Sex lethal and U2 small nuclear ribonucleoprotein auxiliary factor (U2AF65) recognize polypyrimidine tracts using multiple modes of binding

Abstract

The molecular basis for specific recognition of simple homopolymeric sequences like the polypyrimidine tract (Py tract) by multiple RNA recognition motifs (RRMs) is not well understood. The Drosophila splicing repressor Sex lethal (SXL), which has two RRMs, can directly compete with the essential splicing factor U2AF(65), which has three RRMs, for binding to specific Py tracts. We have combined site-specific photocross-linking and chemical cleavage of the proteins to biochemically map cross-linking of each of the uracils within the Py tract to specific RRMs. For both proteins, RRM1 and RRM2 together constitute the minimal Py-tract recognition domain. The RRM3 of U2AF(65) shows no cross-linking to the Py tract. Both RRM1 and RRM2 of U2AF(65) and SXL can be cross-linked to certain residues, with RRM2 showing a surprisingly high number of residues cross-linked. The cross-linking data eliminate the possibility that shorter Py tracts are bound by fewer RRMs. We present a model to explain how the binding affinity can nonetheless change as a function of the length of the Py tract. The results indicate that multiple modes of binding result in an ensemble of RNA-protein complexes, which could allow tuning of the binding affinity without changing sequence specificity.

FIGURE 1.

Schematics of site-specific cross-linking assay. (A) Sequences of three natural Py tracts used for cross-linking. (B, top) Schematics of SXL(W), SXL(W)ΔC, SXL(W)ΔNΔC, U2AF⁶⁵(1W23), and U2AF⁶⁵(12W3) proteins. Shaded areas represent RRMs. Asterisks indicate the positions of tryptophan substitutions: tyrosine-200, SXL(W); leucine-235, U2AF⁶⁵ (1W23); and alanine-339, U2AF⁶⁵ (12W3). (Bottom) Diagrammatic representation of SDS–polyacrylamide gels showing the hypothetical positions of the radiolabeled protein fragments following the NCS cleavage of SXL(W), SXL(W)ΔC, SXL(W)ΔNΔC, U2AF⁶⁵(1W23), and U2AF⁶⁵(12W3). For SXL (left panel), if an RNA is cross-linked to RRM2, NCS cleavage of SXL(W)ΔC would generate a smaller labeled polypeptide than that observed with SXL(W). However, the size of the polypeptide would remain unaffected if it is cross-linked to RRM1. On the other hand, the NCS cleavage of SXL(W)ΔNΔC would generate a smaller labeled polypeptide fragment than that with SXL(W) regardless of whether RNA is cross-linked to RRM1 or RRM2. If an RNA is cross-linked to both RRMs (RRM1/RRM2), two cleavage products, of varying intensity, would appear in the same lane. For U2AF⁶⁵ (right panel), if an RNA is cross-linked to RRM1, NCS cleavage would generate a smaller polypeptide for U2AF⁶⁵(1W23) and a larger polypeptide for U2AF⁶⁵(12W3). If an RNA is cross-linked to RRM3, NCS cleavage would generate a smaller polypeptide for U2AF⁶⁵(12W3) and a larger polypeptide for U2AF⁶⁵(1W23). If it is cross-linked to RRM2, NCS cleavage would generate a larger polypeptide for both proteins. If it is cross-linked to two RRMs (RRM1/RRM2), two cleavage products, of varying intensity, would appear in the same lane. For simplicity, other double combinations (RRM1/RRM3, RRM2/RRM3) are not shown. This strategy unambiguously identifies whether a given residue is cross-linked to RRM1, RRM2, or RRM3. Under these conditions, a portion of the input protein remains uncleaved (Mirfakhrai and Weiner 1993). Solid rectangles represent cleaved peptides corresponding to RRM1, RRM2, or RRM3, and empty rectangles represent uncleaved proteins.

FIGURE 2.

Site-specific cross-linking of SXL to 5-IU containing tra NSS (A) or AdML (B) Py tracts. Each of the 5′-end-labeled RNAs containing either no 5-IU (None) or a single 5-IU at various positions was cross-linked to SXL(W), SXL(W)ΔC, and SXL(W)ΔNΔC. The cross-linked protein was cleaved with NCS and resolved in an SDS–polyacrylamide gel. The positions of the 5-IU used for the cross-linking are shown below, and the percentage relative cross-linking to either RRM1 or RRM2 is shown above the autoradiograms; (–) weak cross-linking. For reference, the tra (NSS) and the AdML Py-tract sequences are shown. Lanes a, b, and c correspond to various SXL derivatives. The positions of the uncleaved protein (curly brackets) and of the cleaved fragments corresponding to RRM1 and RRM2 (arrows) are shown. (C) Summary of the X-ray structure of SXL and the NSS Py tract of tra (Handa et al. 1999). The boxes refer to RRM1 and RRM2, and lines below the sequence refer to the nucleotides that were either degraded or did not contact SXL in the X-ray structure.

