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Proteintech control igg
Control Igg, supplied by Proteintech, used in various techniques. Bioz Stars score: 93/100, based on 17 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Average 93 stars, based on 17 article reviews

control igg - by Bioz Stars, 2026-08

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(A) The 5-methylcytosine (m 5 C) RNA methyltransferases add a methyl group at the carbon 5 position of cytosine using S-adenosyl methionine (sam) as a methyl donor producing S-adenosylhomocysteine (sah) as a byproduct. (B) The m 5 C-RNA methyltransferase proteins in humans and their closest homologue in Drosophila melanogaster (Drosophila) were identified using the NCBI BLAST online alignment tool ( https://blast.ncbi.nlm.nih.gov/Blast.cgi ). (C) Phylogenetic tree of the m 5 C-RNA methyltransferases in humans and Drosophila. The dendogram was generated using clustal omega ( https://www.ebi.ac.uk/Tools/msa/clustalo/ ) and reconstructed using PRESTO ( http://www.atgc-montpellier.fr/presto ). The main RNA subtype for each enzyme is indicated. (D) Nsun1 down-regulation by siRNA (si. Nsun1 ) in adult head and thorax tissue with Daughterless-geneswitch (GS)-GAL4 ( DaGS-GAL4 ) led to a significant reduction in Nsun1 mRNA versus the control (si.mCherry). All data are relative to Tubulin. The graph is the mean (±SD) of three biological replicates; each data point is a technical replicate, three from each biological repeat, and a t test. (E) Expression of siRNAs to Nsun2 , Nsun5 , Nsun6 , and by 50% loss of function of Nsun4 causes a significant reduction in the respective genes versus the control (si.mCherry). Data are relative to Tubulin. The graph is the mean (±SD) of three biological replicates, each data point is a technical replicate, three from each biological repeat, and a t test, Genotypes: (D) Control: y, sc, v, sev/w 1118 ; Daughterless-GAL4-geneswitch/+; si.mCherry 3578 5 /+ , and si. Nsun1 : y, sc, v, sev/w 1118 ; Daughterless-GAL4-geneswitch/si.Nsun1 TRiP.HMC04440 ; +/+ . (E) Control: w 1118 ; +/+; Daughterless-GAL4/si.mCherry 35785 , si.Nsun2 : si.Nsun2 TRiP.HMJ24019 /+; Daughterless-GAL4/+ , Nsun4 : w 1118 ; cn[1] l(2)10685[10685]/+; Daughterless-GAL4/+ , Nsun5 : w 1118 ; +/+; Daughterless-GAL4/si.Nsun5 TRiP.HMS00438 , Nsun6 : w 1118 ; +/+; Daughterless-GAL4/si.Nsun6 TRiP.HMC04118 , Mt2 : w 1118 ; si.mt2 TRiP . HMS0166 /+; Daughterless-GAL4/+ .

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 5-methylcytosine (m 5 C) RNA methyltransferases add a methyl group at the carbon 5 position of cytosine using S-adenosyl methionine (sam) as a methyl donor producing S-adenosylhomocysteine (sah) as a byproduct. (B) The m 5 C-RNA methyltransferase proteins in humans and their closest homologue in Drosophila melanogaster (Drosophila) were identified using the NCBI BLAST online alignment tool ( https://blast.ncbi.nlm.nih.gov/Blast.cgi ). (C) Phylogenetic tree of the m 5 C-RNA methyltransferases in humans and Drosophila. The dendogram was generated using clustal omega ( https://www.ebi.ac.uk/Tools/msa/clustalo/ ) and reconstructed using PRESTO ( http://www.atgc-montpellier.fr/presto ). The main RNA subtype for each enzyme is indicated. (D) Nsun1 down-regulation by siRNA (si. Nsun1 ) in adult head and thorax tissue with Daughterless-geneswitch (GS)-GAL4 ( DaGS-GAL4 ) led to a significant reduction in Nsun1 mRNA versus the control (si.mCherry). All data are relative to Tubulin. The graph is the mean (±SD) of three biological replicates; each data point is a technical replicate, three from each biological repeat, and a t test. (E) Expression of siRNAs to Nsun2 , Nsun5 , Nsun6 , and by 50% loss of function of Nsun4 causes a significant reduction in the respective genes versus the control (si.mCherry). Data are relative to Tubulin. The graph is the mean (±SD) of three biological replicates, each data point is a technical replicate, three from each biological repeat, and a t test, Genotypes: (D) Control: y, sc, v, sev/w 1118 ; Daughterless-GAL4-geneswitch/+; si.mCherry 3578 5 /+ , and si. Nsun1 : y, sc, v, sev/w 1118 ; Daughterless-GAL4-geneswitch/si.Nsun1 TRiP.HMC04440 ; +/+ . (E) Control: w 1118 ; +/+; Daughterless-GAL4/si.mCherry 35785 , si.Nsun2 : si.Nsun2 TRiP.HMJ24019 /+; Daughterless-GAL4/+ , Nsun4 : w 1118 ; cn[1] l(2)10685[10685]/+; Daughterless-GAL4/+ , Nsun5 : w 1118 ; +/+; Daughterless-GAL4/si.Nsun5 TRiP.HMS00438 , Nsun6 : w 1118 ; +/+; Daughterless-GAL4/si.Nsun6 TRiP.HMC04118 , Mt2 : w 1118 ; si.mt2 TRiP . HMS0166 /+; Daughterless-GAL4/+ .

