The multiple functions of TRBP, at the hub of cell responses to viruses, stress, and cancer

Abstract

The TAR RNA binding protein (TRBP) has emerged as a key player in many cellular processes. First identified as a cellular protein that facilitates the replication of human immunodeficiency virus, TRBP has since been shown to inhibit the activation of protein kinase R (PKR), a protein involved in innate immune responses and the cellular response to stress. It also binds to the PKR activator PACT and regulates its function. TRBP also contributes to RNA interference as an integral part of the minimal RNA-induced silencing complex with Dicer and Argonaute proteins. Due to its multiple functions in the cell, TRBP is involved in oncogenesis when its sequence is mutated or its expression is deregulated. The depletion or overexpression of TRBP results in malignancy, suggesting that the balance of TRBP expression is key to normal cellular function. These studies show that TRBP is multifunctional and mediates cross talk between different pathways. Its activities at the molecular level impact the cellular function from normal development to cancer and the response to infections.

Fig 1

Structural organization of TRBP. (A) Structural organization of TRBP1 and TRBP2. dsRBDs mediate RNA binding and are represented in red. Dark red represents the RNA binding motif, which is fully functional only in dsRBD2 and has been named the KR-helix motif. The Medipal domain is involved in protein-protein interactions. The orange domain (also called C4) has a weak homology with dsRBDs and does not bind RNA but mediates Dicer and PACT interactions. Sites of homo- and heterodimerizations are indicated. The dark red and dark orange domains are basic domains with an α-helix structure. (B) Comparison of the dsRNA binding surfaces of the dsRBDs of TRBPs. (Top) Sequence of dsRBD1 and dsRBD2 of TRBP (amino acid numbers refer to TRBP1). The dsRNA binding residues are indicated with arrowheads. The conserved dsRNA binding residues are shaded in yellow. (Bottom left) Docking model of the TRBP dsRBD1 with dsRNA. The residues with side chains that interact directly with dsRNA are shown in stick representations. The hydrogen bonds are shown by dashed lines. (Bottom right) Structure of the dsRBD2-dsRNA complex. (Adapted from reference with permission of Wiley-Blackwell [copyright 2010 The Protein Society].)

Fig 2

Evolution and phylogeny of TRBP. (A) Homologies between mammalian TRBP2 proteins. Alignments were performed by using BLASTP (http://www.ncbi.nlm.nih.gov/blast/) and COBALT multiple alignments (110). Different amino acids compared to human TRBP2 are shown in red. GenBank accession numbers are indicated on the left. The name of the species used is shown on the right. The human clone in HeLa cells is the first identified protein (52). Mouse PRBP is the first identified murine protein (89). Underlined are dsRBD1 (aa 31 to 96), dsRBD2 (aa 160 to 226), and the C4 domain of Medipal (aa 298 to 366), with amino acid numbers referring to TRBP2. (B) Phylogenetic tree of TRBP2 obtained from COBALT multiple alignments. The phylogenetic tree was created by using the fast minimum-evolution method (33), showing evolutionary distance according to data reported previously by Grishin (61). The branches on the right of the PACT homologs represent proteins that have more homology with PACT than with TRBP2. The branches on the right of the TRBP homologs are more closely related to TRBP2. Proteins between sea urchins and tunicates are related to both PACT and TRBP2.

Fig 3

Role of TRBP in regulating HIV-1 expression and replication through PKR regulation. (A) Control of HIV replication in astrocytes compared to HIV-permissive cells through TRBP expression. In HIV-permissive cells, the NF-Y transcription factors activate the TRBP1 promoter, which results in the production of TRBP1 and TRBP2. TRBP binds to PKR and inhibits its activation. This results in the translation of HIV-1 proteins and high-level HIV-1 replication. Astrocytes express low levels of NF-Y transcription factors, which results in low TRBP promoter expression and low TRBP protein production levels. Low TRBP protein expression levels can also be obtained by using siRNAs against TRBP mRNA. Low TRBP expression levels result in high levels of PKR activation, a block of HIV-1 protein production, and low levels of HIV-1 replication (3, 4, 24, 109, 127). (B) Control of PKR activation by TRBP, ADAR1, and PACT during HIV-1 expression. PKR is activated by HIV-1 TAR RNA. This activation is inhibited by TRBP and ADAR1 but enhanced by PACT. TRBP also inhibits PACT activity, which can be restored by oxidative stress (25, 26, 29, 30, 84). (Adapted from reference .)