FIGURE 2.

Site-specific cross-linking of SXL to 5-IU containing tra NSS (A) or AdML (B) Py tracts. Each of the 5′-end-labeled RNAs containing either no 5-IU (None) or a single 5-IU at various positions was cross-linked to SXL(W), SXL(W)ΔC, and SXL(W)ΔNΔC. The cross-linked protein was cleaved with NCS and resolved in an SDS–polyacrylamide gel. The positions of the 5-IU used for the cross-linking are shown below, and the percentage relative cross-linking to either RRM1 or RRM2 is shown above the autoradiograms; (–) weak cross-linking. For reference, the tra (NSS) and the AdML Py-tract sequences are shown. Lanes a, b, and c correspond to various SXL derivatives. The positions of the uncleaved protein (curly brackets) and of the cleaved fragments corresponding to RRM1 and RRM2 (arrows) are shown. (C) Summary of the X-ray structure of SXL and the NSS Py tract of tra (Handa et al. 1999). The boxes refer to RRM1 and RRM2, and lines below the sequence refer to the nucleotides that were either degraded or did not contact SXL in the X-ray structure.

FIGURE 2.

Site-specific cross-linking of SXL to 5-IU containing tra NSS (A) or AdML (B) Py tracts. Each of the 5′-end-labeled RNAs containing either no 5-IU (None) or a single 5-IU at various positions was cross-linked to SXL(W), SXL(W)ΔC, and SXL(W)ΔNΔC. The cross-linked protein was cleaved with NCS and resolved in an SDS–polyacrylamide gel. The positions of the 5-IU used for the cross-linking are shown below, and the percentage relative cross-linking to either RRM1 or RRM2 is shown above the autoradiograms; (–) weak cross-linking. For reference, the tra (NSS) and the AdML Py-tract sequences are shown. Lanes a, b, and c correspond to various SXL derivatives. The positions of the uncleaved protein (curly brackets) and of the cleaved fragments corresponding to RRM1 and RRM2 (arrows) are shown. (C) Summary of the X-ray structure of SXL and the NSS Py tract of tra (Handa et al. 1999). The boxes refer to RRM1 and RRM2, and lines below the sequence refer to the nucleotides that were either degraded or did not contact SXL in the X-ray structure.

FIGURE 3.

Site-specific cross-linking of U2AF⁶⁵ to 5-IU containing tra NSS (A), AdML (B), and tra FS (C) Py tracts. The 5-IU RNAs were cross-linked to U2AF⁶⁵(1W23) (lane a′) and U2AF⁶⁵(12W3) (lane b′). Positions of various fragments are indicated; an asterisk represents positions of variant size band(s), which is prominent in lane 8a′, of unknown identity. For reference, the tra (FS) Py-tract sequence is shown in panel C. For further details, see legend to Figure 2 ▶.

FIGURE 4.

RNA binding of SXL to wild type and various mutants of the tra NSS Py tract. Molar concentrations of recombinant GST–SXL are shown on the X-axis, and the fraction of RNA bound on the Y-axis. The sequences of various mutants are shown at the bottom, and the mutations are underlined. For simplicity, only the Py tracts are shown.

FIGURE 5.

3′-splice-site switching by SXL. (Top) Schematics of the sex-specific alternative splicing of the wild-type pre-mRNA substrate. The boxes represent exons, and the lines represent introns. The alternative 3′-splice sites (NSS and FS), the NSS Py tract (SXL-binding site), and the translation stop codon (STOP) are shown. (Bottom) SXL-mediated splice-site switching for the wild-type (M-tra), and the U1,3,5C and U2,4C mutants (underlined) of the NSS Py tract of tra. Precursor RNAs were spliced in an HeLa nuclear extract in the absence (−) or presence of different concentrations of GST–SXL (0.08 μM, 0.25 μM, 0.76 μM, and 2.3 μM), and the spliced products were analyzed by primer extension assay using radiolabeled NSS or FS splice junction primers indicated by the arrows.

FIGURE 6.

Model for Py-tract recognition—multiple modes of binding. Both RRM1 and RRM2 contact adjacent uridine stretches (A). In comparison to complex A, only RRM2 binds in a different register in complex B, and both RRMs bind in different registers in complex C. Triangles represent nucleotides or uridines. Double arrows indicate that these complexes are in equilibrium. For simplicity, only three binding sites are shown for each RRM. The actual number of complexes will depend on the length of the Py tract and the number of nucleotides that interact with each RRM.