Features of Double-stranded RNA-binding Domains of RNA Helicase A Are Necessary for Selective Recognition and Translation of Complex mRNAs

Ranji, Arnaz; Shkriabai, Nikolozi; Kvaratskhelia, Mamuka; Musier‐Forsyth, Karin; Boris‐Lawrie, Kathleen

doi:10.1074/jbc.m110.176339

Cited by 44 publications

(56 citation statements)

References 36 publications

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“…The α-β-β-β-α dsRBD facilitates interaction with retroviral RNA sequences, in particular the 5’ UTR (Agbottah et al, 2007; Fujii et al, 2001; Mouland et al, 2000; Ranji et al, 2011). …”

Section: Resultsmentioning

confidence: 99%

HIV-1 and two avian retroviral 5′ untranslated regions bind orthologous human and chicken RNA binding proteins

Stake

Singh

et al. 2015

Virology

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Essential host cofactors in retrovirus replication bind cis-acting sequences in 5’untranslated region (UTR). Although host RBPs are crucial to all aspects of virus biology, elucidating their roles in replication remains a challenge to the field. Here RNA affinity-coupled-proteomics generated a comprehensive, unbiased inventory of human and avian RNA binding proteins (RBPs) co-isolating with 5’UTRs of HIV-1, spleen necrosis virus and Rous sarcoma virus. Applying stringent biochemical and statistical criteria, we identified 185 RBP; 122 were previously implicated in retrovirus biology and 63 are new to the 5’UTR proteome. RNA electrophoretic mobility assays investigated paralogs present in the common ancestor of vertebrates and one hnRNP was identified central node to the biological process-anchored networks of HIV-1, SNV, and RSV 5’ UTR-proteomes. This comprehensive view of the host constituents of retroviral RNPs is broadly applicable to investigation of viral replication and antiviral response in both human and avian cell lineages.

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Section: Resultsmentioning

confidence: 99%

HIV-1 and two avian retroviral 5′ untranslated regions bind orthologous human and chicken RNA binding proteins

Stake

Singh

et al. 2015

Virology

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“…The N-terminal regions of RHA/MLE contain tandem dsRBDs, and although their relative contributions to RNA-binding activity have not been fully resolved, deletion of dsRBD2 abrogates productive engagement of RHA and MLE with their specific RNA substrates (e.g., refs. [20][21][22]. Deletion of the dsRBD in DHX29 abrogated ribosomal association and ribosomal stimulation of its NTPase activity and abolished its function in 48S complex formation, even though it did not affect stimulation of DHX29's NTPase activity by RNA.…”

Section: Discussionmentioning

confidence: 99%

Roles of individual domains in the function of DHX29, an essential factor required for translation of structured mammalian mRNAs

Dhote

Sweeney

Kim

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

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On most eukaryotic mRNAs, initiation codon selection involves baseby-base inspection of 5′ UTRs by scanning ribosomal complexes. Although the eukaryotic initiation factors 4A/4B/4G can mediate scanning through medium-stability hairpins, scanning through more stable structures additionally requires DHX29, a member of the superfamily 2 DEAH/RNA helicase A (RHA) helicase family that binds to 40S subunits and possesses 40S-stimulated nucleoside triphosphatase (NTPase) activity. Here, sequence alignment and structural modeling indicated that DHX29 comprises a unique 534-aa-long N-terminal region (NTR), central catalytic RecA1/RecA2 domains containing a large insert in the RecA2 domain, and the C-terminal part, which includes winged-helix, ratchet, and oligonucleotide/oligosaccharide-binding (OB) domains that are characteristic of DEAH/RHA helicases. Functional characterization revealed that specific ribosomal targeting is required for DHX29's activity in initiation and is determined by elements that map to the NTR and to the N-terminal half of the winged-helix domain. The ribosome-binding determinant located in the NTR was identified as a putative double-stranded RNAbinding domain. Mutational analyses of RecA1/RecA2 domains confirmed the essential role of NTP hydrolysis for DHX29's function in initiation and validated the significance of a β-hairpin protruding from RecA2. The large RecA2 insert played an autoinhibitory role in suppressing DHX29's intrinsic NTPase activity but was not essential for its 40S-stimulated NTPase activity and function in initiation. Deletion of the OB domain also increased DHX29's basal NTPase activity, but more importantly, abrogated the responsiveness of the NTPase activity to stimulation, which abolished DHX29's function in initiation. This finding suggests that the OB domain, which is specific for DEAH/RHA helicases, plays an important role in their NTPase cycle.DEAH/RHA family | translation initiation T ranslation initiation on the majority of eukaryotic mRNAs occurs by the scanning mechanism (1). First, 43S preinitiation complexes comprising 40S subunits, Met-tRNA i Met /eIF2/GTP, and eukaryotic initiation factors (eIFs) 3, 1, and 1A attach to the cap-proximal region of mRNA in a process that is mediated by eIF4A, eIF4B, and eIF4F. eIF4F consists of three subunits: eIF4E (a cap-binding protein), eIF4A (a DEAD-box RNA helicase), and eIF4G (a scaffold for eIF4E and eIF4A, which also binds to eIF3). eIFs 4A/4B/4G cooperatively unwind the cap-proximal region of mRNA allowing attachment of 43S complexes. After attachment, 43S complexes scan to the initiation codon where they form 48S initiation complexes with established codon-anticodon base-pairing. In addition to promoting attachment, eIFs 4A/4B/4G assist 43S complexes during scanning. Although eIFs 4A/4B/4G can mediate scanning through stems of medium stability, scanning through more stable secondary structures additionally requires the DExH-box protein DHX29 (2). DHX29 is also essential for initiation on several viral mRNAs (3, 4). Con...

