Abstract:DHX9 is member of the DExD/H-box family of helicases with a “DEIH” sequence at its eponymous DExH-box motif. Initially purified from human and bovine cells and identified as a homologue of the Drosophila Maleless (MLE) protein, it is an NTP-dependent helicase consisting of a conserved helicase core domain, two double-stranded RNA-binding domains at the N-terminus, and a nuclear transport domain and a single-stranded DNA-binding RGG-box at the C-terminus. With an ability to unwind DNA and RNA duplexes, as well … Show more
“…A comparison of these data sets shows significant overlap in the identity of gene products affected by the depletion of either protein (287 genes with altered expression upon DHX9 or Nup98 depletion, p=3.24×10 −36 ; see Material and methods), consistent with the idea that these proteins form a functional complex. Interestingly, a significant number (p-value 2.38 × 10 −4 ) of those genes exhibiting altered expression upon Nup98 depletion contain a putative cAMP-response element (CRE) (Zhang et al, 2005), a regulatory element whose transcriptional activity can be regulated by DHX9 (Aratani et al, 2001; Fidaleo et al, 2016; Lee and Pelletier, 2016). 10.7554/eLife.18825.024Figure 11.Nup98 or DHX9 depletion alters the abundance of target mRNAs.HEK293T cells were transduced with a control shRNA or an shRNA targeting Nup98 or DHX9.…”
Beyond their role at nuclear pore complexes, some nucleoporins function in the nucleoplasm. One such nucleoporin, Nup98, binds chromatin and regulates gene expression. To gain insight into how Nup98 contributes to this process, we focused on identifying novel binding partners and understanding the significance of these interactions. Here we report on the identification of the DExH/D-box helicase DHX9 as an intranuclear Nup98 binding partner. Various results, including in vitro assays, show that the FG/GLFG region of Nup98 binds to N- and C-terminal regions of DHX9 in an RNA facilitated manner. Importantly, binding of Nup98 stimulates the ATPase activity of DHX9, and a transcriptional reporter assay suggests Nup98 supports DHX9-stimulated transcription. Consistent with these observations, our analysis revealed that Nup98 and DHX9 bind interdependently to similar gene loci and their transcripts. Based on our results, we propose that Nup98 functions as a co-factor that regulates DHX9 and, potentially, other RNA helicases.DOI:
http://dx.doi.org/10.7554/eLife.18825.001
“…A comparison of these data sets shows significant overlap in the identity of gene products affected by the depletion of either protein (287 genes with altered expression upon DHX9 or Nup98 depletion, p=3.24×10 −36 ; see Material and methods), consistent with the idea that these proteins form a functional complex. Interestingly, a significant number (p-value 2.38 × 10 −4 ) of those genes exhibiting altered expression upon Nup98 depletion contain a putative cAMP-response element (CRE) (Zhang et al, 2005), a regulatory element whose transcriptional activity can be regulated by DHX9 (Aratani et al, 2001; Fidaleo et al, 2016; Lee and Pelletier, 2016). 10.7554/eLife.18825.024Figure 11.Nup98 or DHX9 depletion alters the abundance of target mRNAs.HEK293T cells were transduced with a control shRNA or an shRNA targeting Nup98 or DHX9.…”
Beyond their role at nuclear pore complexes, some nucleoporins function in the nucleoplasm. One such nucleoporin, Nup98, binds chromatin and regulates gene expression. To gain insight into how Nup98 contributes to this process, we focused on identifying novel binding partners and understanding the significance of these interactions. Here we report on the identification of the DExH/D-box helicase DHX9 as an intranuclear Nup98 binding partner. Various results, including in vitro assays, show that the FG/GLFG region of Nup98 binds to N- and C-terminal regions of DHX9 in an RNA facilitated manner. Importantly, binding of Nup98 stimulates the ATPase activity of DHX9, and a transcriptional reporter assay suggests Nup98 supports DHX9-stimulated transcription. Consistent with these observations, our analysis revealed that Nup98 and DHX9 bind interdependently to similar gene loci and their transcripts. Based on our results, we propose that Nup98 functions as a co-factor that regulates DHX9 and, potentially, other RNA helicases.DOI:
