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. 2022 May 20;50(9):5299-5312.
doi: 10.1093/nar/gkac287.

Pre-mRNA splicing factor U2AF2 recognizes distinct conformations of nucleotide variants at the center of the pre-mRNA splice site signal

Eliezra Glasser  1 , Debanjana Maji  1 , Giulia Biancon  2 , Anees Mohammed Keedakkatt Puthenpeedikakkal  1 , Chapin E Cavender  1 , Toma Tebaldi  2   3 , Jermaine L Jenkins  1 , David H Mathews  1 , Stephanie Halene  2   4   5 , Clara L Kielkopf  1   6
Affiliations

Pre-mRNA splicing factor U2AF2 recognizes distinct conformations of nucleotide variants at the center of the pre-mRNA splice site signal

Eliezra Glasser et al. Nucleic Acids Res. .

Abstract

The essential pre-mRNA splicing factor U2AF2 (also called U2AF65) identifies polypyrimidine (Py) tract signals of nascent transcripts, despite length and sequence variations. Previous studies have shown that the U2AF2 RNA recognition motifs (RRM1 and RRM2) preferentially bind uridine-rich RNAs. Nonetheless, the specificity of the RRM1/RRM2 interface for the central Py tract nucleotide has yet to be investigated. We addressed this question by determining crystal structures of U2AF2 bound to a cytidine, guanosine, or adenosine at the central position of the Py tract, and compared U2AF2-bound uridine structures. Local movements of the RNA site accommodated the different nucleotides, whereas the polypeptide backbone remained similar among the structures. Accordingly, molecular dynamics simulations revealed flexible conformations of the central, U2AF2-bound nucleotide. The RNA binding affinities and splicing efficiencies of structure-guided mutants demonstrated that U2AF2 tolerates nucleotide substitutions at the central position of the Py tract. Moreover, enhanced UV-crosslinking and immunoprecipitation of endogenous U2AF2 in human erythroleukemia cells showed uridine-sensitive binding sites, with lower sequence conservation at the central nucleotide positions of otherwise uridine-rich, U2AF2-bound splice sites. Altogether, these results highlight the importance of RNA flexibility for protein recognition and take a step towards relating splice site motifs to pre-mRNA splicing efficiencies.

