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Research article
Juan Manuel Iglesias-Pedraz
Universidad Cientifica del Sur
Corresponding Author
ORCiD: https://orcid.org/0000-0003-0070-8116
License:
This work is licensed under a CC BY 4.0 License. Read Full License
Background
The Werner syndrome protein (WRN) belongs to the RecQ family of helicases and its loss of function results in the premature aging disease Werner syndrome (WS). We previously demonstrated that an early cellular change induced by WRN depletion is a posttranscriptional decrease in the levels of enzymes involved in metabolic pathways that control macromolecular synthesis and protect from oxidative stress. This metabolic shift is tolerated by normal cells but causes mitochondria dysfunction and acute oxidative stress in rapidly growing cancer cells, thereby suppressing their proliferation.
Results
To identify the mechanism underlying this metabolic shift, we examined global protein synthesis and mRNA nucleocytoplasmic distribution after WRN knockdown. We determined that WRN depletion in HeLa cells attenuates global protein synthesis without affecting the level of key components of the mRNA export machinery. We further observed that WRN depletion affects the nuclear export of mRNAs and demonstrated that WRN interacts with mRNA and the Nuclear RNA Export Factor 1 (NXF1).
Conclusions
Our findings suggest that WRN influences the export of mRNAs from the nucleus through its interaction with the NXF1 export receptor thereby affecting cellular proteostasis. In summary, we identified a new partner and a novel function of WRN, which is especially important for the proliferation of cancer cells.
Keywords
Werner syndrome protein, mRNA export, NXF1 export receptor, mRNA export, cancer, senescence
Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6
This is a list of supplementary files associated with this preprint. Click to download.
- SuppFig1.png
Supplementary Fig 1. RT-qPCR analysis of metabolic genes, stress granules formation and oxidative DNA damage. (A) Quantitative RT-qPCR analysis of G6PD and IDH1 using B2M as the internal control were performed as described in(25). The results of four biological replicates are plotted using GraphPad Prism. Two-way ANOVA followed by Sidak's multiple comparisons test was used to calculate the significance. The error bars represent the mean ± SEM (n=4). ** p value < 0.005; ns, no significant differences. (B) Western blot analysis showing WRN depletion in HeLa cells that were used for the SG formation experiment. Molecular size markers (in KiloDaltons) are shown. (C) WRN-depleted and control HeLa cells were seeded in an 8-well chamber slide. After fixation and permeabilization, the cells were incubated with the respective antibodies (see Supplemental Information and Supplementary Table 1) and counterstain solutions. The samples were analyzed by immunofluorescence using confocal microscopy. Treatment with 3 mM sodium arsenite for 2 h was used to induce stress granule formation in both cell lines (Scale bar = 50 µm). Expanded boxed regions are shown on the right (Scale bar = 20 µm). (D) Representative Western blot analysis showing reduced levels of WRN after dox treatment. (Right panel) bands quantification. (E) The extracts were assayed for protein content using the Bradford method and same amount of proteins were loaded on a polyacrylamide gel. The samples were probed for phosphor--H2AX and tubulin was used as the loading control. Two different amounts of extracts were used to better visualize potential changes in phosphor--H2AX. (Right panel) bands quantification. Molecular size markers (in KiloDaltons) are shown.
- SuppFig2.png
Supplementary Fig 2. Nuclear/cytoplasmic fractionation and polysomes purification. Differential centrifugation followed by ultracentrifugation on a 30% sucrose cushion bed was used to generate five fractions. (A) A schematic representation of the procedure is shown. (B) Equal volumes of each fraction were loaded onto each lane. No cross contamination was observed in the fractions using PARP1 (nuclear), G6PD (Cytosol), Cox6b1 (mitochondria), RPL7a (ribosomes) and actin. The lack of detection of any of these markers in the Polysome Enriched Fraction (PEF) indicates the purity of this fraction which was used for the analysis of the 5.8S, 18S and 28S RNA by qPCR (Fig. 2C). This experiment was performed several times with identical results.
- SuppFig3.png
Supplementary Fig 3. Analysis of mTOR and its downstream target P70S6K1 in WRN depleted and control HeLa cells. Western blot analysis of the dox time course experiment in shWRN and shCTR HeLa cells. Whole cell extracts were resolved by SDS-PAGE and immunoblotted against the indicated antibodies. The same extracts were loaded in three different gels and actin was used as a control in each blot. Bands intensities were quantitated using Image J and plotted into a graph.