Article Snippet: Antibodies for co-immunoprecipitation were mouse anti-TDP-43 (#60019-2-IG, RRID:AB_2200520; Proteintech), sheep GFP (#S268B; MRC-PPU), mouse GFP (#A-11120, RRID:AB_221568; Thermo Fisher Scientific), rabbit NSUN1 (10448-1-AP, RRID:AB_2282772; Proteintech), normal mouse IgG (#sc-2025; Santa Cruz Biotechnology), normal sheep IgG (#12-515; EMD Millipore), normal rabbit IgG (#2729s; Cell Signaling Technology).

Techniques: Generated, Control, Expressing

(A) Schematic of the Nsun1 locus which is in the first intron of Dgt5 and expressed from the opposite strand. The siRNA used is indicated and Nsun1 arrows indicate the primers for measuring Nsun1 and Dgt5 mRNA levels. **** P < 0.0001; ns, not significant. (B) Nsun1 down-regulation by siRNA (si. Nsun1 ) with Daughterless-geneswitch (GS)-GAL4 ( DaGS-GAL4 ) causes a significant reduction in Nsun1 mRNA compared with control (si. mCherry ), whereas it had no effect on the levels of Dgt5 . Total RNA was isolated form head and thorax. The graph is the mean (±SD) of three independent experiments and an unpaired t test ( P < 0.005). (C) Expression of human TDP-43-Q331K causes degeneration of the external eye (area in white hatched line, upper panel) and reduced retinal width (white arrow, lower panel) compared with the normal control. Reduction in Nsun1 by co-expression of a siRNA directed to Nsun1 (si. Nsun1 ) reduced TDP-43-Q331K-induced degeneration of the external eye (upper panel) and internal retinal width (white arrow lower panel). Scale bars: upper panel is 25 μm, lower panel is 40 μm. (D) Nsun1 down-regulation by siRNA (si. Nsun1 ) significantly reduces TDP-43–induced degeneration of the external eye. Each datapoint represents one animal and all are taken from three independent repeats. Control is si.mCherry. Graph is the mean ± SD, one-way ANOVA ( P < 0.0001) and a Tukey’s test. **** P < 0.0001. (E) Nsun1 down-regulation by siRNA (si. Nsun1 ) significantly improves retinal width. Each datapoint represents one animal and all are taken across three independent repeats. Control is si.mCherry. Graph is the mean ± SD, one-way ANOVA ( P < 0.0001), and a Tukey’s test. **** P < 0.0001. (F) Nsun1 down-regulation by siRNA (si. Nsun1 ) had no effect on the levels of the TDP-43 protein. Total protein was isolated from heads of 1-day-old female Drosophila, and protein was immunoblotted for TDP-43 (upper panel) and Tubulin (lower panel). (G) TDP-43 protein levels relative to Tubulin (E). The mean (±SD) from four biological repeats and an unpaired and two-tailed t test is presented. ns, not significant. (H) Nsun1 down-regulation (si. Nsun1 ) had no effect on the levels of the β-galactosidase indicating that si. Nsun1 does not affect the GAL4-binary system. Total protein isolated from heads of 1-day-old females was immunoblotted for β-galactosidase (top panel) and Tubulin (lower panel). (E, I) β-galactosidase relative to Tubulin (E). The mean (±SD) from four biological repeats and an unpaired and two-tailed t test is presented. ns, not