The structure of human cleavage factor I(m) hints at functions beyond UGUA-specific RNA binding: a role in alternative polyadenylation and a potential link to 5' capping and splicing

Abstract

3'-end cleavage and subsequent polyadenylation are critical steps in mRNA maturation. The precise location where cleavage occurs (referred to as poly(A) site) is determined by a tripartite mechanism in which a A(A/U)UAAA hexamer, GU rich downstream element and UGUA upstream element are recognized by the cleavage and polyadenylation factor (CPSF), cleavage stimulation factor (CstF) and cleavage factor I(m) (CFI(m)), respectively. CFI(m) is composed of a smaller 25 kDa subunit (CFI(m)25) and a larger 59, 68 or 72 kDa subunit. CFI(m)68 interacts with CFI(m)25 through its N-terminal RNA recognition motif (RRM). We recently solved the crystal structures of CFI(m)25 bound to RNA and of a complex of CFI(m)25, the RRM domain of CFI(m)68 and RNA. Our study illustrated the molecular basis for UGUA recognition by the CFI(m) complex, suggested a possible mechanism for CFI(m) mediated alternative polyadenylation, and revealed potential links between CFI(m) and other mRNA processing factors, such as the 20 kDa subunit of the cap binding protein (CBP20), and the splicing regulator U2AF65.

Figure 1

A model for how CFI_m might facilitate alternative polyadenylation.

Figure 2

Sequence and structure comparison of CFI_m68 and CFI_m59. (A) Sequence alignment of the CFI_m68 and CFI_m59 RRM domains. Identical residues are highlighted in red. (B) Conservation of the CFI_m68 RRM was calculated using the ConSurf server and displayed with PyMOL. (C) The electrostatic surface potential of the CFI_m68 (left) and CFI_m59 (right) RRM domains was calculated with Delphi and colored according to the electrostatic potential (blue, positive; red, negative). Models are in the same orientation as the model on the right in (B). (D) Superposition of the CFI_m25/CFI_m59 (PDB ID: 3N9U) complex with CFI_m25/CFI_m68 (3Q2S). Both complexes are shown as cartoon models. CFI_m25 monomers complexed with CFI_m59 (CFI_m25₅₉) are colored in purple and pink, whereas CFI_m25 monomers in complex with CFI_m68 (CFI_m25₆₈) are colored in green and light green. The CFI_m59 RRMs are colored in gold and yellow and CFI_m68 RRMs are colored in teal and blue. Cleft1 on the left side of the picture is narrower due to a movement in the RRM domain, indicated with the orange curved arrow. The region delineated by a gray rectangle is shown in more detail in (E). (E) A close-up view of cleft1. Arg68 of CFI_m25 participates in a salt bridge with Glu112 of CFI_m59 (Glu112₅₉), but the same arginine residue is too far away to make contact with the analogous glutamate in CFI_m68 (Glu111₆₈). Residues are shown as stick models and colored according to the molecules they belong to. Ionic bonds are shown as red dashed lines. Helices are shown as cylinders for clarity.

Figure 3

Superposition of CFI_m68 with RRMs harboring a loop3 of similar shape. The CFI_m25 monomers are colored in green and light green. The CFI_m68 RRM (PDB ID: 3Q2S18) is colored in teal, CBP20 (1H2V51) in red, U2AF65 (2G4B55) in olive, PAPBC (1CVJ54) in purple and Rna15 (2X1A40) in salmon.