http://dx.doi.org/10.7554/eLife.18825.001
“…Nonetheless, it is intriguing that DHX9, an NTP-dependent DNA and RNA helicase, was identified. DHX9 reportedly interacts with G quadruplexes and antiparallel DNA triple helices (24). Thus, DHX9 may contribute to the unwinding of the MALAT1 triple helix, possibly the first step in the 3′-5′ decay of MALAT1.…”
; reviewed by Brenda L. Bass and Eric M. Phizicky)Metastasis-associated lung adenocarcinoma transcript 1 (MALAT1), a cancer-promoting long noncoding RNA, accumulates in cells by using a 3′-triple-helical RNA stability element for nuclear expression (ENE). The ENE, a stem-loop structure containing a U-rich internal loop, interacts with a downstream A-rich tract (ENE+A) to form a blunt-ended triple helix composed of nine U • A-U triples interrupted by a C • G-C triple and C-G doublet. This unique structure prompted us to explore the possibility of protein binding. Native gel-shift assays revealed a shift in radiolabeled MALAT1 ENE+A RNA upon addition of HEK293T cell lysate. Competitive gel-shift assays suggested that protein binding depends not only on the triple-helical structure but also its nucleotide composition. Selection from the lysate using a biotinylated-RNA probe followed by mass spectrometry identified methyltransferase-like protein 16 (METTL16), a putative RNA methyltransferase, as an interacting protein of the MALAT1 ENE+A. Gel-shift assays confirmed the METTL16-MALAT1 ENE+A interaction in vitro: Binding was observed with recombinant METTL16, but diminished in lysate depleted of METTL16, and a supershift was detected after adding anti-METTL16 antibody. Importantly, RNA immunoprecipitation after in vivo UV cross-linking and an in situ proximity ligation assay for RNA-protein interactions confirmed an association between METTL16 and MALAT1 in cells. METTL16 is an abundant (∼5 × 10 5 molecules per cell) nuclear protein in HeLa cells. Its identification as a triple-stranded RNA binding protein supports the formation of RNA triple helices inside cells and suggests the existence of a class of triple-stranded RNA binding proteins, which may enable the discovery of additional cellular RNA triple helices.RNA binding protein | RNA triple helix | noncoding RNA
“…One of these helicases is DHX9 (also known as RHA), a highly abundant and multifunctional RNA/DNA helicase involved in DNA replication, transcription, and posttranscriptional and translational regulation (201). It contains two tandem repeats of dsRBDs followed by the DExH-motif helicase domain, and it displays an ATP-driven dsRNA-unwinding activity (201). Earlier studies showed that DHX9 is packaged into HIV-1 virions and is a host factor necessary for HIV-1 replication (202).…”
Section: Helicases (Besides Rlrs and Dicer)mentioning
Detection of double-stranded RNAs (dsRNAs) is a central mechanism of innate immune defense in many organisms. We here discuss several families of dsRNA-binding proteins involved in mammalian antiviral innate immunity. These include RIG-I-like receptors, protein kinase R, oligoadenylate synthases, adenosine deaminases acting on RNA, RNA interference systems, and other proteins containing dsRNA-binding domains and helicase domains. Studies suggest that their functions are highly interdependent and that their interdependence could offer keys to understanding the complex regulatory mechanisms for cellular dsRNA homeostasis and antiviral immunity. This review aims to highlight their interconnectivity, as well as their commonalities and differences in their dsRNA recognition mechanisms.
KeywordsdsRNA; RIG-I-like receptor; protein kinase R; oligoadenylate synthase; dsRNA-dependent adenosine deaminase; RNA interference HHS Public Access
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