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, 10 (3), 1216-27

Proteome-wide Identification of WRN-interacting Proteins in Untreated and Nuclease-Treated Samples

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Proteome-wide Identification of WRN-interacting Proteins in Untreated and Nuclease-Treated Samples

Sophie Lachapelle et al. J Proteome Res.

Abstract

Werner syndrome (WS) is characterized by the premature onset of several age-associated pathologies. The protein defective in WS patients (WRN) is a helicase/exonuclease involved in DNA repair, replication, telomere maintenance, and transcription. Here, we present the results of a large-scale proteome analysis to determine protein partners of WRN. We expressed fluorescent tagged-WRN (eYFP-WRN) in human 293 embryonic kidney cells and detected interacting proteins by co-immunoprecipitation from cell extract. We identified by mass spectrometry 220 nuclear proteins that complexed with WRN. This number was reduced to 40 when broad-spectrum nucleases were added to the lysate. We consider these 40 proteins as directly interacting with WRN. Some of these proteins have previously been shown to interact with WRN, whereas most are new partners. Among the top 15 hits, we find the new interactors TMPO, HNRNPU, RPS3, RALY, RPS9 DDX21, and HNRNPM. These proteins are likely important components in understanding the function of WRN in preventing premature aging and deserve further investigation. We have confirmed endogenous WRN interaction with endogenous RPS3, a ribosomal protein with endonuclease activities involved in oxidative DNA damage recognition. Our results suggest that the use of nucleases during cell lysis severely restricts interacting protein partners and thus enhances specificity.

Figures

Figure 1
Figure 1
Cellular and enzymatic activities of the eYFP-WRN protein. (A) Time course localization of the eYFP-WRN protein in a representative HEK 293 cell by live imaging after microlaser beam application. The path of the laser is indicated by a rectangle box in the image at 0 s. Magnification is 40×. (B) Recruitment kinetics of eYFP-WRN shown by the fluorescence intensity to sites of DNA damage after microlaser beam application. (C) Helicase activity of the eYFP-WRN protein. Immunoprecipitated eYFP (lanes 3–6) or YFP-WRN (lanes 7–10) were diluted in reaction buffer 1/25, 1/50, 1/100, and 1/200, as indicated. The position of the displaced strand is indicated on the right. Autoradiogram represents a 6 h exposition. Lane 1, heated splayed arm substrate; lane 2, no protein. (D) Exonuclease activity of the eYFP-WRN protein. Immunoprecipitated eYFP (lanes 3–5) or YFP-WRN (lanes 6–8) were diluted in reaction buffer 1/10, 1/30, and 1/50 to better see the exonuclease activity. Nuclease fragments are indicated on the right. Lane 1, heated splayed arm substrate; lane 2, no protein. Autoradiogram represents 18 h of exposition.
Figure 2
Figure 2
Co-immunoprecipitation of proteins from cells transfected with eYFP or eYFP-WRN constructs. (A) SYPRO Ruby-staining pattern of the co-immunoprecipitated proteins from transfected cells with the anti-YFP antibody. The immunoprecipitation was performed a second time (panel on the right) to show the reproducibility of the experiment. All immunoprecipitations were performed 24 h after the transfection reactions. Molecular-mass sizes are indicated in kilodaltons (kDa). (B) Levels of eYFP-WRN in transfected cells. HEK 293 cells were transfected with eYFP or eYFP-WRN contructs. Twenty-four hours later, whole cell lysates were analyzed on 6% SDS-PAGE followed by Western blotting with an antibody against WRN protein. The positions of the EYFP-WRN and of the endogenous WRN proteins are indicated on the right. (C) Agarose gel showing the presence of nucleic acids without or with Benzonase and RNase A in the lysis buffer prior to the immunoprecipitation step. An aliquot (representing one 150-mm Petri dish of HEK 293 cells) was treated with phenol-chloroform and the nucleic acids were precipitated in ethanol. The whole precipitate were analyzed on a 1% agarose gel.
Figure 3
Figure 3
Co-immunoprecipitation of selected proteins with the eYFP-WRN construct. Human 293 embryonic kidney cells were transfected with an eYFP or a eYFP-WRN expression vector. Whole cell extracts (WCE) were immunoprecipitated with an anti-eYFP antibody (IP α-YFP). The immunoprecipitate was analyzed by immunoblotting with antibodies against POLR2A, POLR2B, HNRPK, MSH2, XRCC1 Lamin B1, and nucleophosmin. Panels on the left represent the immunoprecipitation performed in the absence of nuclease. Panels on the right represent the immunoprecipitation performed in the presence of Benzonase and RNase A in the lysis buffer.
Figure 4
Figure 4
Interaction of RPS3 with the WRN protein. (A) Co-precipitation of RPS3 with the eYFP-WRN protein in HEK 293 cells. (WCE represents the whole cell extract.) (B) Co-immunoprecipitation of human RPS3 protein with endogenous WRN protein. Approximately 2 mg of proteins from HEK 293 cells was immunoprecipitated with antibodies against the C-terminus region of human WRN protein H300 from Santa Cruz Biotechnology. Control antibodies were of the same IgG species. The immunoprecipitates were analyzed by Western blotting with the anti-WRN antibody (WRN; top panel) and an antibody against RPS3. Proteins were revealed with an ECL kit. The anti-WRN antibody for the immunoblot is from Novus Biologicals. (C) Interaction of RPS3 with different domains of WRN in whole cell extract. Immunoblot against RPS3 protein bound to different GST-WRN affinity Sepharose beads. HEK 293 whole cell extracts were incubated with 50 µg of the GST-WRN fragments or GST linked glutathione-sepharose beads overnight. Proteins bound to the affinity beads were analyzed by SDS-PAGE with antibodies against RPS3. (D) Interaction of WRN with RPS3 in whole cell extract. Immunoblot against WRN protein bound to GST-RPS3 but not GST affinity Sepharose beads. HEK 293 whole cell extracts were incubated with 50 µg of the GST-RPS3 or GST linked glutathione-sepharose beads overnight. Proteins bound to the affinity beads were analyzed by SDS-PAGE with antibodies against WRN. (E) Schematic representation of different WRN fragments that were used in the WRN affinity chromatography experiments. Each domain of the WRN protein is indicated on the full WRN protein figure. The amino acid residues of the WRN fragments used in this study are indicated on the left of each construct. Binding of RPS3 is indicated on the right by the “+” sign. The “−” sign indicates no binding detected.

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