Interaction between the double-stranded RNA-binding protein Xlrbpa and the double stranded RNA-dependent Kinase PKR

The Xenopus double stranded RNA-binding protein Xlrbpa is associated with ribosomes and hnRNAs (Eckmann and Jantsch, 1997). Mammalian homologues of this protein have been isolated from mouse and human by their ability to bind protamine or HIV-TAR RNA, respectively (Lee et al., 1996; Gatignol et al., 1991). However, association with those RNAs most likely reflects the structured nature of these RNAs and thus might not be the only interacting RNAs in the cell. In fact, the ubiquitous expression of XIrbpa and its homologues suggests a more general function for this group of proteins. Recent data indicate that TRBP, the human homologue of Xlrbpa, might act as a cellular regulator of the double- stranded RNA activated kinase PKR (Park et al., 1994; Cosentino, et al., 1995; Benkirane et al., 1997). PKR is a major player in the cellular anti viral defense mechanism. The kinase can be activated by viral RNAs and subsequently phosphorylates translation factor eIF2a which in turn leads to a shut down in protein synthesis (for review see Park, 1995). Surprisingly, PKR is not activated by cellular RNAs. It seems possible, however, that TRBP (or any of its homologues) prevents inadvertent activation of PKR by cellular RNAs by binding and sequestering them (Cosentino et al., 1995). Alternatively, it has been proposed that direct interaction between TRBP and PKR might prevent activation of the kinase (Benkirane et al., 1997). The recent finding that TRBP can interact with HIV Tax protein further supports the idea that this double- stranded RNA-binding protein can interact with other cellular proteins via direct protein-protein interaction (Donzeau et al., 1997). Here we propose to study the interaction of Xlrbpa and PKR kinase in vivo and in vitro. Xlrbpa interacting proteins should be isolated by means of a yeast two-hybrid screen. Finally, to investigate the proposed interaction of Xlrbpa and PKR in Xenopus we propose to isolate the Xenopus homologue of the double-stranded RNA dependent kinase PKR.

RNA-binding proteins determine the fate of an RNA and are thus important regulators of cellular functions such as RNA-transport, processing or translation. Given the diversity of functions that are intrinsic to RNA-binding proteins, proper substrate recognition is very important and to date several protein motifs that can specifically recognize different RNAs have been described. While some RNA-binding motifs read the sequence of an RNA others only identify the structure of a particular RNA-segment or require additional cofactors to facilitate and stabilize specific RNA-interactions. Members of the dsRBD family of proteins interact with structured RNAs by means of the so called double- stranded RNA-binding domain (dsRBD) that recognizes the structure but not the sequence of an RNA. It is therefore enigmatic how members of this diverse group of RNA-binding proteins specifically recognize and interact with their substrates. The interferon inducible kinase PKR is activated by double-stranded RNA which is bound by two dsRBDs in the protein. Interestingly, PKR is selectively activated by viral but not cellular RNAs. Using baker`s yeast as a model system we have tested the interaction of PKR with cellular RNAs in competition with other dsRBD proteins. We could show, that Xlrbpa, another dsRBD protein can prevent activation of PKR by competing for structured RNAs. Based on this finding we hypothesize that substrate specificity of dsRBD proteins is governed through competition amongst various dsRBD proteins. We also studied intracellular interaction partners of dsRBD proteins. Here we could show that dsRBDs can serve as protein interaction domains which is a novel function of this motif.