Fig 2: Aortic arch abnormalities in Matr3Gt-ex13 heterozygotes. (A and B) Newborn wild-type aortic arch vasculature, showing pre- (A) and post-corrosion (B) cast analysis. (C–L) Matr3 heterozygous newborns with various outflow tract defects. (C and D) Tubular hypoplasia and CoA. The deformed aortic arch is uniformly narrowed (segment between arrowheads), and a CoA (arrow) lies distal to the LSA near a closed DAo. (E and F) CoA (arrow) just distal to the LSA and at the level of the closed DAo also called a ‘juxaductal CoA’. (G and H) Interrupted aortic arch just distal to the LSA, with a strand of tissue joining the two segments (‘atretic aortic arch’; arrow). A VSD with left to right shunting is also present, as evident by red polymer in both ventricles. A large PDA (arrowhead) is the sole source of blood to the lower half of the body. (I and J) A wide PDA (arrowhead) and VSD are present. Following LV injection, both ventricles and the PT contain red polymer; the PT is connected to the PDA that joins the DAo. (K and L) Dual-color corrosion casting shows admixture of red (injected into LV) and blue polymers (injected into RV) in both ventricles, confirming the presence of a VSD (arrow, K). Both polymers are also present in the pulmonary trunk and aorta. A small PDA is present (arrowheads, K and L). AAo, ascending aorta; BA, brachiocephalic artery; DAo, descending aorta; IAA, interrupted aortic arch; LV, left ventricle; PT, pulmonary trunk; RCC/LCC, right/left common carotid arteries; RSA/LSA, right/left subclavian arteries; RV, right ventricle; VSD, ventricular septal defect.

Fig 3: Both RRM domains of Matr3 are required for the interaction with pY RNA1-s2. A. Schematic of Matr3 indicating the Zinc finger binding domains and the RNA Recognition Motifs (RRMs) from within Matr3. B. Schematic of constructs created as N-terminal Glutathione S-transferase (GST) fusion proteins with the RRM from Matr3, used in this binding study. C. Immobilized pY RNA1-s2 was incubated with the indicated GST-RRM proteins and SDS-PAGE followed by Coomassie blue staining; this demonstrates that RRM1+2, but not RRM1 or RRM2 alone bind to pY RNA1-s2. “Input” lanes represent the total amount of each protein (2 µg) that was incubated with either beads alone (no oligo lanes) or with 100 pmols of pY RNA1-s2-biotin pre-coupled to streptavidin M280 beads (Biotin-pY RNA1-s2 lanes). D. Alignment of crystal structures of the Matr3 RRM domains (PDB ID: 1WEX and 1X4F) obtained from the RCSB database [39] reveal an RMSD of 1.03 Å.

Fig 4: Caspase cleavage of proteins coded by genes linked to ALS. Western immunoblot analysis of TDP-43, C9orf72 (C9ORF72) p62/sequestosome-1 (p62), matrin-3 (MATR3), VCP, FUS, SOD1 and profilin-1 (PFN1) evaluated, as endogenous protein, in SK-N-BE(2) cell lysate incubated with 2 U of recombinant active human caspases-3, 6, 7 or -8, for 3 h at 37 °C. Blots are representative of three independent experiments. The arrowhead indicates the immunoreactive band corresponding to the full-length protein.

Fig 5: Phosphorylation enhances the interaction between Matr3 and pY RNA1-s2.Pre-conjugated biotinylated pY RNA1-s2: streptavidin beads (indicated with “pY RNA1-s2”) were incubated with retina lysates prepared from six animals, as described in “Materials and Methods”. Negative control binding assays are labeled “control”. Load controls are indicated with “2% of lysate”. The recovered proteins were subjected to SDS-PAGE and western blotting using an antibody raised against a generic phosphorylated substrate (α-PKA substrate) followed by re-probing against Matr3. Prior to the binding assay, retina lysates were prepared in the absence (−) or presence (+) of phosphatase inhibitors (Ppase inhibitors) or recombinant protein kinase A (PKA), to enhance phosphorylation of endogenous Matr3. See “Materials and Methods” for details of inhibitors and PKA used. The lower panel shows a re-probe using an anti-Matr3 antibody of the upper blot.