IN-VITRO SELECTION OF RNA LIGANDS FOR THE RIBOSOMAL L22 PROTEIN ASSOCIATED WITH EPSTEIN-BARR VIRUS-EXPRESSED RNA BY USING RANDOMIZED AND CDNA-DERIVED RNA LIBRARIES

The Epstein-Barr virus (EBV) expressed RNA 1 (EBER1) associates tightl y with the ribosomal protein L22, We determined the general requiremen ts for an RNA to bind L22 in a SELEX experiment, selecting RNA ligands for L22 from a randomized pool of RNA sequences by using an L22-gluta thione S-transferase fusion protein, The selected sequences all contai ned a stem-loop motif similar to that of the region of EBER1 previousl y shown to interact with L22, The nucleotides were highly conserved at three positions within the stem-loop and identical to the correspondi ng nucleotides in EBER1, Two independent binding sites for L22 could b e identified in EBER1, and mobility shift assays indicated that two L2 2 molecules can interact with EBER1 simultaneously, To search for a ce llular L22 ligand, we constructed a SELEX library from cDNA fragments derived from RNA that was coimmunoprecipitated with L22 from an EBV-ne gative whole-cell lysate, After four rounds of selection and amplifica tion, most of the clones that were obtained overlapped a sequence corr esponding to the stem-loop between nucleotides 302 and 317 in human 28 S ribosomal RNA, This stem-loop fulfills the criteria for optimal bind ing to L22 that were defined by SELEX, suggesting that human 28S ribos omal RNA is likely to be a cellular L22 ligand. Additional L22 binding sites were found in 28S ribosomal RNA, as well as within 18S ribosoma l RNA and in RNA segments not present in sequence databases, The metho dology described for the conversion of a preselected cellular RNA pool into a SELEX library might be generally applicable to other proteins for the identification of cellular RNA ligands.