Fig 4

TRBP in the exogenous and endogenous RNAi pathways. (A) Exogenous pathway mediated by siRNAs and shRNAs. Perfectly matched dsRNAs are bound by Dicer and TRBP and cleaved by Dicer to form 21- to 23-nucleotide (nt) siRNAs. The complex then recruits Ago2 and forms the RISC. After strand separation, the complex hybridizes to the complementary sequence in an mRNA. Ago2 mediates the cleavage of the mRNA. (B) Endogenous pathway mediated by miRNAs. Primary mRNAs (pri-miRNAs) are synthesized in the nucleus. They are bound by Drosha and DGCR8. Drosha cleaves them to form the precursor miRNA (pre-miRNA), which is an imperfectly matched dsRNA. Pre-miRNAs are exported to the cytoplasm via exportin5 (Exp5), where they are bound by TRBP and Dicer. Dicer cleaves them to form miRNAs. The RISC is then formed with Ago2. After strand separation, one miRNA strand targets the corresponding mRNA. The translation of this mRNA is generally repressed by a process that involves the recruitment of additional factors and the formation of processing bodies, but in some cases, cleavage of the target mRNA may occur (dotted arrow).

Fig 5

TRBP involvement in malignant phenotypes. (Left) In cells, TRBP can be bound to Merlin, Dicer, PKR, and PACT. These interactions are reversible and regulated. (Top) When TRBP is overexpressed, it binds Dicer for increased RNAi activity, it fully represses PACT and PKR, and it is not fully inactivated by Merlin. This results in a transformed phenotype. (Middle) When the nf2 gene, coding for Merlin, is disrupted in brain cancers, TRBP becomes overexpressed by a lack of degradation, which results in increased RNAi and increased PKR inhibition. (Bottom) When TRBP is truncated in its C terminus, as seen in some cancer cells, it no longer binds to Dicer, which results in decreased RNAi. It still binds to PKR and inactivates it, but its affinity for PACT is decreased. It no longer interacts with Merlin, which cannot degrade it. As a consequence, TRBP overexpression, Merlin inactivation, or TRBP truncation results in malignant phenotypes by different mechanisms.

Fig 6

Known and possible interactions and functions of TRBP under normal conditions, in HIV-infected cells, and in cancer cells. (A) Known and possible interactions and functions of TRBP under normal conditions. TRBP interacts with cellular RNAs and miRNAs as well as with Dicer, PKR, and PACT to regulate translation, RNAi, and PKR activation. Its binding to Merlin mediates its degradation and control of cell growth. (B) Known and possible interactions and functions of TRBP in HIV-1-infected cells. TRBP interacts with HIV-1 TAR RNA to increase the translation of HIV-1 mRNAs, while it still binds to cellular mRNAs and miRNAs. TRBP binds to Dicer, PKR, and PACT to regulate RNAi and PKR activation. Its interaction with Merlin has not been tested in infected cells. (C) Known and possible interactions and functions of TRBP in cancer cells. The disruption between TRBP and Merlin leads to an increased TRBP concentration and increased cell growth. The overexpression or truncation of TRBP results in increased binding to cellular mRNA and miRNA, which disturbs translational control. Increased or decreased binding to Dicer may disturb RNAi activity and lead to cancer. The small molecule enoxacin enhances miRNA processing by TRBP and inhibits cancer growth (100). Increased binding to PKR and PACT inhibits PKR activation.