TDP-43 mediated splicing does not predict whole transcript abundance. (A) Schematic of primers used in analysis, showing their binding sites on an example transcript. 6D junctions between splice donor in exon 6 and downstream acceptor site in exon 7 assayed using primers in red, 7D junctions between exon 7 and downstream acceptor sites in green. A third set of primers (blue) assayed specific transcripts by incorporating both junctions. (B) qPCR to assay abundance of junctions in neuro2a cells transiently transfected with myc-Tdp-43 or empty plasmid vector for 24 h (N = 5 per group, NRQ = Normalized Relative Quantity). (C) qPCR to assay abundance of splice junctions in cortex of female nontransgenic mice (NT) and female mice overexpressing human wild type or M337V TDP-43 (Tg, N = 3 per genotype). (D) Relative abundance of indicated junctions in either DMSO treated cells or cells exposed to cycloheximide (Chx) as outlined in Materials and Methods; an NMD-sensitive exon in Nol5 was used as positive control (N = 5 per group). (E) Relative abundance of indicated junctions in HeLaS3 cells transiently transfected with human TDP-43-myc or empty plasmid for 24 h (N = 5 per group). (F) Western blotting of SDS lysate from HeLaS3 cells transfected with empty vector control (C) or TDP-43-myc (T), run alongside lysate from murine cortex (MC). Antibodies to myc tag, TDP-43 N-terminus (N-term), or the RRM domains of TDP-43 (RRM) were used. Asterisks denote C-terminal fragment immunopositive for myc and RRM antibodies; arrowheads denote N-terminal and RRM positive alternative isoform. Exogenous full-length TDP-43-myc (myc) and endogenous TDP-43 (TDP) are labeled. (G) 3′ RACE of HeLaS3 cells transfected with empty vector as control (C) or TDP-43-myc (T), using primers specific to full-length TDP-43 encoding transcript. Quantification of the ratio of unspliced TDP-43γ to α-spliced TDP-43α is below. In B, C, D, E, and G: (*) P < 0.05, (**) P < 0.01, (#) P < 0.001, Student unpaired two-tailed t-test, error bars are SEM.
