N-terminal sequences in matrin 3 mediate phase separation into droplet-like structures that recruit TDP43 variants lacking RNA binding elements.

RNA binding proteins associated with amyotrophic lateral sclerosis (ALS) and muscle myopathy possess sequence elements that are low in complexity, or bear resemblance to yeast prion domains. These sequence elements appear to mediate phase separation into liquid-like membraneless organelles. Using fusion proteins of matrin 3 (MATR3) to yellow fluorescent protein (YFP), we recently observed that deletion of the second RNA recognition motif (RRM2) caused the protein to phase separate and form intranuclear liquid-like droplets. Here, we use fusion constructs of MATR3, TARDBP43 (TDP43) and FUS with YFP or mCherry to examine phase separation and protein colocalization in mouse C2C12 myoblast cells. We observed that the N-terminal 397 amino acids of MATR3 (tagged with a nuclear localization signal and expressed as a fusion protein with YFP) formed droplet-like structures within nuclei. Introduction of the myopathic S85C mutation into NLS-N397 MATR3:YFP, but not ALS mutations F115C or P154S, inhibited droplet formation. Further, we analyzed interactions between variants of MATR3 lacking RRM2 (ΔRRM2) and variants of TDP43 with disabling mutations in its RRM1 domain (deletion or mutation). We observed that MATR3:YFP ΔRRM2 formed droplets that appeared to recruit the TDP43 RRM1 mutants. Further, coexpression of the NLS-397 MATR3:YFP construct with a construct that encodes the prion-like domain of TDBP43 produced intranuclear droplet-like structures containing both proteins. Collectively, our studies show that N-terminal sequences in MATR3 can mediate phase separation into intranuclear droplet-like structures that can recruit TDP43 under conditions of low RNA binding.

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

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.