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

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.