Crystal structure of a human cleavage factor CFI(m)25/CFI(m)68/RNA complex provides an insight into poly(A) site recognition and RNA looping

Abstract

Cleavage factor I(m) (CFI(m)) 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 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 CFI(m)25 is both necessary and sufficient for sequence-specific binding of the poly(A) site upstream element UGUA. Here, we report the crystal structure of CFI(m)25 complexed with the RRM domain of CFI(m)68 and RNA. The CFI(m)25 dimer is clasped on opposite sides by two CFI(m)68 RRM domains. Each CFI(m)25 subunit binds one UGUA element specifically. Biochemical analysis indicates that the CFI(m)68 RRMs serve to enhance RNA binding and facilitate RNA looping. The intrinsic ability of CFI(m) to direct RNA looping may provide a mechanism for its function in the regulation of alternative poly(A) site selection.

Figure 1

Overall structure of the CFI_m complex (A) Cartoon diagram of the CFI_m complex. The CFI_m25 homodimer, colored in dark green and lime, is flanked by the RRM domain of two CFI_m68 monomers colored in lavender and blue. In CFI_m68, loops connecting β₁/α₁ (residues 87-93) and β₂/β₃ (residues 115-125) are shown as thicker tubes and colored in magenta and brown, respectively. The RRM C-terminal α-helix (α₃, residues 161-172) is highlighted in gold. See also Figure S1 (B) CFI_m complex rotated 90° compared to A. (C) Surface representation of the CFI_m complex in the same orientation as in A. (D) Topological representation of the secondary structure elements of the CFI_m68 RRM. A sequence comparison between CFI_m68 and consensus RNP is shown.

Figure 2

Interactions between the CFI_m25 homodimer and one CFI_m68 subunit. Close-up view of interactions between the CFI_m25 dimer and a CFI_m68 monomer (Mol D) mediated by (A) hydrophobic stacking and (B) hydrogen bonding. The proteins are colored as in Figure 1. Residues involved in protein-protein interactions are shown as stick model and colored according to the molecule and domain they belong to. See also Figure S2. (C) Schematic representation of the CFI_m25-CFI_m68 interactions. CFI_m25 residues are represented with ellipses, CFI_m68 residues with rectangles. Stacking interactions are represented by black wavy lines and hydrogen bonds by red dashed lines. Filled boxes represent main chain interactions, whereas open boxes indicate side chain interactions. Residues are colored as in A and B. (D) A sequence alignment of the CFI_m68 RRM homologs from various species listed below. Consensus RNPs are delineated by the blue boxes. Residues with more than 70% conservation are colored in red. Strictly conserved residues are in the filled red boxes. Species abbreviations are as follows: Hs, Homo sapiens; Gg, Gallus gallus; Mm, Mus musculus; Xl, Xenopus laevis; Dr, Danio rerio; Is, Ixodes scapularis; Ci, Ciona intestinalis; Am, Apis mellifera; Dm, Drosophila melanogaster; Ag, Anopheles gambiae; Sm, Schistosoma mansoni. See also Figure S6 for a more complete alignment.

Figure 3

The CFI_m complex binds two UGUA elements simultaneously. (A) Overview of the CFI_m-UAUUUUGUA complex. RNA molecules are shown as stick model and colored in yellow and salmon. Simulated annealing omit map for the RNA molecules is shown as a blue mesh and contoured a 2.5σ. (B) A close up view of the bound RNA molecules. Density was observed for the UGUA elements and the ribose of the base preceding the first U (U0). (C) A close up view of CFI_m25 interacting with UGUA elements in Mol A. Residues participating in RNA binding are shown in stick model representation. Hydrogen bonds are represented by red dashed lines. (D) Electrophoretic mobility shift assays (EMSA) of CFI_m variants in complex with various PAPOLA RNA sequence variants. An asterisk (*) indicates that the protein underwent reductive methylation. A single prime (′) represents the first UGUA element, and double prime (″) represents the second UGUA element. See also Figure S3.

Figure 4

The CFI_m68 RRM facilitates RNA looping (A) A close up view of the CFI_m68 RRM (Mol D). The β-sheet is colored in blue, α₁ and α₂ in green, C-terminal helix α₃ in gold, loop β₁/α₁ in pink, loop β₂/β₃ in brown and the other loops in white. Mutated residues are shown as stick models. (B) EMSA data of CFI_m in complex with PAPOLA RNA variants with spacers of various lengths. UGUA elements are highlighted in red. Inserted nucleotides and nucleotides moved from their original position are shown in green. EMSA data were plotted based on bound fractions relative to the wild type PAPOLA 21mer. Error bars represent the standard deviation from triplicate experiments. (C) Bar graph representation of the EMSA of CFI_m68 RRM variants in complex with wild type CFI_m25 and 21 mer PAPOLA RNA. Error bars represent the standard deviation from triplicate experiments. (D) EMSA of CFI_m68 RRM variants in complex with wild type CFI_m25 and PAPOLA RNA variants with spacers of various lengths, or PAPOLG. Error bars represent the standard deviation from duplicate experiments. All bound fractions in figure (B), (C) and (D) were plotted relative to the wild type complex and wild type 21mer PAPOLA RNA. The error bars represent the standard deviation. See also Figure S5. (E) and (F) Protein residues that were subjected to alanine substitution are shown in surface representation. The red color represents a decrease greater than 80% in RNA binding efficiency by CFI_m68 E111A, W90A/W91A/D94A and W90A/W91A/N117A/R118A; magenta represents a 50% decrease by CFI_m25 E154A, and pink represents a less than 40% decrease by CFI_m68 Y84A/L128A. Green indicates an increase in RNA binding efficiency by CFI_m68 F126A.

Figure 5

Model illustrating how CFI_m68 may facilitate RNA looping. RNA is shown as a red line. UGUA elements are highlighted by ovals. Dashed lines indicate that the RNA is below the RRM surface. See also Figure S4

Figure 6

Model illustrating how CFI_m may mediate alternative polyadenylation. (A) Cleavage reaction occurs at poly(A) site 1. (B) Cleavage reaction occurs at poly(A) site 2.