Fig 2: TDP-43 binds the 3′ UTR of messenger RNAs with high affinity in vitro. A, representative image of multiplexed EMSA for TDP-43 binding to 3′ UTR fragment of CSNK1E mRNA, positive control TARDBP mRNA region, and negative control BARD1 mRNA region. B, Hill’s plot for TDP-43 binding with CSNK1E, TARDBP, and BARD1 RNA transcripts. At least three experimental replicate assays for TDP-43 binding to each RNA fragment were performed, and data are plotted with SD. C, representative image of multiplexed EMSA for TDP-43 binding to mRNA 3′ UTR fragments from HMGB2, SLC1A5, and CSNK1E. D, Hill’s plot for TDP-43 binding with 3′ UTR fragments from HMGB2, SLC1A5, and CSNK1E transcripts. At least three experimental replicate assays for TDP-43 binding to each RNA fragment were performed, and data are plotted with SD. E, competitive EMSA for TDP-43 RRMs bound to Cy5-labeled TARDBP RNA with addition of excess unlabeled TARDBP RNA. F, competitive mEMSA for TDP-43 RRMs bound to Cy5-labeled TARDBP positive control with addition of excess unlabeled BARD1 negative control RNA. G, quantitation of TARDBP RNA fraction bound in competition assays for each sample pair. N = 2 biological replicates for BARD1 negative control. N = 3 biological replicates for TARDBP positive control. Data are plotted with SD.

Fig 3: Reduced level of MALAT1 RNA results in relocalization of TDP-43 binding to messenger RNA transcripts in HEK293 cells.A, IP-qPCR analysis of TDP-43 enrichment of HMGB2, SLC1A5, CSNK1E, and BARD1 mRNA transcripts, MALAT1 RNA, and TARDBP mRNA control compared to IgG enrichment of each transcript. B, representative Western blot of TDP-43 protein recovery after immunoprecipitation with TDP-43 or IgG control antibody, from negative control (NC) GapmeR– or MALAT1 GapmeR–treated cells. C, IP-qPCR analysis of RNA quantification for TDP-43 enrichment of each RNA transcript after knockdown of MALAT1 RNA, compared to TDP-43 enrichment of each RNA transcript in NC GapmeR–treated cells. D, gene expression changes in 3′ UTR target genes after knockdown of MALAT1 RNA or TDP-43 protein, compared to negative control (NC) GapmeR treatment. E, Western blot for CSNK1E, SLC1A5, and HMGB2 protein levels after 48 h of transfection with MALAT1 GapmeR or NC GapmeR. F, quantification of protein levels from Western blots in (E). All experiments in this figure were performed in HEK293 cells with N = 3 biological replicates. T tests are conducted with two tailed unpaired equal variance conditions. ∗ = p < 0.05, ∗∗ = p < 0.01, ns = p > 0.05. All data are plotted with SD.

Fig 4: Depletion of MALAT1 RNA protects against MPP + toxicity in a cell culture model of neurodegeneration.A, quantification of MALAT1 RNA levels in WT- or MPP+-treated SH-SY5Y cells. B, quantification of MALAT1 RNA levels in MPP+ cells treated with either negative control (NC) GapmeR or MALAT1 GapmeR. C, relative expression of mRNA transcripts HMGB2, SLC1A5, and CSNK1E after MPP+ treatment, compared to expression levels in untreated (WT) SH-SY5Y cells. D, relative expression of mRNA transcripts HMGB2, SLC1A5, and CSNK1E in MPP+ condition after treatment with negative control (NC) GapmeR or MALAT1 GapmeR. E, IP-qPCR for TDP-43 binding to MALAT1 RNA, HMGB2, CSNK1E, SLC1A5, and BARD1 mRNA in MPP+-treated cells compared to untreated (WT) SH-SY5Y cells. N = 2. F, viability of SH-SY5Y cells after MPP+ treatment compared to untreated (WT) cells. G, viability of SH-SY5Y cells after treatment with the indicated combinations of MPP+, TDP-43 GapmeR, MALAT1 GapmeR, pcEV empty vector control, pcMALAT1 overexpression construct, or pcTDP-43 overexpression construct. All experiments in this figure were performed in SH-SY5Y cells. N = 3 for RNA expression experiments, N = 3 for IP experiments, and N = 6 for cell viability experiments. T tests are conducted with two tailed unpaired equal variance conditions. ∗ = p < 0.05, ∗∗ = p < 0.01 All data are plotted with SD.