Journal: Life Science Alliance

Article Title: Aberrant NSUN1 activity connects m 5 C-RNA modification to TDP-43 neurotoxicity in ALS/FTD

doi: 10.26508/lsa.202503297

Figure Lengend Snippet: (A) The proteins that co-immunoprecipitated with either endogenous TDP-43 or the IgG control were subjected to LC-MS/MS and MaxQuant analysis. The experiment was performed in triplicate. (B) A total of 1,027 proteins selectively co-immunoprecipitated with TDP-43. Only proteins that co-immunoprecipitated with TDP-43 and not IgG in all three repeats were included in the analysis (783 proteins). Depicted is a full string network built upon experimental and text mining data. Only interactions with confidence score of >0.9 are shown and with a maximum of five interactors in the first shell. The network was clustered using a kmeans clustering of five. The KEGG pathways associated with each cluster are listed. Each cluster is presented in supplemental as larger figures ( , , , , and ). (C) The gene ontology (GO) analysis shows the enriched molecular functions. With pathways linked to RNA methylation highlighted in green. (D) TDP-43 interacts with proteins that have functions in the nucleus, cytoplasm, and nucleolus. Source data are available for this figure.

Article Snippet: For bisulphite sequencing, 1,000 ng of total RNA was bisulphite treated according to the EZ RNA methylation kit (R5001; Zymo Research) and eluted in 13 μl of water by incubating water on column for 4 min at RT before elution by centrifugation.

Techniques: Immunoprecipitation, Control, Liquid Chromatography with Mass Spectroscopy, Methylation

Journal: Life Science Alliance

Article Title: Aberrant NSUN1 activity connects m 5 C-RNA modification to TDP-43 neurotoxicity in ALS/FTD

doi: 10.26508/lsa.202503297

Figure Lengend Snippet: (A) The pre-47S pre-rRNA is depicted; the two Nsun1 proteins show sites of the rRNA that are regulated by Nsun1. The 5′ end shows the region of Nsun1 regulation of rRNA processing and the 3′ end shows the region of Nsun1 cytosine methylation. The primer positions for real-time PCR and bisulphite sequencing are presented. (B, C) TDP-43 expression causes a Nsun1-dependent increase in 5-methylcytosine (m 5 C) in total RNA. Top blot is immunoblotted for m 5 C and lower blot is stained with methylene blue. BR, biological repeat. (C) TDP-43 expression causes a Nsun1-dependent increase in 5-methylcytosine (m 5 C). (C) Graph is mean (±SD) of m 5 C levels in RNA relative to methylene blue (C) from ∼100 heads per genotype for each of the two biological repeats, a one-way ANOVA and a Tukey’s test is presented. * P < 0.05; ns, not significant. (D) The Nsun1-target cytosine and its methylation is conserved in Drosophila. Total RNA from WT Drosophila was bisulphite treated and sequenced to show the methylated cytosine (C3402). Dm, Drosophila melanogaster ; Hs, Homo sapiens ; Ce, Caenorhabditis elegans . (E) Bisulphite followed by Illumina sequencing showed that C3402 is nearly always methylated and is unaltered by TDP-43, or TDP-43 with si. Nsun1 . Graph is mean (±SD), a one-way ANOVA, and a Tukey’s test is presented; ns, not significant. (F) The levels of the 47S rRNA precursor are unaltered by TDP-43 expression or by TDP-43 with reduction in Nsun1 (si. Nsun1 ). Graph is mean (±sem) of six biological repeats, one-way ANOVA, and a Tukey’s test. Each data point is the mean of three technical replicates. (G) The levels of the 18S, 28S and 5.8S subunits are unaltered by TDP-43 expression or by TDP-43 with reduction in Nsun1 (si. Nsun1 ), measured by real-time PCR. Graph is mean (±sem) of six biological repeats, one-way ANOVA, and a Tukey’s test. Genotypes are gmr-GAL4 control: +/+; gmr-GAL4 (YH3)/si.mCherry 35785 , gmr-GAL4 + TDP-43 + control: TDP-43 (37M)/+; gmr-GAL4 (YH3)/ si.mCherry 35785 , gmr-GAL4 + TDP-43 + si. Nsun1 : TDP-43 (37M)/si.Nsun1 TRiP.HMC04440 ; gmr-GAL4 (YH3)/+. Source data are available for this figure.

Article Snippet: For bisulphite sequencing, 1,000 ng of total RNA was bisulphite treated according to the EZ RNA methylation kit (R5001; Zymo Research) and eluted in 13 μl of water by incubating water on column for 4 min at RT before elution by centrifugation.

Techniques: Methylation, Real-time Polymerase Chain Reaction, Bisulfite Sequencing, Expressing, Staining, Illumina Sequencing, Control