significant. (J) Expression of UAS-CAG 78 , UAS-ATXN1-CAG 82 and UAS-(G 4 C 2 ) 48 with gmr-GAL4 causes external degeneration of the Drosophila eye. Down-regulation of Nsun1 by siRNA (si. Nsun1 ) had little effect on UAS-CAG 78 but enhanced the toxicity of ATXN1-CAG 82 and UAS-(G 4 C 2 ) 48 compared with the control (si.mCherry). Down-regulation of Nsun1 by si.RNA (si. Nsun1 ) with gmr-GAL4 , and in the absence of a neurodegenerative disease protein, had no effect on the external retina compared with the control (si.mCherry), indicating that the enhancement observed with ATXN1-CAG 82 or UAS-(G 4 C 2 ) 48 is not additive. Scale bar is 25 μm. (K) Nsun1 is required for development. Nsun1 down-regulation compared with the control (si.mCherry) is lethal when down-regulated globally ( Daughterless-GAL4 ), in all neurons ( elav-GAL4 ), in glia ( Repo-GAL4 ) and in muscle ( 24B-GAL4 ). Expression of si. Nsun1 is semi-lethal in motor neurons when expressed with D42-GAL4 at 25°C and lethal at 29°C. Expression of si. Nsun1 in the eye ( gmr-YH3-GAL4I ) is not lethal at 25°C or 29°C. Genotypes: (C, D, E) gmr > control: y, sc, v, sev/w 1118 ; +/+; gmr-GAL4 (YH3)/si.mCherry 35785 , gmr > TDP-43 Q331K + control: y, sc, v, sev/w 1118 ; UAS-TDP-43 Q331K /+; gmr-GAL4 (YH3)/si.mCherry 35785 , gmr > TDP-43 Q331K + si. Nsun 1: y, sc, v, sev/w 1118 ; UAS-TDP-43 Q331K /si.Nsun1 TRiP.HMC04440 ; gmr-GAL4 (YH3)/+ . (D) Control: w-;+/+; daGS-GAL4/si.mCherry 35785 . si. Nsun1 : w-; si.Nsun1 TRiP.HMC04440 /+; daGS-GAL4/+ . (F, G) gmr > control: y, sc, v, sev/w 1118 ; +/+; gmr-GAL4 (YH3)/si.mCherry 35785 , gmr > TDP-43 + control: y, sc, v, sev/w 1118 ; UAS-TDP-43/+; gmr-GAL4 (YH3)/si.mCherry 35785 , gmr > TDP-43 + si. Nsun1 : y, sc, v, sev/w 1118 ; UAS-TDP-43 (37M)/si.Nsun1 TRiP.HMC04440 ; gmr-GAL4 (YH3)/+ . (H, I) gmr > lacZ + control: w*; gmr-GAL4, UAS-LacZ/+; +/si.mCherry 35785 , gmr > lacZ + si .Nsun1 : w*; gmr-GAL4, UAS-LacZ/si.Nsun1 TRiP.HMC04440 ; +/+ . (J) gmr-GAL4 > control: y, sc, v, sev/w 1118 ; +/+; gmr-GAL4 (YH3)/si.mCherry 35785 . gmr-GAL4 > si .Nsun1 : y, sc, v, sev/w 1118 ; +/si.Nsun1 TRiP.HMC04440 ; gmr-GAL4 (YH3)/+ , gmr-GAL4 > UAS-CAG78 + control: y, sc, v, sev/w 1118 /+; gmr-GAL4 (YH3), UAS-CAG78/ si.mCherry 35785 , gmr-GAL4 > UAS-CAG78 + si. Nsun1 : y, sc, v, sev/w 1118 ; si.Nsun1 TRiP.HMC04440 /+; gmr-GAL4 (YH3), UAS-CAG78/+, gmr-GAL4 > UAS-CAG78 + si. Nsun1 : y, sc, v, sev/w 1118 ; si.Nsun1 TRiP.HMC04440 /+; gmr-GAL4 (YH3), UAS-CAG78/+, gmr-GAL4 > UAS-ATXN1-CAG82 + control: y, sc, v, sev/w 1118 ; +/+; gmr-GAL4 (YH3), UAS-ATXN1-CAG82/ si.mCherry 35785 , gmr-GAL4 > UAS-ATXN1-CAG82 + si. Nsun1 : y, sc, v, sev/w 1118 ; si.Nsun1 TRiP.HMC04440 /+; gmr-GAL4 (YH3), UAS-ATXN1-CAG82/+, gmr-GAL4 > UAS-(G4C2) 48 + control: y, sc, v, sev/w 1118 ; +/+; gmr-GAL4 (YH3), UAS-(G4C2) 48 / si.mCherry 35785 , gmr-GAL4 > UAS-(G4C2) 48 + si. Nsun 1 : y, sc, v, sev/w 1118 ; si. Nsun1 TRiP.HMC04440 /+; gmr-GAL4 (YH3), UAS-(G4C2) 48 /+. ( K) elav3A > control: y, sc, v, sev/w 1118 ; +/+; elav3A-GAL4/si.mCherry 