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“…3), favoring the hypothesis that the dsRBDs are the main domains that mediate the binding of RHA to substrate RNA and that the main target for RHA binding would be a duplex RNA region. The importance of dsRBDs for the interaction of RHA with RNA is suggested by the fact that dsRBDs are critical for RHA to promote the translational efficiency of HIV-1 mRNA (45), and, in contrast to wild-type RHA, mutant RHAs lacking either one or both dsRBDs are unable to be packaged into virus particles or to promote the annealing of tRNA 3 Lys to viral RNA (Fig. 4).…”

Section: Discussionmentioning

confidence: 99%

“…The residues N terminal to the core helicase domain contain two dsRNA binding domains (dsRBDs). The C terminus of RHA is characterized by a stretch of repeated arginine and glycine-glycine (RGG) residues that have a higher binding affinity for single-stranded RNA (ssRNA) than for dsRNA (10,45,57). Between the core helicase and the RGG domains are the helicase-associated domain (HA2) and the oligonucleotide/ oligosaccharide binding (OB-fold) that was defined by its homology with the Saccharomyces cerevisiae protein Prp43p (51).…”

mentioning

confidence: 99%

In Vitro and In Vivo Analysis of the Interaction between RNA Helicase A and HIV-1 RNA

Xing

Niu

Kleiman

2012

J Virol

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bRNA helicase A (RHA) promotes multiple steps of HIV-1 RNA metabolism during viral replication, including transcription, translation, and the annealing of primer tRNA 3 Lys to the viral RNA. RHA is a member of the DExH subclass of RNA helicases that uniquely contains two double-stranded RNA binding domains (dsRBDs) at its N terminus. Here, we performed a genome-wide analysis of the interaction of RHA with HIV-1 RNA both in vitro, using fluorescence polarization, and during viral replication, using an RNA-protein coprecipitation assay. In vitro, RHA binds to all the isolated regions of the HIV-1 RNA genome tested, with K d (equilibrium dissociation constant) values ranging from 44 to 178 nM. In contrast, during viral replication, RNA-protein coprecipitation assays detected only a major interaction of RHA with the 5=-untranslated region (5=-UTR) and a minor interaction with the Rev response element (RRE) of HIV-1 RNA. Since RHA does not associate well with all the highly structured regions of HIV-1 RNA tested in vivo, the results suggest that other viral or cellular factors not present in vitro may modulate the direct interaction of RHA with HIV-1 RNA during virus replication. Nevertheless, a role for duplex RNA as a target for RHA binding in vivo is suggested by the fact that the deletion of either one or both dsRBDs eliminates the in vivo interaction of RHA with HIV-1 RNA. Furthermore, these mutant RHAs do not promote the in vivo annealing of tRNA 3Lys to viral RNA, nor are they packaged into virions, demonstrating that the dsRBDs are essential for the role of RHA in HIV-1 replication. RNA helicases are ubiquitous in nature and have been shown to be involved in all aspects of RNA metabolism. They are classified into five different superfamilies, of which superfamily 2 (SF2) is the largest (7). RNA helicase A (RHA), also known as DHX9, is an SF2 DExH box protein that can unwind both doublestranded RNA (dsRNA) and double-stranded DNA (dsDNA) by utilizing any of the four ribo-or deoxyribonucleotide triphosphates (31, 57). RHA contains 7 domains (depicted in Fig. 3A). The core helicase domain is located at the center and is composed of two subdomains, the DExH subdomain (named after a signature amino acid sequence in RNA helicases, which is DEIH in RHA) and the HELICc subdomain (35). The core helicase domain can bind and hydrolyze nucleotide triphosphates (NTPs) and is composed of 8 motifs that are conserved in all members of the DExD/H box helicases (14). The residues N terminal to the core helicase domain contain two dsRNA binding domains (dsRBDs). The C terminus of RHA is characterized by a stretch of repeated arginine and glycine-glycine (RGG) residues that have a higher binding affinity for single-stranded RNA (ssRNA) than for dsRNA (10,45,57). Between the core helicase and the RGG domains are the helicase-associated domain (HA2) and the oligonucleotide/ oligosaccharide binding (OB-fold) that was defined by its homology with the Saccharomyces cerevisiae protein Prp43p (51).RHA was reported to participate in a d...

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Features of Double-stranded RNA-binding Domains of RNA Helicase A Are Necessary for Selective Recognition and Translation of Complex mRNAs

Cited by 44 publications

References 36 publications

HIV-1 and two avian retroviral 5′ untranslated regions bind orthologous human and chicken RNA binding proteins

HIV-1 and two avian retroviral 5′ untranslated regions bind orthologous human and chicken RNA binding proteins

Roles of individual domains in the function of DHX29, an essential factor required for translation of structured mammalian mRNAs

In Vitro and In Vivo Analysis of the Interaction between RNA Helicase A and HIV-1 RNA

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