INDIVIDUAL RNA RECOGNITION MOTIFS OF TIA-1 AND TIAR HAVE DIFFERENT RNA-BINDING SPECIFICITIES

TIA-1 and TIAR are two closely related RNA recognition motif (RRM) pro teins which possess three RRM-type RNA binding domains (RRMs 1, 2, and 3). Although both proteins have been implicated as effecters of apopt otic cell death, the specific functions of TIA-1 and TIAR are not know n, We have performed in vitro selection/amplification from pools of ra ndom RNA sequences to identify RNAs to which TIA-1 and TIAR bind with high affinity. Both proteins selected RNAs containing one or several s hort stretches of uridylate residues suggesting that the two proteins have similar RNA binding specificities. Replacement of the uridylate s tretch with an equal number of cytidine residues eliminates the protei n-RNA interaction. Mutational analysis indicates that, for both TIA-1 and TIAR, it is the second RNA binding domain (RRM 2) which mediates t he specific binding to uridylate-rich RNAs. Although RRM 2 is both nec essary and sufficient for this interaction, the affinity for the selec ted RNA (as determined by filter binding assays) does increase when th e second domain of TIAR is expressed together with the first and third domains (K-d = 2 x 10(-8) M) rather than alone (K-d = 5 x 10(-8) M). Although RRM 3 (of either TIA-1 or TIAR) does not interact with the ur idylate-rich sequences selected by the full-length proteins, it is a b ona fide RNA binding domain capable of affinity-precipitating a popula tion of cellular RNAs ranging in size from 0.5 to 5 kilobases. In cont rast, RRM 1 does not affinity-precipitate cellular RNA. The inability of RRM 1 to interact with RNA may be due to the presence of negatively charged amino acids within the RNP 1 octamer.