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Dissecting the roles of TRBP and PACT in double‐stranded RNA recognition and processing of noncoding RNAs

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HIV TAR RNA‐binding protein (TRBP) and Protein Activator of PKR (PACT) are double‐stranded (ds) RNA‐binding proteins that participate in both small regulatory RNA biogenesis and the response to viral dsRNA. Despite considerable progress toward understanding the structure–function relationship of TRBP and PACT, their specific roles in these seemingly distinct cellular pathways remain unclear. Both proteins are composed of three copies of the double‐stranded RNA‐binding domain, two of which interact with dsRNA, while the C‐terminal copy mediates protein–protein interactions. PACT and TRBP are found in a complex with the endonuclease Dicer and facilitate processing of immature microRNAs. Their precise contribution to the Dicing step has not yet been defined: possibilities include precursor recruitment, rearrangement of dsRNA within the complex, loading the processed microRNA into the RNA‐induced silencing complex, and distinguishing different classes of small dsRNA. TRBP and PACT also interact with the viral dsRNA sensors retinoic acid‐inducible gene I (RIG‐I) and double‐stranded RNA‐activated protein kinase (PKR). Current models suggest that PACT enables RIG‐I to detect a wider range of viral dsRNAs, while TRBP and PACT exert opposing regulatory effects on PKR. Here, the evidence that implicates TRBP and PACT in regulatory RNA processing and viral dsRNA sensing is reviewed and discussed in the context of their molecular structure. The broader implications of a link between microRNA biogenesis and the innate antiviral response pathway are also considered. WIREs RNA 2015, 6:271–289. doi: 10.1002/wrna.1272 This article is categorized under: RNA Interactions with Proteins and Other Molecules > Protein–RNA Interactions: Functional Implications RNA Turnover and Surveillance > Turnover/Surveillance Mechanisms Regulatory RNAs/RNAi/Riboswitches > Biogenesis of Effector Small RNAs
Functions and domain composition of TAR RNA‐binding protein (TRBP) and Protein Activator of PKR (PACT). (a) Precursor (pre‐)microRNAs (miRNAs) are RNA hairpins that are produced in the nucleus, and exported to the cytoplasm. They contain the ∼22 nt sequence of the mature miRNA, indicated in red. The endonuclease Dicer removes the terminal loop to give an RNA duplex, one strand of which is loaded into an Argonaut (Ago) protein to form RNA‐induced silencing complex (RISC). TRBP and PACT are implicated in both Dicing and RISC loading. (b) PACT and TRBP have roles in at least two viral response pathways. First, PACT can facilitate activation of retinoic acid‐inducible gene I (RIG‐I) by viral double‐stranded RNA (dsRNA) (distinguished from cellular dsRNA by distinct molecular features, discussed in section TRBP and PACT mediate innate immune surveillance). This begins a signaling cascade that results in the production of interferon and other antiviral genes. PACT and TRBP also regulate PKR, a kinase that targets the translation initiation factor eIF2α to inhibit protein production and promote apoptosis. TRBP inhibits PKR, while PACT can activate it in response to cellular stress. (c) Both PACT and TRBP contain three double‐stranded RNA‐binding domains (dsRBDs). The first two domains can bind dsRNA, while the third cannot. Interactions with many other proteins have been documented, particularly for the third domain. Solid lines indicate direct protein–protein interactions, while dashed lines indicate interactions that may be mediated via dsRNA. The third dsRBD potentially has an N‐terminal extension, based on sequence conservation. Each protein has a number of phosphorylation sites (marked by yellow triangles) that regulate function under certain conditions. A region of TRBP implicated in cancers exhibiting microsatellite instability is indicated in red.
