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. 2019 Sep 26;105(4):869–878. doi: 10.1016/j.ajhg.2019.09.007

Figure 2.

Figure 2

Frameshift Variants in METTL5 Impact the Predicted Domains and Conformation of the Protein, Ubiquitously Expressed in the Brain

(A) Alternative splicing leads to three isoforms of human METTL5. Non-coding segments and coding regions of exons are denoted by gray and black boxes, respectively. Regions coding for the S-adenosyl-L-methionine-dependent-methyltransferase domain are colored in orange. Variants analyzed in this study are depicted in red (PKMR43M), purple (F47949), and blue.11 The numbering of the position of variants c.182G>A (p.Gly61Asp), c.344_345delGA (p.Arg115Asnfs∗19), and c.571_572delAA (p.Lys191Valfs∗10) is based on accession number GenBank: NM_014167.2. The three isoforms lead to a unique protein, predicted to contain an S-adenosyl-L-methionine-dependent-methyltransferase (orange, amino acids 12–161), a methyltransferase small domain (brown box, amino acids 46–146), S-adenosylmethionine binding sites (dark brown box, amino acids 58L, 59G, 60C, 61G, 62C, 63G, 64V, 81D, 82I, 107C, 108D, 109V, 126V), and a DNA-methylase n-6-adenine-specific conserved site (red box, amino acids 123–129). The localization of the peptide used to produce the METTL5 antibody described in the following analysis is shown as a green bar (Novus Biologicals Cat# NBP1-56640, RRID:AB_11039697, amino acids 35–83).

(B) Protein modeling of the WT and the two novel mutant proteins, using PHYRE2 software,16 shows that the overall conformation of the protein is not affected by the different disease-causing variants. However, due to early termination codon, 2 α helix and 2 β sheet are missing in METTL5R115Nfs∗19 and 2 β sheet are missing in METTL5L191Vfs∗10. The red and purple arrows represent the site of truncation in variant p.Arg115Asnfs∗19 and p.Lys191Valfs∗10, respectively, and the blue arrow highlights the amino acid at position 61. Cyan, N terminus of the protein; yellow, C terminus of the protein.

(C) Representative immunofluorescent labeling images of endogenous METTL5 (green) highlight the partial co-localization of the WT protein with pre-synaptic (Bassoon, Enzo Life Sciences Cat# SAP7F407, RRID:AB_2313990, red) and post-synaptic (PSD95, UC Davis/NIH NeuroMab Facility Cat# 75-028, RRID:AB_2292909, red) markers in rat hippocampal neurons in culture. All images are projection of confocal optical sections stack. Scale bars: 10 μm.