Human Nup98 regulates the localization and activity of DExH/D-box helicase DHX9
- University of Alberta, Canada
- University of California, United states
Figures
Figure 1
Identification of Nup98-interacting proteins.
Plasmids encoding GFP-Nup98 or GFP alone were transfected into HEK293T cells. These proteins were immunoprecipitated from whole cell lysates using an antibody directed against GFP. …
Figure 2 with 1 supplement
Nup98 protein-protein interaction network identified by IP-MS.
(A) Curated protein-protein interactions (PPI) among identified Nup98 binding partners are represented in a PPI network. Edge thickness indicates the confidence score for the interaction and node …
Figure 2—figure supplement 1
Immunoprecipitation of endogenous Nup98 with DHX9 and hnRNP U.
Endogenous DHX9, hnRNP U, and Nup98 were immunoprecipitated from whole cell lysates (Input) using specific antibodies as indicated above the panels. Anti-GFP antibodies were used to assess …
Figure 3
Localization of Nup98 with DHX9 and hnRNP U.
The cellular distribution of Nup98, DHX9, and hnRNP U in HEK293T cells was examined by indirect immunofluorescence using antibodies directed against each protein as indicated. The positions of …
Figure 4 with 2 supplements
Nup98 depletion alters the intranuclear distribution of DHX9, but not hnRNP U.
HEK293T cells were transfected with shRNA targeting Nup98 or with a control shRNA. Four days later the cellular distributions of Nup98 and either DHX9 or hnRNP U were examined by indirect …
Figure 4—figure supplement 1
DHX9 depletion does not alter Nup98 localization in the cell.
HEK293T cells were transfected with a control shRNA or an shRNA targeting DHX9. Four days later the cellular distribution of Nup98 and DHX9 were examined by indirect immunofluorescence. Merged …
Figure 4—figure supplement 2
Immunoblotting of cell extracts following shRNA-mediated protein depletion.
Four days after shRNA transfection, lysates from cells depleted of the indicated protein (listed above top panel) were analyzed by western blotting using antibodies directed against Nup98, DHX9, and …
Figure 5 with 3 supplements
DHX9 interacts with intranuclear Nup98.
(A) HEK293T cells expressing GFP-NUP98 were used to compare DHX9 and GFP-Nup98 localization by immunofluorescence microscopy. Two magnifications are shown, each showing that upon GFP-NUP98 …
Figure 5—figure supplement 1
GFP expression does not alter the localization of DHX9, nor does GFP-Nup98 alter hnRNP U localization.
HEK293T cells expressing GFP (A) or GFP-NUP98 (B) were used to compare their localization to DHX9 or hnRNP U detected by indirect immunofluorescence microscopy using specific antibodies. Merged …
Figure 5—figure supplement 2
GFP or GFP-Nup98 expression does not alter cellular levels of DHX9 or hnRNP U.
Western blots of proteins derived from HEK293T cells lysates expressing GFP or GFP-Nup98 are shown. Antibodies were used for immunoblotting to detect the proteins indicated to the right of the …
Figure 5—figure supplement 3
DHX9 is recruited to intranuclear GFP-Nup981–497 foci.
(A) HEK293T cells expressing GFP-NUP981–497 were used to assay the localization of DHX9 detected by immunofluorescence microscopy. Two magnifications are shown and examples of GFP-Nup981–497 …
Figure 6 with 1 supplement
Nup98 binds directly to DHX9.
(A) Nup98 was affinity purified from HEK293T cell lysates. Bead-bound protein complexes were then incubated with or without RNase A, and proteins remaining bound to Nup98 were analyzed by western …
Figure 6—figure supplement 1
In vitro interaction of Nup98 and DHX9.
(A) Anti-DHX9 antibodies coupled to beads were used to immobilize recombinant DHX9. Bead-bound DHX9 was then incubated with recombinant Nup98 or GST in the presence or absence of RNA (poly I:C), …
Figure 7 with 1 supplement
Nup98 stimulates DHX9 ATPase activity.
