Volume 23, Issue 1, Pages 49-59 (July 2006) Structural Basis for Polypyrimidine Tract Recognition by the Essential Pre-mRNA Splicing Factor U2AF65  E. Allen Sickmier, Katherine E. Frato, Haihong Shen, Shanthi R. Paranawithana, Michael R. Green, Clara L. Kielkopf  Molecular Cell  Volume 23, Issue 1, Pages 49-59 (July 2006) DOI: 10.1016/j.molcel.2006.05.025 Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 1 Sequence Alignment of Py Tract Binding Domains from Representative U2AF65 Homologs Human (accession code X64044), mouse (AAH43071), frog (AAH44032), fly (L23404), worm (AAL00879), plant (NP_176287), and fission yeast (NP_595396) sequences are colored in a gradient from white (≤50% identity) to dark green (100% identity). Human U2AF65 secondary structure elements are indicated above the aligned sequences, where primed italics distinguish RRM2 secondary structures from those belonging to RRM1. The U2AF65 ribonucleoprotein consensus motifs (RNP1 and RNP2) are shown with red font, and consensus sequences are given below. Residues below dashes were absent from the dU2AF651,2 variant used for crystallization. Protein/RNA interactions are indicated above the sequences as follows: s, sidechain contact; m, mainchain contact; w, water-mediated contact. Underscores distinguish residues tested for contribution to RNA affinity by site-directed mutagenesis. Molecular Cell 2006 23, 49-59DOI: (10.1016/j.molcel.2006.05.025) Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 2 The In Vitro Splicing Activity of the dU2AF65 Variant Used for Structural Analysis Is Comparable to that of Wild-Type U2AF65 The concentrations of U2AF65 proteins added to the splicing extract were 0, 0.062, 0.125, 0.25, 0.50, and 1.0 μM, increasing left to right. The products of the splicing reaction are shown schematically to the right. Molecular Cell 2006 23, 49-59DOI: (10.1016/j.molcel.2006.05.025) Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 3 Overall Structure of the Human dU2AF651,2/rU7 Complex (A) View of the crystal packing interactions of the dU2AF651,2/rU7 complex. The symmetry-related protein molecules are shown as green or blue backbone worms, and the bound nucleic acids are shown as purple or yellow ball-and-stick models. One asymmetric unit contains one protein molecule and one rU7 oligonucleotide, so that each rU7 oligonucleotide is bound by RRM1 and RRM2 donated by distinct, symmetry-related protein molecules. (B) View of the integrated structural unit of RRM1 and RRM2 interacting with the rU7 strand, into the RNA binding surface (top panel) or rotated 180° about the vertical axis (lower panel). Secondary structure elements of the dU2AF651,2 protein are labeled (primed italics distinguish RRM2 secondary structures) on a ribbon model that is colored by domain as follows: RRM1 (residues 148–237), green; and RRM2 (residues 258–336), blue. A composite omit electron density map at 1σ contour level surrounds a stick representation of the oligonucleotides (purple). (C) Electrostatic surface representation of dU2AF651,2, shown bound to a stick representation of the RNA in a similar orientation as the lower panel of (B) was generated using the program Pymol (http://www.pymol.org). Red areas, highly negatively charged residues; blue areas, highly positively charged residues. Molecular Cell 2006 23, 49-59DOI: (10.1016/j.molcel.2006.05.025) Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 4 Comparison of dU2AF651,2/rU7 RNA Conformation with Available Structures of Tandem RRMs Bound to Continuous, Single RNA Strands Superposition of N-terminal RRMs shows a range of bound RNA conformations (proteins not shown for clarity). Coil representations of the RNA ligands are colored by structure: HuD/(AU)-rich RNA (ARE) (Wang and Tanaka-Hall, 2001), blue; SXL/tra Py tract (Handa et al., 1999), purple; PAB/polyadenosine (polyA) (Deo et al., 1999), pink; and dU2AF651,2/polyuridine (polyU), green. Molecular Cell 2006 23, 49-59DOI: (10.1016/j.molcel.2006.05.025) Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 5 Uridine Recognition in the dU2AF651,2/rU7 Complex (A) Schematic diagram of dU2AF651,2/rU7 interactions. Residue names are boxed and colored as in (B)–(H). Filled boxes indicate main chain contacts, and open boxes indicate side chain interactions. Dashed lines represent hydrogen bonds, dashed lines ending with a filled circle represent stacking interactions, and solid lines represent ionic interactions. (B–H) Ball-and-stick representations of the interactions between dU2AF651,2 and each bound uridine are shown. Residues belonging to RRM1 are colored green, residues from RRM2 are colored blue, and residues altered by site-directed mutagenesis are colored yellow. RNA bonds are colored purple, with a lighter shade distinguishing the C5-C6 bond of the base edges. The oxygen atoms of the stick representations are colored red, and nitrogen atoms are blue. Dashed lines indicate hydrogen bonds. Recognition of (B) Uri1, (C) Uri2, (D) Uri3, (E) Uri4, (F) Uri5, (G) Uri6, and (H) Uri7. Molecular Cell 2006 23, 49-59DOI: (10.1016/j.molcel.2006.05.025) Copyright © 2006 Elsevier Inc. Terms and Conditions