- Additionalfile1.pdf
Original images. Original uncropped images used to generate the figures shown in the manuscript.
- SuppTable1.pdf
Supplementary Table 1. List of the primary and secondary antibodies used in this study.
- SuppTable2.pdf
Supplementary Table 2. List of the primers for qPCR used in this study.
- SupplementalInformation.docx
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View the published article at BMC Molecular and Cell Biology.
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On 22 Jun, 2020
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A new preprint design is coming!
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See the published version of this preprint at BMC Molecular and Cell Biology.
This is a preprint, a preliminary version of a manuscript that has not completed peer review at a journal. Research Square does not conduct peer review prior to posting preprints. The posting of a preprint on this server should not be interpreted as an endorsement of its validity or suitability for dissemination as established information or for guiding clinical practice.
Learn more about In Review
Research article
Juan Manuel Iglesias-Pedraz
Universidad Cientifica del Sur
Corresponding Author
ORCiD: https://orcid.org/0000-0003-0070-8116
Badges
Prescreen
This manuscript passed our Prescreen assessment
Complete author information
Appropriate declaration statements
Potential risks to human health
Prescreen
Peer Review Timeline
CURRENT STATUS: Published
View the published article at BMC Molecular and Cell Biology.
Version 5
Posted 06 Oct, 2020
No community comments so far
Editorial decision: Accept
On 30 Sep, 2020
Editor assigned
On 29 Sep, 2020
Submission checks complete
On 28 Sep, 2020
Editor invited
On 28 Sep, 2020
No comments provided
Submission checks complete
On 25 Sep, 2020
Editorial decision: Minor revision
On 25 Sep, 2020
No comments provided
Review #2 received
Received 18 Sep, 2020
Reviewers invited
Invitations sent on 17 Sep, 2020
Reviewer #1 agreed
On 17 Sep, 2020
Reviewer #2 agreed
On 17 Sep, 2020
Review #1 received
Received 17 Sep, 2020
Editor assigned
On 16 Sep, 2020
Submission checks complete
On 15 Sep, 2020
Editor invited
On 15 Sep, 2020
No comments provided
Review #3 received
Received 02 Sep, 2020
Editorial decision: Major revision
On 02 Sep, 2020
Review #5 received
Received 31 Aug, 2020
Review #4 received
Received 26 Aug, 2020
Reviewer #5 agreed
On 16 Aug, 2020
Reviewer #4 agreed
On 14 Aug, 2020
Editor assigned
On 13 Aug, 2020
Reviewers invited
Invitations sent on 13 Aug, 2020
Reviewer #1 agreed
On 13 Aug, 2020
Reviewer #2 agreed
On 13 Aug, 2020
Reviewer #3 agreed
On 13 Aug, 2020
Review #1 received
Received 13 Aug, 2020
Review #2 received
Received 13 Aug, 2020
Submission checks complete
On 12 Aug, 2020
Editor invited
On 12 Aug, 2020
No comments provided
Editorial decision: Major revision
On 14 Jul, 2020
Review #4 received
Received 13 Jul, 2020
Review #3 received
Received 13 Jul, 2020
Review #5 received
Received 12 Jul, 2020
Review #2 received
Received 12 Jul, 2020
Review #1 received
Received 25 Jun, 2020
Reviewer #5 agreed
On 23 Jun, 2020
Reviewers invited
Invitations sent on 22 Jun, 2020
Reviewer #1 agreed
On 22 Jun, 2020
Reviewer #2 agreed
On 22 Jun, 2020
Reviewer #3 agreed
On 22 Jun, 2020
Reviewer #4 agreed
On 22 Jun, 2020
Editor assigned
On 15 Jun, 2020
Submission checks complete
On 14 Jun, 2020
Editor invited
On 14 Jun, 2020
First submitted
On 12 Jun, 2020
Metrics
Comments: 0
PDF Downloads: ...
HTML Views: ...
License:
This work is licensed under a CC BY 4.0 License. Read Full License
View the published article at BMC Molecular and Cell Biology.
