Abstract:The RNA helicase A (RHA) is involved in several steps of RNA metabolism, such as RNA processing, cellular transit of viral molecules, ribosome assembly, regulation of transcription and translation of specific mRNAs. RHA is a multifunctional protein whose roles depend on the specific interaction with different molecular partners, which have been extensively characterized in physiological situations. More recently, the functional implication of RHA in human cancer has emerged. Interestingly, RHA was shown to coo… 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
“…Given the functional relevance of DHX9 in genomic instability and in cancer [12], we asked whether its expression represents a prognostic factor in Ewing sarcoma. Analysis of event-free survival and overall survival in a dataset comprising 64 Ewing sarcoma, 4 Askin, and 20 perypheral primitive neuroectodermal tumors [25], which all belong to the family of Ewing tumors, showed that high DHX9 expression correlates with worse prognosis of the patients ( Figure 1A,B).…”
Section: Dhx9 Expression Correlates With Worse Prognosis In Ewing Sarmentioning
confidence: 99%
“…However, reduction in the RNAPII elongation rate within the DHX9 transcription unit favors exon 6A inclusion and targets the transcript to NMD [11]. Both UV light irradiation and etoposide treatment induced this event by slowing down the RNAPII [11], with the consequent decrease in DHX9 expression, thus leading to higher sensitivity of Ewing sarcoma cells to genotoxic stress [11,12]. Nevertheless, the mechanism by which exon 6A inclusion is normally repressed in Ewing sarcoma cells is currently unknown.DHX9 is a member of the DExH subgroup of RNA helicases, which play important roles in several aspects of RNA metabolism [12].…”
mentioning
confidence: 99%
“…Both UV light irradiation and etoposide treatment induced this event by slowing down the RNAPII [11], with the consequent decrease in DHX9 expression, thus leading to higher sensitivity of Ewing sarcoma cells to genotoxic stress [11,12]. Nevertheless, the mechanism by which exon 6A inclusion is normally repressed in Ewing sarcoma cells is currently unknown.DHX9 is a member of the DExH subgroup of RNA helicases, which play important roles in several aspects of RNA metabolism [12]. DHX9 is involved in the regulation of gene expression by acting as a scaffold for the interaction of breast cancer 1 (BRCA1) [13] and cyclic adenosine monophosphate (AMP) response element-binding protein-binding protein (CBP) [14] with the RNAPII holoenzyme, thus modulating their activity and regulating transcription.…”
mentioning
confidence: 99%
“…DHX9 is a member of the DExH subgroup of RNA helicases, which play important roles in several aspects of RNA metabolism [12]. DHX9 is involved in the regulation of gene expression by acting as a scaffold for the interaction of breast cancer 1 (BRCA1) [13] and cyclic adenosine monophosphate (AMP) response element-binding protein-binding protein (CBP) [14] with the RNAPII holoenzyme, thus modulating their activity and regulating transcription.…”
Alternative splicing is a combinatorial mechanism by which exons are joined to produce multiple mRNA variants, thus expanding the coding potential and plasticity of eukaryotic genomes. Defects in alternative splicing regulation are associated with several human diseases, including cancer. Ewing sarcoma is an aggressive tumor of bone and soft tissue, mainly affecting adolescents and young adults. DHX9 is a key player in Ewing sarcoma malignancy, and its expression correlates with worse prognosis in patients. In this study, by screening a library of siRNAs, we have identified splicing factors that regulate the alternative inclusion of a poison exon in DHX9 mRNA, leading to its downregulation. In particular, we found that hnRNPM and SRSF3 bind in vivo to this poison exon and suppress its inclusion. Notably, DHX9 expression correlates with that of SRSF3 and hnRNPM in Ewing sarcoma patients. Furthermore, downregulation of SRSF3 or hnRNPM inhibited DHX9 expression and Ewing sarcoma cell proliferation, while sensitizing cells to chemotherapeutic treatment. Hence, our study suggests that inhibition of hnRNPM and SRSF3 expression or activity could be exploited as a therapeutic tool to enhance the efficacy of chemotherapy in Ewing sarcoma.
A robust body of work has demonstrated that a reduction in cAMP‐specific 3',5'‐cyclic phosphodiesterase 4D isoform 7 (PDE4D7) is linked with negative prostate cancer outcomes; however, the exact molecular mechanism that underpins this relationship is unknown. Epigenetic profiling has shown that the PDE4D gene can be hyper‐methylated in transmembrane serine protease 2 (TMPRSS2)–ETS transcriptional regulator ERG (ERG) gene‐fusion‐positive prostate cancer (PCa) tumours, and this inhibits messenger RNA (mRNA) expression, leading to a paucity of cellular PDE4D7 protein. In an attempt to understand how the resulting aberrant cAMP signalling drives PCa growth, we immunopurified PDE4D7 and identified binding proteins by mass spectrometry. We used peptide array technology and proximity ligation assay to confirm binding between PDE4D7 and ATP‐dependent RNA helicase A (DHX9), and in the design of a novel cell‐permeable disruptor peptide that mimics the DHX9‐binding region on PDE4D7. We discovered that PDE4D7 forms a signalling complex with the DExD/H‐box RNA helicase DHX9. Importantly, disruption of the PDE4D7–DHX9 complex reduced proliferation of LNCaP cells, suggesting the complex is pro‐tumorigenic. Additionally, we have identified a novel protein kinase A (PKA) phosphorylation site on DHX9 that is regulated by PDE4D7 association. In summary, we report the existence of a newly identified PDE4D7–DHX9 signalling complex that may be crucial in PCa pathogenesis and could represent a potential therapeutic target.
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