1. Volume 11, Issue 4, Pages 965-976 (April 2003)
Structural Basis for the Molecular Recognition between Human Splicing Factors U2AF65 and SF1/mBBP 
Philipp Selenko, Goran Gregorovic, Remco Sprangers, Gunter Stier, Zakaria Rhani, Angela Krämer, Michael Sattler 
Molecular Cell 
Volume 11, Issue 4, Pages (April 2003)
DOI: /S (03)

2. Figure 1 Multiple Sequence Alignments of U2AF65 and SF1 Proteins
(A) Structure-based multiple sequence alignment of U2AF65 RRMs 1, 2, and 3. Sequences of U2AF65 of Homo sapiens (hs, acc. no A), Mus musculus (mm, S22646), Drosophila melanogaster (dm, NP_476891), Caenorhabditis elegans (ce, AAM44400), Arabidopsis thaliana (at, CAB16828), Schizosaccharomyces pombe (sp, AAA03578), and Mud2p of Saccharomyces cerevisiae (sc, AAA64215) were aligned with ClustalX (Jeanmougin et al., 1998). Based on the available three-dimensional structures (Ito et al., 1999; this work), secondary structure elements of the three RRMs (boxes) were manually grouped and realigned. RNP1 and RNP2 signature sequences in RRMs 1, 2, and 3 are indicated on top. Residues that are conserved in all three RRMs are colored green for hydrophobic, blue for basic, turquoise for aromatic, and red for acidic side chains. The sequence numbering and secondary structure elements of human U2AF65-RRM3 are indicated above and below, respectively. Yellow circles designate residues that form the hydrophobic binding pocket accommodating Trp22 of SF1. Blue circles indicate positions of charge-reverting mutations (see Figure 3E). Magenta circles indicate hydrophobic residues involved in packing of helix C against the β sheet of U2AF65-RRM3 (Figure 5B).
(B) Multiple sequence alignment of N-terminal residues of SF1. Sequences of SF1 (hs, acc. no. CAA03883; mm, CAA59797; ce, CAB64866; dm, CAB64937; at_1 and at_2, BAA97393 [the N terminus of atSF1 contains two putative U2AF65 interaction sequences]; sp, AAF02214; sc, NP_013217) were aligned with ClustalX. Conserved positively charged residues are colored in blue, and positions of point mutations (Figure 3D) are indicated by red circles. Ser20 and Trp22 are shown in yellow and white on black, respectively.
Molecular Cell  , DOI: ( /S (03) )

3. Figure 2
Definition of the U2AF65/SF1 Interface by Competition Experiments and NMR
(A) GST pull-down experiments in the presence of increasing concentrations of His6-SF1Δ1-28 (top) or the SF1(11-25) peptide (bottom) during the binding reaction. Numbers above the figure indicate the x-fold molar excess of these proteins over SF1 (see Experimental Procedures for details).
(B) 1H,15N correlation spectrum of 0.5 mM 15N-labeled recombinant human SF1(1-25) in the free form (black) and complexed with 2 mM unlabeled U2AF65-RRM3 (red and blue). “A*” is an additional N-terminal residue resulting from the TEV cleavage site.
(C) Summary of NMR data for SF1(1-25) bound to U2AF65-RRM3. Chemical shift changes (Δδ) of the amide groups of SF1(1-25) upon U2AF65-RRM3 binding are shown together with secondary chemical shifts (ΔδCα-Cβ) and {1H}-15N heteronuclear NOE values for the U2AF65-RRM3/SF1 complex.
(D) Summary of NMR data for free and SF1-bound U2AF65-RRM3. Fast/medium/slow exchange of amide protons in D2O solution is shown by open/half-open/closed circles. Chemical shift changes Δδ = √(δ15N2 + δ1H2) of the amide groups of U2AF65-RRM3 upon SF1 binding are shown together with a comparison of secondary chemical shifts (Δδ(Cα-Cβ) and {1H}-15N heteronuclear NOE values for the free [uncplx] and bound [cplx] U2AF65-RRM3). Secondary structure elements are indicated on top.
Molecular Cell  , DOI: ( /S (03) )