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Figures

Graphical Abstract
Graphical Abstract
Splice site RNA sequence variants adapt to U2AF2 scaffold.
Figure 1.
Figure 1.
The specificity of the U2AF2 RRM-containing region for centrally-substituted Py tract oligonucleotides. The boundary of the U2AF212L construct (blue) used for RNA binding and structure determination is inset in panel (A). Fluorescence anisotropy measurements of U2AF212L titrated into the given RNAs, including (A) an AdML Py tract (blue) and its central cytidine (mustard/yellow), guanosine (salmon), or adenosine (green) substitutions, (B) a nine-uridine tract (blue) and substituted with cytidine (mustard/yellow), adenosine (green), or guanosine at G5 (salmon), or (C) a nine-uridine tract (dashed blue line shown for reference) substituted with G4, (light gray), G5 (salmon), G4/G5 (orange-yellow), G6 (dark grey), or G5/G6 (maroon). The average data points and standard deviations of three experiments are overlaid with the fitted binding curves. The sequences of the 5���-fluorescein-labeled RNA oligonucleotides are inset, alongside average apparent equilibrium dissociation constants (KD) with standard deviations of three replicates. (D) Bar graph of U2AF212L binding affinities for the RNAs shown in B and C. The KD’s of U2AF212L for binding the A5 RNAs are estimates due to the very low affinities. The significance of the changes in the average apparent binding affinities compared to the G5-substituted oligonucleotide were calculated using two-tailed unpaired t-tests with Welch's correction in GraphPad Prism: P-values: n.s., not significant, *, <0.05; **, <0.005. The differences between the U2AF212L binding affinities for the G4 and G4/G5 RNAs, or between the G6 and G5/G6 RNAs, were not significant. The U2AF212L binding affinities for modified oligonucleotides used for co-crystallization are shown in Supplementary Figure 1.
Figure 2.
Figure 2.
The ternary complex of U2AF2 with SF1 and MBP-tagged U2AF1 has subtle preferences for G-substitutions in the Py tract of the AdML 3′ splice site sequence. (A) Domains of subunits in the ternary complex with construct boundaries indicated by double-headed arrows. (B) Sequences of 5′ fluorescein-labeled oligonucleotides used for RNA binding. (C) Fitted binding curves and (D) bar graph of average binding affinities and standard deviations, with the significance indicated as for Figure 1. The guanosine-substitutions of the wild-type AdML (blue) are numbered in reverse from the splice site junction following the AG consensus (underlined): –8G, black; –9G, salmon; –10G, dark grey; –11G, light grey.
Figure 3.
Figure 3.
Crystal structures of U2AF212L recognizing AdML Py tract variants that differ in the identities of the central nucleotide. The amino (N)/carboxy (C)-termini of the polypeptide and 5′/3′ termini of the oligonucleotide are labeled in italics. The nucleotide positions are numbered on panel A. (A–D) Overall ribbon diagrams of the protein (blue) bound to oligonucleotides (grey) substituted with (A) uridine (U5, magenta, PDB ID 6XLW), (B) cytidine (C5, yellow, PDB ID 7S3A, this study), (C) guanosine (G5, salmon, PDB ID 7S3B, this study), or (D) adenosine (A5, green, PDB ID 7S3C, this study). On panels A–D, the temperature factors (mobility) of the inter-RRM linker (residues 230–260) are represented using cartoon putty in PyMOL, which scales the size of the coil proportionately to the temperature factors of the residues. Residues 230–247 are pale blue and residues 248–260 are dark blue (boundary residues labeled on panel A). Ranges of linker residues that unresolved in the G5 and A5 structures are labeled. (E) Superposition by matching Cα atoms in the four structures shows conformational changes at the fifth and sixth nucleotide positions (numbered according to the nine nucleotide-binding sites of PDB ID 5EV4). (F) Closer view of the fifth and sixth nucleotides shown in (E) following a 90° clockwise rotation about the y-axis relative to (E).
Figure 4.
Figure 4.
U2AF212L interactions with the fifth and sixth nucleotides of bound Py tracts (numbered with the convention of PDB ID 5EV4). Variants of the fifth nucleotide include (A) uridine (U5, magenta), (B) cytidine (C5, yellow), (C) guanosine (G5, salmon) or (D) adenosine (A5, green). Perspectives are similar to Figure 3F. Panel C is rotated 10° into the plane of the page for clarity of the interactions. Nitrogens (blue) and oxygens (red) are colored; interacting water molecules (red) and sodium ions (lime green) are indicated by spheres. Hydrogen bonds are indicated by dashed lines. Mutated residues are bold and marked by an asterisk. Representative electron density is shown for the nucleotides in Supplementary Figure S3.
Figure 5.
Figure 5.
Molecular dynamics studies of conformational flexibility. (A) Root mean squared fluctuations (RMSF) of U2AF2 Cα by residue numbers. The RRM1 and RRM2 regions are relatively static. (B) Inset showing RMSF of the inter-RRM linker Cαs (residues 230–260). (C) RMSF values of six-membered rings of the RNA in the U2AF2-RNA complex simulation (2 μs). (D) RMSF values of six-membered rings of the RNA in the oligonucleotide simulations (1 μs). The oligonucleotide simulations have higher fluctuations than the U2AF2–RNA simulations. Supplementary Figures S4 and S5 show RMSDs to demonstrate stability of the protein during simulation and convergence of the simulation.
Figure 6.
Figure 6.
U2AF2 residues at the RNA interface influence its specificity for the central nucleotide. (A–C) Average fluorescence anisotropy data points and standard deviations from three replicates of the indicated U2AF212L mutants titrated into 5′-fluorescein-labeled RNA oligonucleotides. The fitted curves are overlaid. The RNA sequences comprising nine-uridines (blue) or its C5, G5 or A5 variants (mustard, salmon, or green), are inset alongside the apparent equilibrium dissociation constants (KD) and standard deviations. (D) Scatter graph of the ratios of the wild-type or mutant U2AF212L binding affinities for U5 to the affinities for the C5 (square), G5 (inverted triangle), or A5 (triangle) variants of the central nucleotide. The KD’s and specificities of the U2AF2 variants binding the G5 and A5 RNAs are estimates due to the very low affinities. Supplementary Figure S2 shows penalties of K225E or R227E mutations on U2AF212L–RNA binding.
Figure 7.
Figure 7.
U2AF2–RNA binding adapts to the local uridine content of the 3′ splice site in vivo. (A) Classification of U2AF2-bound splice site junctions according to the number of uridines (Us) in the –11 to –4 region of the 3′��splice site. Shadowed areas distinguish the three classes of junctions: blue, poor uridine content (0–2 Us); grey, medium uridine content (3–5 Us); red, high uridine content (6–8 Us). This analysis is focused on internal and last exons of all spliced transcripts. (B) 3′ splice site sequence logos for each class. N, number of junctions per class. (C) Binding metaprofiles (y-axis, mean ± SEM of the percentage of crosslinking events) for each class of uridine-containing junctions in U2AF2 eCLIP-seq (top panel, n = 2) and in U2AF2 eCLIP-seq in the presence of modestly overexpressed (OE) U2AF1, at 1.7X in comparison to endogenous levels (36) (bottom panel, n = 4). Yellow area: –11 to –4 region. Supplementary Figure S7 shows steps and results of the U2AF2 eCLIP-seq sample preparation. Supplementary Figure S8 shows binding profiles of representative junctions from each class of uridine content.
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