The ATPase activity (ATP hydrolysis rate) of purified recombinant GST-DHX9 in the presence of RNA alone (no addition) or following the addition of increasing concentrations of GST-Nup98 or GST (show …
Figure 7—figure supplement 1
DHX9 ATPase assay.
(A) The ATP hydrolysis rate (ATP/sec) of purified recombinant GST-DHX9 or GST alone in the presence or absence of RNA was examined. Error bars indicate standard deviation. Results from three …
Figure 8 with 1 supplement
Nup98 directly interacts with target mRNA molecules.
Following crosslinking of HEK293T cells to preserve protein/RNA complexes, cell lysates were incubated with beads coupled to a control IgG (α-GFP) or beads coupled to Nup98 or DHX9 specific …
Figure 8—figure supplement 1
Analysis of protein immunoprecipitation.
Western blot analysis of protein immunoprecipitation (IP) fractions described in Figure 8 to detect associated RNA. The indicated IP samples (IP IgG) were analyzed by western blotting using …
Figure 9 with 2 supplements
The association of Nup98 or DHX9 with specific mRNAs is altered by depletion of its binding partner.
HEK293T cells were transfected with a control shRNA or an shRNA targeting Nup98 or DHX9. RNA immunopurified with Nup98 or DHX9 was reverse transcribed and used in qPCR reactions to assess the levels …
Figure 9—figure supplement 1
Analysis of protein immunoprecipitation from HEK293T cells depleted of Nup98 or DHX9.
Western blot analysis of cell lysates and protein immunoprecipitation (IP) fractions described in Figure 9 to detect associated RNA. The indicated cell lysates and IP samples (list above the panels) …
Figure 9—figure supplement 2
Nup98 or DHX9 depletion has no significant impact on the nuclear or cytoplasmic abundance of target mRNAs.
(A) HEK293T cells were transduced with a control shRNA or an shRNA targeting Nup98 or DHX9. Cells were fractionated into nuclear and cytoplasmic samples and the levels of the indicated gene …
Figure 10 with 1 supplement
Nup98 and DHX9 associate with similar gene loci and their binding is interdependent.
HEK293T cells stably expressing Dam-GFP, Dam-Nup98 or Dam-DHX9 were transduced with lentivirus encoding a control shRNA (white) or an shRNA targeting Nup98 or DHX9 (gray) and DamID analysis was …
Figure 10—figure supplement 1
The N-terminal FG/GLFG region of Nup98 associates with specific gene loci and is altered by depletion of DHX9.
HEK293T cells stably expressing Dam-GFP or Dam-Nup981–504 were transduced with lentivirus encoding a control shRNA (white) or an shRNA targeting DHX9 (gray), and Dam-ID analysis was performed. The …
Figure 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. RNA was purified and transcript levels from the indicated genes (x-axis) were reverse transcribed and …
Figure 12 with 1 supplement
Nup98 stimulates the transcriptional activity of DHX9.
HEK293T cells transfected with the luciferase gene under control of a cAMP-regulatory element (CRE) were co-transfected with two plasmids, one containing GFP-NUP98 or GFP and another containing …
Figure 12—figure supplement 1
DHX9 point mutant constructs are expressed at levels similar to WT.
Western blots of proteins derived from HEK293T cell lysates expressing the indicated DHX9 constructs were performed. These constructs contain a C-terminal HA-tag, allowing detection and comparison …
Figure 13
Nup98 or DHX9 depletion affects alternative splicing of E1A mRNA.
HEK293T cells were transfected with a control shRNA or an shRNA targeting Nup98 or DHX9. RNA from these cells was reverse transcribed and cDNAs used as template in qPCR reactions containing primers …
Additional files
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Supplementary file 1
Supplemental information.
(A) Complete mass spectrometry data exported from PEAKS software. (B) List of plasmids. (C) List of shRNAs. (D) List of primers.
- https://doi.org/10.7554/eLife.18825.028
