ANALYSIS OF RNA-BINDING PROTEINS BY IN-VITRO GENETIC SELECTION - IDENTIFICATION OF AN AMINO-ACID RESIDUE IMPORTANT FOR LOCKING U1A ONTO ITSRNA TARGET

An in vitro genetic system was developed as a rapid means for studying the specificity determinants of RNA-binding proteins. This system was used to investigate the origin of the RNA-binding specificity of the mammalian spliceosomal protein U1A. The U1A domain responsible for bin ding to U1 small nuclear RNA was locally mutagenized and displayed as a combinatorial library on filamentous bacteriophage. Affinity selecti on identified four U1A residues in the mutagenized region that are imp ortant for specific binding to Ill hairpin II. One of these residues ( Leu-49) disproportionately affects the rates of binding and release an d appears to play a critical role in locking the protein onto the RNA. Interestingly, a protein variant that binds more tightly than U1A eme rged during the selection, showing that the affinity of U1A for Ill RN A has not been optimized during evolution.