TARGET DISCRIMINATION BY RNA-BINDING PROTEINS - ROLE OF THE ANCILLARYPROTEIN U2A' AND A CRITICAL LEUCINE RESIDUE IN DIFFERENTIATING THE RNA-BINDING SPECIFICITY OF SPLICEOSOMAL PROTEINS U1A AND U2B''

The spliceosomal proteins U1A and U2B'' each use a homologous RRM doma in to bind specifically to their respective snRNA targets, U1hpll and U2hpIV, two stem-loops that are similar yet distinct in sequence. Prev ious studies have shown that binding of U2B'' to U2hpIV is facilitated by the ancillary protein U2A', whereas specific binding of U1A to U1h pll requires no cofactor. Here we report that U2A' enables U2B'' to di stinguish the loop sequence of U2hpIV from that of U1hpll but plays no role in stem sequence discrimination. Although U2A' can also promote heterospecific binding of U1A to U2hpIV, a much higher concentration o f the ancillary protein is required due to the similar to 500-fold gre ater affinity of U2A' for U2B''. Additional experiments have identifie d a single leucine residue in U1A(Leu-44) that is critical for the int rinsic specificity of this protein for the loop sequence of U1hpll in preference to that of U2hpIV. Our data suggest that most of the differ ence in RNA-binding specificity between U1A and U2B'' can be accounted for by this leucine residue and by the contribution of the ancillary protein U2A' to the specificity of U2B''.