Crystal Structure of a Human Cleavage Factor CFIm25/CFIm68/RNA Complex Provides an Insight into Poly(A) Site Recognition and RNA Looping  Qin Yang, Molly Coseno, Gregory M. Gilmartin, Sylvie Doublié  Structure  Volume 19, Issue 3, Pages 368-377 (March 2011) DOI: 10.1016/j.str.2010.12.021 Copyright © 2011 Elsevier Ltd Terms and Conditions

Structure 2011 19, 368-377DOI: (10.1016/j.str.2010.12.021) Copyright © 2011 Elsevier Ltd Terms and Conditions

Figure 1 Overall Structure of the CFIm Complex (A) Cartoon diagram of the CFIm complex. The CFIm25 homodimer, colored in dark green and lime, is flanked by the RRM domain of two CFIm68 monomers colored in lavender and blue. In CFIm68, 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) CFIm complex rotated 90° compared to (A). (C) Surface representation of the CFIm complex in the same orientation as in (A). (D) Topological representation of the secondary structure elements of the CFIm68 RRM. A sequence comparison between CFIm68 and consensus RNP is shown. Structure 2011 19, 368-377DOI: (10.1016/j.str.2010.12.021) Copyright © 2011 Elsevier Ltd Terms and Conditions

Figure 2 Interactions between the CFIm25 Homodimer and One CFIm68 Subunit (A and B) Close-up view of interactions between the CFIm25 dimer and a CFIm68 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 a stick model and colored according to the molecule and domain they belong to. See also Figure S2. (C) Schematic representation of the CFIm25-CFIm68 interactions. CFIm25 residues are represented with ellipses and CFIm68 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 CFIm68 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. Structure 2011 19, 368-377DOI: (10.1016/j.str.2010.12.021) Copyright © 2011 Elsevier Ltd Terms and Conditions

Figure 3 The CFIm Complex Binds Two UGUA Elements Simultaneously (A) Overview of the CFIm-UAUUUUGUA complex. RNA molecules are shown as a stick model and colored in yellow and salmon. Simulated annealing omit map for the RNA molecules is shown as a blue mesh and contoured at 2.5 σ. (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 CFIm25 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) EMSAs of CFIm 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. Structure 2011 19, 368-377DOI: (10.1016/j.str.2010.12.021) Copyright © 2011 Elsevier Ltd Terms and Conditions

Figure 4 The CFIm68 RRM Facilitates RNA Looping (A) A close-up view of the CFIm68 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 CFIm 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 21-mer. Error bars represent the standard deviation from triplicate experiments. (C) Bar graph representation of the EMSA of CFIm68 RRM variants in complex with wild-type CFIm25 and 21-mer PAPOLA RNA. Error bars represent the standard deviation from triplicate experiments. (D) EMSA of CFIm68 RRM variants in complex with wild-type CFIm25 and PAPOLA RNA variants with spacers of various lengths, or PAPOLG. Error bars represent the standard deviation from duplicate experiments. All bound fractions in (B)–(D) were plotted relative to the wild-type complex and wild-type 21-mer 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 CFIm68 E111A, W90A/W91A/D94A, and W90A/W91A/N117A/R118A; magenta represents a 50% decrease by CFIm25 E154A, and pink represents a less than 40% decrease by CFIm68 Y84A/L128A. Green indicates an increase in RNA-binding efficiency by CFIm68 F126A. Structure 2011 19, 368-377DOI: (10.1016/j.str.2010.12.021) Copyright © 2011 Elsevier Ltd Terms and Conditions

Figure 5 Model Illustrating How CFIm68 May Facilitate RNA Looping RNA is shown as a black line. UGUA elements are highlighted by ovals. Dashed lines indicate that the RNA is below the RRM surface. See also Figure S4. Structure 2011 19, 368-377DOI: (10.1016/j.str.2010.12.021) Copyright © 2011 Elsevier Ltd Terms and Conditions

Figure 6 Model Illustrating How CFIm May Mediate Alternative Polyadenylation (A) Cleavage reaction occurs at poly(A) site 1. (B) Cleavage reaction occurs at poly(A) site 2. Structure 2011 19, 368-377DOI: (10.1016/j.str.2010.12.021) Copyright © 2011 Elsevier Ltd Terms and Conditions