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) proteins which possess three RRM-type RNA binding domains (RRMs 1, 2, and 3). Although both proteins have been implicated as effectors of apoptotic cell death, the specific functions of TIA-1 and TIAR are not known. We have performed in vitro selection/amplification from pools of random RNA sequences to identify RNAs to which TIA-1 and TIAR bind with high affinity. Both proteins selected RNAs containing one or several short stretches of uridylate residues suggesting that the two proteins have similar RNA binding specificities. Replacement of the uridylate stretch with an equal number of cytidine residues eliminates the protein-RNA interaction. Mutational analysis indicates that, for both TIA-1 and TIAR, it is the second RNA binding domain (RRM 2) which mediates the specific binding to uridylate-rich RNAs. Although RRM 2 is both necessary and sufficient for this interaction, the affinity for the selected RNA (as determined by filter binding assays) does increase when the second domain of TIAR is expressed together with the first and third domains (Kd = 2 x 10(-8) M) rather than alone (Kd = 5 x 10(-8) M). Although RRM 3 (of either TIA-1 or TIAR) does not interact with the uridylate-rich sequences selected by the full-length proteins, it is a bona fide RNA binding domain capable of affinity-precipitating a population of cellular RNAs ranging in size from 0.5 to 5 kilobases. In contrast, 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.
Dember
L M
LM
Division of Tumor Immunology, Dana-Farber Cancer Institute, Boston, MA 02115, USA.
Kim
N D
ND
Liu
K Q
KQ
Anderson
P
P
D001483:Base Sequence; D007700:Kinetics; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D016296:Mutagenesis; D039102:Poly(A)-Binding Proteins; D011485:Protein Binding; D011506:Proteins; D012313:RNA / Q000737:chemistry / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D000074019:T-Cell Intracellular Antigen-1; D014529:Uridine / Q000378:metabolism
D008565:Membrane Proteins; D039102:Poly(A)-Binding Proteins; D011506:Proteins; D016601:RNA-Binding Proteins; D000074019:T-Cell Intracellular Antigen-1; C071558:TIA1 protein, human; C076751:TIAL1 protein, human; D012313:RNA; D014529:Uridine