35785 , elav3A > si. Nsun 1: y, sc, v, sev/w 1118 ; si.Nsun1 TRiP.HMC04440 /+; elav3A-GAL4/+ , D42-GAL4 > control: y, sc, v, sev/w 1118 ; +/+;D42-GAL4/si.mCherry 35785 , D42-GAL4 > si. Nsun1 : y, sc, v, sev/w 1118 ; si.Nsun1 TRiP.HMC04440 /+; D42-GAL4/+ , repo-GAL4 > control: y, sc, v, sev/w 1118 ; +/+;repo-GAL4/si.mCherry 35785 , repo-GAL4 > si. Nsun1 : y, sc, v, sev/w 1118 ; si. Nsun1 TRiP.HMC04440 /+; repo-GAL4/+ , 24B-GAL4 > control: y, sc, v, sev/w 1118 ; +/+; 24B -GAL4/si.mCherry 35785 , 24B-GAL4 > si. Nsun1 : y, sc, v, sev/w 1118 ; si. Nsun1 TRiP.HMC04440 /+; 24B -GAL4/+ , gmr-GAL4 > control: y, sc, v, sev/w 1118 ; +/+; gmr-GAL4 (YH3)/si.mCherry 35785 , gmr-GAL4 > si. Nsun1 : y, sc, v, sev/w 1118 ; si. Nsun1 TRiP.HMC04440 /+; gmr-GAL4 (YH3)/+ .

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) Schematic of the Nsun1 locus which is in the first intron of Dgt5 and expressed from the opposite strand. The siRNA used is indicated and Nsun1 arrows indicate the primers for measuring Nsun1 and Dgt5 mRNA levels. **** P < 0.0001; ns, not significant. (B) Nsun1 down-regulation by siRNA (si. Nsun1 ) with Daughterless-geneswitch (GS)-GAL4 ( DaGS-GAL4 ) causes a significant reduction in Nsun1 mRNA compared with control (si. mCherry ), whereas it had no effect on the levels of Dgt5 . Total RNA was isolated form head and thorax. The graph is the mean (±SD) of three independent experiments and an unpaired t test ( P < 0.005). (C) Expression of human TDP-43-Q331K causes degeneration of the external eye (area in white hatched line, upper panel) and reduced retinal width (white arrow, lower panel) compared with the normal control. Reduction in Nsun1 by co-expression of a siRNA directed to Nsun1 (si. Nsun1 ) reduced TDP-43-Q331K-induced degeneration of the external eye (upper panel) and internal retinal width (white arrow lower panel). Scale bars: upper panel is 25 μm, lower panel is 40 μm. (D) Nsun1 down-regulation by siRNA (si. Nsun1 ) significantly reduces TDP-43–induced degeneration of the external eye. Each datapoint represents one animal and all are taken from three independent repeats. Control is si.mCherry. Graph is the mean ± SD, one-way ANOVA ( P < 0.0001) and a Tukey’s test. **** P < 0.0001. (E) Nsun1 down-regulation by siRNA (si. Nsun1 ) significantly improves retinal width. Each datapoint represents one animal and all are taken across three independent repeats. Control is si.mCherry. Graph is the mean ± SD, one-way ANOVA ( P < 0.0001), and a Tukey’s test. **** P < 0.0001. (F) Nsun1 down-regulation by siRNA (si. Nsun1 ) had no effect on the levels of the TDP-43 protein. Total protein was isolated from heads of 1-day-old female Drosophila, and protein was immunoblotted for TDP-43 (upper panel) and Tubulin (lower panel). (G) TDP-43 protein levels relative to Tubulin (E). The mean (±SD) from four biological repeats and an unpaired and two-tailed t test is presented. ns, not significant. (H) Nsun1 down-regulation (si. Nsun1 ) had no effect on the levels of the β-galactosidase