Characterization of gene regulation and protein interaction networks for Matrin 3 encoding mutations linked to amyotrophic lateral sclerosis and myopathy

Abstract

To understand how mutations in Matrin 3 (MATR3) cause amyotrophic lateral sclerosis (ALS) and distal myopathy, we used transcriptome and interactome analysis, coupled with microscopy. Over-expression of wild-type (WT) or F115C mutant MATR3 had little impact on gene expression in neuroglia cells. Only 23 genes, expressed at levels of >100 transcripts showed ≥1.6-fold changes in expression by transfection with WT or mutant MATR3:YFP vectors. We identified ~123 proteins that bound MATR3, with proteins associated with stress granules and RNA processing/splicing being prominent. The interactome of myopathic S85C and ALS-variant F115C MATR3 were virtually identical to WT protein. Deletion of RNA recognition motif (RRM1) or Zn finger motifs (ZnF1 or ZnF2) diminished the binding of a subset of MATR3 interacting proteins. Remarkably, deletion of the RRM2 motif caused enhanced binding of >100 hundred proteins. In live cells, MATR3 lacking RRM2 (ΔRRM2) formed intranuclear spherical structures that fused over time into large structures. Our findings in the cell models used here suggest that MATR3 with disease-causing mutations is not dramatically different from WT protein in modulating gene regulation or in binding to normal interacting partners. The intra-nuclear localization and interaction network of MATR3 is strongly modulated by its RRM2 domain.

Figure 1

A graphic representation of the interactome for WT-MATR3 in human HEK293 cells. Proteomic mass spectrometry data from Supplemental Table S4 and the software program Cytoscape (version 3.5.1) were used to generate a graphic representation of MATR3 interaction. The intensity of shading of the oval containing the gene name is linked to the spectral counting data in Supplemental Table 4; the lighter the shading the higher the fold increase in spectral counts for a given protein relative to controls (untransfected cells; no expression of Avi-tagged MATR3). Proteins marked with an asterisk are interactors also identified by other studies (see Supplemental Table S4). The localization or function of the identified proteins was determined by three sources {www.uniprot.org} and^,. Using quantification by label-free spectral counting, HNRNPM and numerous RNA processing factors including ADAR, PDBP3, SFRS14, and PTBP1 appear to be the strongest interactors with MATR3.

Figure 2

Distribution of MATR3:YFP in nuclear compartments of C2C12 cells. Mouse C2C12 cells were grown on glass cover slips and transiently transfected with pEF.Bos vectors encoding MATR3:YFP variants noted on the figure. At 24 hours post-transfection, the cells were fixed and imaged. The images shown are representative of images seen in 3 independent transfections, viewing 30 to 50 cells on each cover slip (data quantified in Table 3). WT MATR3 and MATR3 lacking ZnF1, ZnF2, or RRM1 sequences show a predominantly diffuse distribution within the nucleus with variable levels of small puncta. MATR3 lacking RRM2 organizes into large uniformly spherical structures. Scale bar = 100 µm.

Figure 3

Images from time-lapse video demonstrates fusion of MATR3:YFPΔRRM2 spheres in C2C12 cells. An example of the timing of fusion is illustrated in this panel of images. Two independent fusion events can clearly be seen to occur over a period of 15–30 minutes in each case. The example shown is representative of observations made in 3 separate transfection experiments tracking 20–30 cells in each field of view. Original magnification 20X.

Figure 4

Lack of WT-MATR3:YFP and WT-MATR3:YFP ΔRRM2co-localization with TDP-43:mCherry in C2C12 cells. Mouse C2C12 cells were grown on glass cover slips and transiently transfected with pEF.Bos vectors encoding MATR3:YFP variants noted on the figure. At 24 hours post-transfection, the cells were fixed and imaged. The images shown are representative of what was found in at least two separate transfection experiments analyzing 50–100 cells in each experiment (original magnification 60X with 2X digital enlargement). Each image is from a single z-plane captured on a confocal microscope (see Methods). WT-MATR3:YFP and WT-TDP-43:mCherry show limited overlap, for example TDP-43:mCherry is not seen in puncta of WT-MATR3:YFP or the spherical structures formed by MATR3:YFPΔRRM2.

Figure 5

Minimal co-localization of MATR3 with lamin A/C or histones. Mouse C2C12 cells were grown on glass cover slips and transiently transfected with pEF.Bos vectors encoding MATR3:YFP variants noted on the figure. At 24 hours post-transfection, the cells were fixed, immunostained with antibodies to lamin A/C (Sigma-Aldrich #SAB4200236) or histone H3 (Abcam #ab8898) and imaged. The images shown are representative of images seen in 3 independent transfections, viewing 30 to 50 cells on each cover slip. Each image is from a single z-plane captured on a confocal microscope (see Methods). Nikon elements software was used to determine the extent of interaction between MATR3 and lamin A/C or histone H3 (Supplemental Figures S16 and S17). MATR3 shows limited co-localization with either lamin A/C or histone H3.