Comparative Interactome Analysis of Emerin, MAN1 and LEM2 Reveals a Unique Role for LEM2 in Nucleotide Excision Repair

Abstract

LAP2-Emerin-MAN1 (LEM) domain-containing proteins represent an abundant group of inner nuclear membrane proteins involved in diverse nuclear functions, but their functional redundancies remain unclear. Here, using the biotinylation-dependent proximity approach, we report proteome-wide comparative interactome analysis of the two structurally related LEM proteins MAN1 (LEMD3) and LEM2 (LEMD2), and the more distantly related emerin (EMD). While over 60% of the relatively small group of MAN1 and emerin interactors were also found in the LEM2 interactome, the latter included a large number of candidates (>85%) unique for LEM2. The interacting partners unique for emerin support and provide further insight into the previously reported role of emerin in centrosome positioning, and the MAN1-specific interactors suggest a role of MAN1 in ribonucleoprotein complex assembly. Interestingly, the LEM2-specific interactome contained several proteins of the nucleotide excision repair pathway. Accordingly, LEM2-depleted cells, but not MAN1- and emerin-depleted cells, showed impaired proliferation following ultraviolet-C (UV-C) irradiation and prolonged accumulation of γH2AX, similar to cells deficient in the nucleotide excision repair protein DNA damage-binding protein 1 (DDB1). These findings indicate impaired DNA damage repair in LEM2-depleted cells. Overall, this interactome study identifies new potential interaction partners of emerin, MAN1 and particularly LEM2, and describes a novel potential involvement of LEM2 in nucleotide excision repair at the nuclear periphery.

Keywords: BioID; DNA repair; LEM-proteins; inner nuclear membrane; nuclear envelope; nucleotide excision repair.

Figure 1

Expression of BirA*-emerin-V5, BirA*-MAN1-V5 and BirA*-LEM2-B-V5 in U2OS cells. (A) Schematic domain organization of LEM2-, MAN1- and emerin. Yellow, transmembrane domain; blue, MSC- (MAN1-SRC1p-C-terminal) domain; red, LEM-domain; green, RNA recognition motif (RRM). (B) BirA* fusion proteins biotinylate proteins in close proximity (10 nm). Cartoon and time course showing the BioID approach. Cells were incubated with medium containing 1 µg/ml doxycycline to induce fusion protein expression, 50 µM biotin was added after 6 h and incubated for another 16 h before cells were lysed. (C) U2OS cells stably expressing doxycycline-inducible BirA*-fusion constructs were incubated without (-Doxycycline) or with (+Doxycycline) doxycycline and biotin and processed for immunofluorescence microscopy using antibodies to the V5 tag and fluorescently labeled streptavidin (biotin). DNA was stained with DAPI. Bar, 10 µm. (D) Total cell lysates of incubated cells were prepared and analyzed by immunoblotting using V5-antibody and horseradish peroxidase (HRP)-conjugated streptavidin. Lanes in Figure 1D were on the same blot (see supplementary Figure S1 for unedited immunoblot) and were generated with identical exposure time.

Figure 2

BioID identifies common and specific interactors for emerin, MAN1 and LAP2-Emerin-MAN1 (LEM2). Mass spectrometric data sets were analyzed using SAINT v. 2.5.0 [43,44] based on an average probability (AvgP) of ≥ 0.45. (A) Venn diagram showing the overlap of interactomes. Numbers indicate identified high-confidence interactors. (B) Bait vs. bait distance similarity analysis revealed a larger similarity between binding partners of MAN1 and LEM2 than the similarity of those proteins with emerin. (C) Gene ontology (GO) enrichment analysis using ENRICHR, considering a fold change ≥ 1 and p-value ≤ 0.05 [47]. ENRICHR identified enriched biological processes for each LEM protein. Blue bars represent p-value (log10) and pink points show GO term fold enrichment. (D) The identified interactors of emerin and MAN1 of indicated GO biological processes were further grouped and analyzed using the STRING database. The fill color of the nodes represents the AvgP and the line width represents the STRING interaction.

Figure 3

The LEM2 interactome contains several proteins involved in the nucleotide excision repair. (A) The identified high confidence interactors of LEM2 of indicated GO processes were further grouped and analyzed using the STRING database. The fill color of the nodes represents the AvgP, and the line width the STRING interaction score. (B) Schematic representation of nucleotide excision repair (NER) pathway. GG-NER, global genomic-NER; TC-NER, transcription coupled NER. Proteins biotinylated by BirA*-LEM2 are colored; proteins not found in the LEM2 interactome are light gray.

Figure 4

LEM2 knockdown impairs DNA damage response after UV-C irradiation. U2OS cell viability was accessed following knockdown of emerin, MAN1, LEM2 or DDB1 by RNA interference. Scrambled RNA (Sc) was used as negative control. (A) Immunofluorescence analyses of U2OS cells 48 h after transfection with indicated siRNAs stained for DNA (blue), LEM2 (red) and emerin (green). Bar, 10 µm. (B) Immunoblot analysis of whole-cell lysates 48 hours after transfection with indicated siRNAs using antibodies as indicated on the top. γ-Tubulin served as loading control. (C) Emerin- MAN1-, LEM2- and DDB1-depleted cells or cells treated with scrambled RNA were left untreated (control) or irradiated with a sublethal dose of UV-C (5 J/m²) (+UV) and cell proliferation was analyzed for 96 h postirradiation. (D) Bar graph representing cell numbers at the 96 h time point. Statistical analysis was performed using two-way ANOVA and Tukey’s multiple comparison, n = 8, *, p-values: ‘*’ for p < 0.05; ‘**’ for p < 0.01; ‘***’ for p < 0.001; ‘****’ for p < 0.0001. Error bars represent standard error of the mean. (E) Whole cell lysates were analyzed by immunoblotting before and 48 h after UV treatment, using antibodies to phosphorylated H2AX (γH2AX). Ponceau S stain served as loading control. (F) Quantification of immunoblot. Graph represents fold increase of γH2AX after UV treatment normalized to total protein.