The TAR RNA-binding Protein, TRBP, Stimulates the Expression of TAR-containing RNAs in Vitro and in VivoIndependently of Its Ability to Inhibit the dsRNA-dependent Kinase PKR

Abstract

TRBP (HIV-1 transactivating response (TAR) RNAbinding protein) and PKR, the interferon-induced dsRNA-regulated protein kinase, contain two dsRNA binding domains. They both bind to HIV-1 TAR RNAs through different sites. Binding to dsRNA activates PKR that phosphorylates the eukaryotic initiation factor eIF-2␣ leading to protein synthesis inhibition. TRBP and PKR can heterodimerize, which inhibits the kinase function of PKR and has a positive effect on HIV-1 expression. In this study, an in vitro reticulocyte assay revealed the poor expression of TAR containing CAT RNAs compared with CAT RNAs. Addition of TRBP restored translation efficiency of TAR-CAT RNA and decreased the phosphorylation status of eIF-2␣, confirming its role as a PKR inhibitor. Unexpectedly, eIF-2␣ was phosphorylated in the presence of TAR-CAT as well as CAT RNA devoid of the TAR structure. TRBP inhibited eIF-2␣ phosphorylation in both cases, suggesting that it restores the translation of TAR-CAT RNA independently and in addition to its ability to inhibit PKR. TRBP activity on gene expression was then analyzed in a PKRfree environment using PKR-deficient murine embryo fibroblasts. In a transient reporter gene assay, TRBP stimulated the expression of a TAR-containing luciferase 3.8-fold whereas the reporter gene with mutated TAR structures or devoid of TAR was stimulated 1.5-to 2.4-fold. Overall, the activity of TRBP2 was higher when the 5-end of the mRNA was structured and was mediated independently by each dsRBD in TRBP. Increasing concentrations of TRBP showed no significant modification of the luciferase RNA levels, suggesting that TRBP stimulates translation of TAR-containing RNAs. Therefore, TRBP is an important cellular factor for efficient translation of dsRNA containing transcripts, both by inhibiting PKR and in a PKR-independent pathway.

ATP-independent diffusion of double-stranded RNA binding proteins

“…TRBP plays many different roles, including inhibition of PKR in antiviral immune signaling (11), activation of HIV-1 gene expression by binding TAR RNA (12), and regulation of cell growth (13). More recently, TRBP was also identified as an integral component of RNA-induced silencing complexes (RISCs) (14) that include TRBP, Dicer, and Argonaute 2 (Ago2).…”

TRBP Homolog Interacts with Eukaryotic Initiation Factor 6 (eIF6) in Fenneropenaeus chinensis

“…TRBP was the first cellular protein identified that strongly binds the HIV-1 encoded leader RNA, a trans-activation response (TAR) protein, and enhances virus expression (1)(2)(3)(4)(5).…”

Emergence of a Complex Relationship between HIV-1 and the microRNA Pathway

“…through a direct block of translation initiation by its secondary structure and by activation of the dsRNA binding protein kinase R (PKR) which, in turn, phosphorylates the eukaryotic initiation factor 2 alpha (eIF2α) leading to an arrest of translation initiation. Both of these negative effects are alleviated by TRBP, which inhibits PKR (53) and releases the translational block due to the TAR structure (54). (For more details about HIV-1 TAR RNA and Tat protein, please refer to these reviews (52,55,56))…”