Human Nup98 regulates the localization and activity of DExH/D-box helicase DHX9

Abstract

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

Hepatitis B virus X protein modulates upregulation of DHX9 to promote viral DNA replication

“…ns, not significant 2.6 | Nup98 is required for DHX9-mediated HBV DNA replication Given that Nup98 interacts with DHX9, and stimulate the helicase activity in vitro (Capitanio & Montpetit, 2017), we wondered whether DHX9 stimulates HBV DNA replication dependent on Nup98. (a) HepAD38 cells were transfected with an increasing amount of Flag-tagged DHX9 plasmids, and the cytoplasmic viral DNA levels were then analyzed by Southern blot (left panel), HBsAg levels in the supernatant were measured by enzyme-linked immunosorbent assays (ELISAs) and HBV 3.5 kb RNA was analyzed by real-time PCR (right panel).…”

“…(b) Huh7 cells were co-transfected with HBV-expressing plasmids (gtD) and an increasing amount DHX9-expressing plasmids. Furthermore, as the region of 1-497 in the C-terminus of Nup98 is required for the interaction of Nup98/DHX9 (Capitanio & Montpetit, 2017), we constructed Nup98 498-920 deletion mutants and co-transfected the DHX9 expression plasmid into Huh7 or HepAD38 cells. (c) Huh7 cells were transfected with siRNA targeting DHX9; the viral DNA levels were measured by Southern blot (SB), and the knockdown effect of DHX9 was confirmed by Western blot (WB).…”

“…These results suggest that DHX9 facilitates viral replication in dependence on Nup98 regulation. Furthermore, as the region of 1-497 in the C-terminus of Nup98 is required for the interaction of Nup98/DHX9 (Capitanio & Montpetit, 2017), we constructed Nup98 498-920 deletion mutants and co-transfected the DHX9 expression plasmid into Huh7 or HepAD38 cells. The results showed that this Nup98 protein from the deletion mutant (Nup98 498-920 ), which lacks the ability to bind DHX9, could not positively stimulate DHX9-mediated HBV replication compared with WT DHX9 (Figure 6e,f).…”

“…Statistical significance was determined by one-way ANOVA with Tukey post hoc test (*p < .05, **p < .01). ns, not significant 2.6 | Nup98 is required for DHX9-mediated HBV DNA replication Given that Nup98 interacts with DHX9, and stimulate the helicase activity in vitro(Capitanio & Montpetit, 2017), we wondered whether DHX9 stimulates HBV DNA replication dependent on Nup98. As shown inFigure 6a,b, silencing of Nup98 expression had no effect on viral DNA levels; however, the promotion of viral DNA replication by DHX9 expression was completely inhibited in the Nup98 knockdown group both in Huh7 and HepAD38 cells.…”

Cellular RNA Helicase DHX9 Interacts with the Essential Epstein-Barr Virus (EBV) Protein SM and Restricts EBV Lytic Replication

“…Ubiquitin-like protein ISG15 X X X DHX9 is a multifunctional ATP-dependent nucleic acid helicase that unwinds DNA and RNA in a 3= to 5= direction and is involved in many cellular processes involving RNA, such as transcriptional activation, posttranscriptional RNA regulation, splicing, mRNA translation, and RNA-mediated gene silencing (46,(51)(52)(53)(54). DHX9 has been reported to promote infectivity of various viruses, including HIV, retrovirus, influenza virus, and foot-and-mouth disease virus (FMDV).…”