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(a) In the absence of appropriate ligands, retinoic acid‐inducible gene I (RIG‐I) has an inactive conformation, in which the helicase domain binds to the caspase recruitment domains (CARDs). 5′‐Triphosphate double‐stranded RNA (dsRNA) binds to the C‐terminal domain (CTD) and helicase domain, which displaces the CARDs and results in signaling. It is less clear how Protein Activator of PKR (PACT) enables RIG‐I activation: one possibility is that it increases RIG‐I binding to additional ligands such as long dsRNA that lacks a 5′‐triphosphate. (b) PKR can bind to long dsRNA through two N‐terminal double‐stranded RNA‐binding domains (dsRBDs). This brings PKR molecules together to form dimers, which can then autophosphorylate and become active. PACT (when phosphorylated during cellular stress) can also activate PKR, although the mechanism is unclear. The two main hypotheses are: PACT‐D3 contacts the kinase domain, somehow favoring activation, or PACT dimers can bind two molecules of PKR, promoting their dimerization and activation. TRBP acts as an inhibitor of PKR, either because its third domain cannot interact with PKR's kinase domain or because its third domain exhibits weaker dimerization.
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(a) Layout of domains within Dicer. (b) Reconstruction of the Dicer–TAR RNA‐binding protein (TRBP) complex from cryo‐electron microscopy (EM) data, with ∼15 Å resolution (EM data bank accession EMD‐1646). The locations of the RNase III and helicase domains are inferred from epitope‐tagged Dicer. The position of TRBP is not resolved. (c) A schematic of a minimal RNA‐induced silencing complex (RISC)‐loading complex of Dicer, Ago2, and TRBP/PACT (Protein Activator of PKR) based on cryo‐EM data. All components are approximately to scale. It is unknown whether all three components assemble prior to double‐stranded RNA (dsRNA) binding, or if the complex is more dynamic. In vivo, it is likely that other proteins associate with the RISC‐loading complex. (d) At least four possible roles for TRBP/PACT can be envisaged (see section How Do TRBP and PACT Affect Processing by Dicer and the Formation of Active RISC?): (1) TRBP/PACT may help recruit dsRNA to Dicer; (2) TRBP/PACT may aid alignment of dsRNA for cleavage by Dicer; (3) TRBP/PACT may help dsRNA unwinding and/or loading into Argonaute proteins; or (4) TRBP/PACT may favor processing and loading of different substrates into RISC (the substrates shown are illustrative only).
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(a) Sequence alignment of the double‐stranded RNA‐binding domains (dsRBDs) of TAR RNA‐binding protein (TRBP) and Protein Activator of PKR (PACT). The top line shows the secondary structure of a ‘typical’ dsRBD, taken from the three‐dimensional (3D) structure of TRBP domain 2 (PDB accession: 3ADL). Residues conserved between all domains are highlighted in black; those conserved between domains 1 and 2 are highlighted in dark gray; while those conserved in the third domain are shown in light gray. The regions of domains 1 and 2 that bind RNA [located in helix α1, the loop between β strands 1 and 2 (loop‐β12), and helix α2] are boxed, as is a conserved region upstream of domain 3, which may represent a structural element additional to the standard dsRBD fold. On the right, the % identity (% similarity) shows that equivalent dsRBDs between TRBP and PACT are more similar than dsRBDs within the same protein. The sequences were aligned using Multalin, and rendered using ESPript (http://espript.ibcp.fr). (b) Two views of TRBP‐D2 bound to two molecules of 10 bp double‐stranded RNA (dsRNA) (PDB accession 3ADL). The RNA‐interacting regions shown in part (a) are highlighted. Several parts of the dsRNA‐binding interface are flexible, most notably loop‐β12 in the second RNA‐interacting region, which contains a highly conserved histidine residue. (c) Structure of a Staufen‐D5 dimer (PDB accession 4DKK). The N‐terminal extension (consisting of two α‐helixes) is thought to interact with the dsRBD core of a second molecule. The linker between the dsRBD core and the extended region is not visible in the crystal structure, and is indicated here with dashed lines.
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RNA Turnover and Surveillance > Turnover/Surveillance Mechanisms
Regulatory RNAs/RNAi/Riboswitches > Biogenesis of Effector Small RNAs
RNA Interactions with Proteins and Other Molecules > Protein–RNA Interactions: Functional Implications

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