA hexamer (yellow). The 5′-end of the RNA (UGUA element) binds to Mol A, while the 3′-end is bound by Mol B of an adjacent symmetry-related dimer (Mol Bs in green). Molecule A and the RNA of the adjacent dimer are shown in orange and pink, respectively. In Mol A and Mol Bs, the conserved Nudix box helix (residues 117–129) is highlighted in purple. Helix α1 and the loop connecting β2 and α1 (residues 51–74) are shown in gold. (B) Close up view of the CFIm25-UUGUAU interface between Mol A and Mol Bs. UUGUAU is shown as a stick model (yellow) with overlaid Fo-Fc electron density map (dark blue) contoured at 3σ. The difference map was calculated immediately after molecular replacement and prior to any refinement, in order to prevent model bias. Convincing density was observed for the entire RNA strand except for the base of the first U (U0). (C) Same view of the CFIm25-UGUAAA complex. UGUAAA is shown as a stick model (salmon), and the Fo-Fc map (3σ) (dark blue) was also calculated before any refinement. Strong density was observed for all six nucleotides.
Fig. 4.
Fig. 4.
CFIm25 is the only Nudix protein of known structure in which the canonical Nudix substrate-binding pocket is occluded. (A) Superposition of the CFIm25-UUGUAU complex (Mol A) with three well-studied Nudix hydrolases (reviewed in ref. 21): MutT pyrophosphohydrolase (PDB ID: 1PPX) in lime, ADP-ribose pyrophosphatase (1V8L) in dark blue, and Ap4A hydrolase (1XSC) in orange. The CFIm25 color scheme is the same as in Fig. 1. The loop connecting β2 and α1 is shown as a thick yellow tube in the CFIm25-UUGUAU complex. (B) Superposition of the ligands from the three Nudix proteins in A onto CFIm25. The ligands (8-oxo-2’-deoxy-GMP, ADP-ribose, and ATP) are shown as stick models and colored as indicated in A.
Fig. 2.
Fig. 2.
Close-up views of the CFIm25-UUGUAU interactions. Close up views of CFIm25 interacting with each base within the UGUA element: (A) U1, (B) G2, (C) U3, and (D) A4. The protein color scheme is the same as in Fig. 1. The RNA backbone is shown in orange. Hydrogen bonds are represented by red dashed lines. Residues involved in RNA binding are shown and colored according to the domain they belong to. Water molecules involved in hydrogen bonding are shown as red spheres.
Fig. 3.
Fig. 3.
CFIm25 specifically recognizes two UGUA elements. (A) Bar graph representation of the electrophoretic mobility shift assay (EMSA) data of CFIm25 variants binding to a 21 nt PAPOLA poly(A) site RNA containing two UGUA elements. (B) EMSA data of wild type CFIm25ΔN21 binding to various RNA sequence variants. A single prime represents the mutation on the first UGUA element, and double prime represents the mutation on the second UGUA element. Experiments were done in triplicate and all the bound fractions were plotted relative to CFIm25ΔN21 and the wild type PAPOLA RNA. The error bars represent the standard deviation.
Fig. 5.
Fig. 5.
Surface presentation of the CFIm25-UUGUAU complex. (A) Electrostatic surface representation of the CFIm25 dimer, colored according to the electrostatic potential (blue, positive; red, negative). The UUGUAU RNA strand is shown as a stick model (yellow). A second UUGUAU molecule (shown in cyan) is modeled in Mol B in the same location as in Mol A. The surface of the RNA molecules is shown in beige. The crystallographic 2-fold axis is represented by a black circle. (B) A close-up view of the RNA binding pocket in Mol A. Superposition of the Ap4A-bound [PDB ID: 3BAP (16)] CFIm25 structure with the UUGUAU-bound structure. Ap4A and Arg63 from 3BAP are shown in white. Hydrogen bond interactions between Arg63 and its ligands are shown as red dashed lines.

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