4. Figure 3
Structure and Biochemical Characterization of the U2AF65-RRM3/SF1 Complex
(A) Stereo view for the backbone atoms (N, Cα, C′) of residues 376–475 in U2AF65-RRM3 and residues 13–25 of SF1 from an NMR ensemble of 10 superimposed lowest-energy structures (out of 100 calculated). Secondary structure elements are colored blue for β strands, green for helices A and B, and orange for the C-terminal helix C. The SF1 peptide is shown in yellow. Residue numbers are indicated.
(B) Ribbon representation of the U2AF65-RRM3/SF1 complex structure closest to the average conformation of the NMR ensemble in the same orientation as in (A). Secondary structure elements and SF1 residues are labeled.
(C) Surface representation of the U2AF65-RRM3 and a stick representation for SF1 residues 15–25. Blue and red colors indicate positive and negative electrostatic surface potential, respectively. SF1 residues are labeled in white, and acidic residues in U2AF65-RRM3 are labeled in black.
(D) Mutational analysis of SF1. His6-SF1 (wt) and His6-SF1 carrying the point mutations indicated above the figure were reacted with glutathione agarose-bound U2AF65-RRM3 in a standard GST pull-down experiment. Western blots of SF1 proteins detected with anti-His6 antibodies are shown in the top panel. The bottom panel shows Western blots of the GST-RRM3 inputs detected with anti-GST antibodies as a control. The first lane represents a negative control in which His6-SF1 was reacted with glutathione agarose-bound GST.
(E) Mutational analysis of U2AF65-RRM3. His6-SF1 was incubated with glutathione agarose-bound U2AF65-RRM3 (wt) or RRM3 carrying point mutations (as indicated above the figure) in a standard GST pull-down experiment. The top and bottom panels are as in (D).
Molecular Cell  , DOI: ( /S (03) )

5. Figure 4 Tryptophan Recognition by the U2AF35 RRM and U2AF65-RRM3
(A) Ribbon representation of the U2AF35 RRM in complex with the proline-rich region in the N terminus of U2AF65 (Kielkopf et al., 2001). Side chains involved in the molecular interface are shown. Trp134, which is not conserved in U2AF65-RRM3, is labeled magenta.
(B) Ribbon representation of the U2AF65-RRM3/SF1 complex. Side chains involved in tryptophan coordination are shown.
(C) Sequence alignment of noncanonical RRMs with an extended and negatively charged helix A. Conserved residues that mediate recognition of SF1 Trp22 and U2AF65 Trp92 by U2AF65-RRM3 and the U2AF35 RRM, respectively, are shown in white on black. Trp134 in the U2AF35 RRM is colored magenta.
(D) Domain organization of proteins that contain noncanonical RRMs. The domain annotations are according to SMART (Schultz et al., 1998). “R/S” is an arginine-serine-rich region, “STY Kc” is a phoshokinase domain, and “Zn” are zinc binding domains.
Molecular Cell  , DOI: ( /S (03) )

6. Figure 5
Helix C in RRMs
(A) Ribbon representation of the structure of the N-terminal RRM of U1A (Nagai et al., 1990). Secondary structure elements are colored blue for the four-stranded β sheet, green for helices A and B, and orange for helix C. Side chains that form the small hydrophobic interface with helix C are shown in yellow. The insert displays the structure of the U1A protein bound to RNA (shown in gold; Oubridge et al., 1994) and indicates the reorientation of helix C upon RNA binding.
(B) Ribbon representation of U2AF65-RRM3. The coloring scheme and orientation are the same as in (A). Residues that participate in the hydrophobic packing of helix C are displayed in yellow.
Molecular Cell  , DOI: ( /S (03) )

7. Figure 6
Structure and Topology of Complex E during Spliceosome Assembly
For a description, see the text. An additional interaction between the R/S domain of U2AF65 and the BPS (Valcárcel et al., 1996) is not shown.
Molecular Cell  , DOI: ( /S (03) )