Background
The Werner syndrome protein (WRN) belongs to the RecQ family of helicases and its loss of function results in the premature aging disease Werner syndrome (WS). We previously demonstrated that an early cellular change induced by WRN depletion is a posttranscriptional decrease in the levels of enzymes involved in metabolic pathways that control macromolecular synthesis and protect from oxidative stress. This metabolic shift is tolerated by normal cells but causes mitochondria dysfunction and acute oxidative stress in rapidly growing cancer cells, thereby suppressing their proliferation.
Results
To identify the mechanism underlying this metabolic shift, we examined global protein synthesis and mRNA nucleocytoplasmic distribution after WRN knockdown. We determined that WRN depletion in HeLa cells attenuates global protein synthesis without affecting the level of key components of the mRNA export machinery. We further observed that WRN depletion affects the nuclear export of mRNAs and demonstrated that WRN interacts with mRNA and the Nuclear RNA Export Factor 1 (NXF1).
Conclusions
Our findings suggest that WRN influences the export of mRNAs from the nucleus through its interaction with the NXF1 export receptor thereby affecting cellular proteostasis. In summary, we identified a new partner and a novel function of WRN, which is especially important for the proliferation of cancer cells.
Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6
This is a list of supplementary files associated with this preprint. Click to download.
- SuppFig1.png
Supplementary Fig 1. RT-qPCR analysis of metabolic genes, stress granules formation and oxidative DNA damage. (A) Quantitative RT-qPCR analysis of G6PD and IDH1 using B2M as the internal control were performed as described in(25). The results of four biological replicates are plotted using GraphPad Prism. Two-way ANOVA followed by Sidak's multiple comparisons test was used to calculate the significance. The error bars represent the mean ± SEM (n=4). ** p value < 0.005; ns, no significant differences. (B) Western blot analysis showing WRN depletion in HeLa cells that were used for the SG formation experiment. Molecular size markers (in KiloDaltons) are shown. (C) WRN-depleted and control HeLa cells were seeded in an 8-well chamber slide. After fixation and permeabilization, the cells were incubated with the respective antibodies (see Supplemental Information and Supplementary Table 1) and counterstain solutions. The samples were analyzed by immunofluorescence using confocal microscopy. Treatment with 3 mM sodium arsenite for 2 h was used to induce stress granule formation in both cell lines (Scale bar = 50 µm). Expanded boxed regions are shown on the right (Scale bar = 20 µm). (D) Representative Western blot analysis showing reduced levels of WRN after dox treatment. (Right panel) bands quantification. (E) The extracts were assayed for protein content using the Bradford method and same amount of proteins were loaded on a polyacrylamide gel. The samples were probed for phosphor--H2AX and tubulin was used as the loading control. Two different amounts of extracts were used to better visualize potential changes in phosphor--H2AX. (Right panel) bands quantification. Molecular size markers (in KiloDaltons) are shown.
- SuppFig2.png
Supplementary Fig 2. Nuclear/cytoplasmic fractionation and polysomes purification. Differential centrifugation followed by ultracentrifugation on a 30% sucrose cushion bed was used to generate five fractions. (A) A schematic representation of the procedure is shown. (B) Equal volumes of each fraction were loaded onto each lane. No cross contamination was observed in the fractions using PARP1 (nuclear), G6PD (Cytosol), Cox6b1 (mitochondria), RPL7a (ribosomes) and actin. The lack of detection of any of these markers in the Polysome Enriched Fraction (PEF) indicates the purity of this fraction which was used for the analysis of the 5.8S, 18S and 28S RNA by qPCR (Fig. 2C). This experiment was performed several times with identical results.
- SuppFig3.png
Supplementary Fig 3. Analysis of mTOR and its downstream target P70S6K1 in WRN depleted and control HeLa cells. Western blot analysis of the dox time course experiment in shWRN and shCTR HeLa cells. Whole cell extracts were resolved by SDS-PAGE and immunoblotted against the indicated antibodies. The same extracts were loaded in three different gels and actin was used as a control in each blot. Bands intensities were quantitated using Image J and plotted into a graph.
- Additionalfile1.pdf
Original images. Original uncropped images used to generate the figures shown in the manuscript.
- SuppTable1.pdf
Supplementary Table 1. List of the primary and secondary antibodies used in this study.
- SuppTable2.pdf
Supplementary Table 2. List of the primers for qPCR used in this study.
- SupplementalInformation.docx
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