indicating that si. Nsun1 does not affect the GAL4-binary system. Total protein isolated from heads of 1-day-old females was immunoblotted for β-galactosidase (top panel) and Tubulin (lower panel). (E, I) β-galactosidase relative to Tubulin (E). The mean (±SD) from four biological repeats and an unpaired and two-tailed t test is presented. ns, not significant. (J) Expression of UAS-CAG 78 , UAS-ATXN1-CAG 82 and UAS-(G 4 C 2 ) 48 with gmr-GAL4 causes external degeneration of the Drosophila eye. Down-regulation of Nsun1 by siRNA (si. Nsun1 ) had little effect on UAS-CAG 78 but enhanced the toxicity of ATXN1-CAG 82 and UAS-(G 4 C 2 ) 48 compared with the control (si.mCherry). Down-regulation of Nsun1 by si.RNA (si. Nsun1 ) with gmr-GAL4 , and in the absence of a neurodegenerative disease protein, had no effect on the external retina compared with the control (si.mCherry), indicating that the enhancement observed with ATXN1-CAG 82 or UAS-(G 4 C 2 ) 48 is not additive. Scale bar is 25 μm. (K) Nsun1 is required for development. Nsun1 down-regulation compared with the control (si.mCherry) is lethal when down-regulated globally ( Daughterless-GAL4 ), in all neurons ( elav-GAL4 ), in glia ( Repo-GAL4 ) and in muscle ( 24B-GAL4 ). Expression of si. Nsun1 is semi-lethal in motor neurons when expressed with D42-GAL4 at 25°C and lethal at 29°C. Expression of si. Nsun1 in the eye ( gmr-YH3-GAL4I ) is not lethal at 25°C or 29°C. Genotypes: (C, D, E) gmr > control: y, sc, v, sev/w 1118 ; +/+; gmr-GAL4 (YH3)/si.mCherry 35785 , gmr > TDP-43 Q331K + control: y, sc, v, sev/w 1118 ; UAS-TDP-43 Q331K /+; gmr-GAL4 (YH3)/si.mCherry 35785 , gmr > TDP-43 Q331K + si. Nsun 1: y, sc, v, sev/w 1118 ; UAS-TDP-43 Q331K /si.Nsun1 TRiP.HMC04440 ; gmr-GAL4 (YH3)/+ . (D) Control: w-;+/+; daGS-GAL4/si.mCherry 35785 . si. Nsun1 : w-; si.Nsun1 TRiP.HMC04440 /+; daGS-GAL4/+ . (F, G) gmr > control: y, sc, v, sev/w 1118 ; +/+; gmr-GAL4 (YH3)/si.mCherry 35785 , gmr > TDP-43 + control: y, sc, v, sev/w 1118 ; UAS-TDP-43/+; gmr-GAL4 (YH3)/si.mCherry 35785 , gmr > TDP-43 + si. Nsun1 : y, sc, v, sev/w 1118 ; UAS-TDP-43 (37M)/si.Nsun1 TRiP.HMC04440 ; gmr-GAL4 (YH3)/+ . (H, I) gmr > lacZ + control: w*; gmr-GAL4, UAS-LacZ/+; +/si.mCherry 35785 , gmr > lacZ + si .Nsun1 : w*; gmr-GAL4, UAS-LacZ/si.Nsun1 TRiP.HMC04440 ; +/+ . (J) gmr-GAL4 > control: y, sc, v, sev/w 1118 ; +/+; gmr-GAL4 (YH3)/si.mCherry 35785 . gmr-GAL4 > si .Nsun1 : y, sc, v, sev/w 1118 ; +/si.Nsun1 TRiP.HMC04440 ; gmr-GAL4 (YH3)/+ , gmr-GAL4 > UAS-CAG78 + control: y, sc, v, sev/w 1118 /+; gmr-GAL4 (YH3), UAS-CAG78/ si.mCherry 35785 , gmr-GAL4 > UAS-CAG78 + si. Nsun1 : y, sc, v, sev/w 1118 ; si.Nsun1 TRiP.HMC04440 /+; gmr-GAL4 (YH3), UAS-CAG78/+, gmr-GAL4 > UAS-CAG78 + si. Nsun1 : y, sc, v, sev/w 1118 ; si.Nsun1 TRiP.HMC04440 /+; gmr-GAL4 (YH3), UAS-CAG78/+, gmr-GAL4 > UAS-ATXN1-CAG82 + control: y, sc, v, sev/w 1118 ; +/+; gmr-GAL4 (YH3), UAS-ATXN1-CAG82/ si.mCherry 35785 , gmr-GAL4 > UAS-ATXN1-CAG82 + si. Nsun1 : y, sc, v, sev/w 1118 ; si.Nsun1 TRiP.HMC04440 /+; gmr-GAL4 (YH3), UAS-ATXN1-CAG82/+, gmr-GAL4 > UAS-(G4C2) 48 + control: y, sc, v, sev/w 1118 ; +/+; gmr-GAL4 (YH3), UAS-(G4C2) 48 / si.mCherry 35785 , gmr-GAL4 > UAS-(G4C2) 48 + si. Nsun 1 : y, sc, v, sev/w 1118 ; si. Nsun1 TRiP.HMC04440 /+; gmr-GAL4 (YH3), UAS-(G4C2) 48 /+. ( K) elav3A > control: y, sc, v, sev/w 1118 ; +/+; elav3A-GAL4/si.mCherry 35785 , elav3A > si. Nsun 1: y, sc, v, sev/w 1118 ; si.Nsun1 TRiP.HMC04440 /+; elav3A-GAL4/+ , D42-GAL4 > control: y, sc, v, sev/w 1118 ; +/+;D42-GAL4/si.mCherry 35785 , D42-GAL4 > si. Nsun1 : y, sc, v, sev/w 1118 ; si.Nsun1 TRiP.HMC04440 /+; D42-GAL4/+ , repo-GAL4 > control: y, sc, v, sev/w 1118 ; +/+;repo-GAL4/si.mCherry 35785 , repo-GAL4 > si. Nsun1 : y, sc, v, sev/w 1118 ; si. Nsun1 TRiP.HMC04440 /+; repo-GAL4/+ , 24B-GAL4 > control: y, sc, v, sev/w 1118 ; +/+; 24B -GAL4/si.mCherry 35785 , 24B-GAL4 > si. Nsun1 : y, sc, v, sev/w 1118 ; si. Nsun1 TRiP.HMC04440 /+; 24B -GAL4/+ , gmr-GAL4 > control: y, sc, v, sev/w 1118 ; +/+; gmr-GAL4 (YH3)/si.mCherry 35785 , gmr-GAL4 > si. Nsun1 : y, sc, v, sev/w 1118 ; si. Nsun1 TRiP.HMC04440 /+; gmr-GAL4 (YH3)/+ .

Techniques: Control, Isolation, Expressing, Two Tailed Test

(A) Expression of human TDP-43 leads to degeneration of the external eye (area in white hatched line, upper panel) and reduced retinal width (white arrow, lower panel) compared with the normal control. Reduction in Nsun1 , Nsun2 or Nsun6 reduced TDP-43–induced degeneration of the external eye (upper panel), whereas only reduction in Nsun1 mitigates TDP-43–induced degeneration of the internal retina (white arrow lower panel). Scale bars: upper panel is 25 μm, lower panel is 35 μm. (B) Reduction in Nsun1 , Nsun2 , Nsun5 , and Nsun6 by siRNA significantly reduces TDP-43–induced degeneration in the external eye. Each datapoint represents one animal and spans three independent biological repeats. Graph is the mean ± SD, one-way ANOVA ( P < 0.0001), and a Tukey’s test; ns, not significant. (C) Reduction Nsun1 significantly mitigates TDP-43–induced reduction in retinal depth. Each datapoint represents one animal from three independent repeats. Graph is the mean ± SD, one-way ANOVA ( P < 0.0001), and a Tukey’s test; ns, not significant. (D) Nsun1 expression levels are unalerted by TDP-43 expression. Expression of the siRNA to Nsun1 is reduced in the eye. TDP-43 was expressed in the Drosophila eye with gmr-GAL4 with either a control (si.mCherry) or a siRNA to Nsun1 (si. Nsun1 ) and both were compared with the normal control (si.mCherry alone). All data are relative to Tubulin. RNA was isolated from the entire head; thus, not all cells will express the siRNA to Nsun1 , and therefore, not all cells will have reduced Nsun1 levels. Graph is the mean of five biological repeats, one-way ANOVA, and a Tukey’s test; ns, not significant. Each data point is a technical replicate, three from each biological repeat. Genotypes: 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)/+ , gmr-GAL4 + TDP-43 + si. Nsun2 : TDP-43 (37M)/si. Nsun2 TRiP.HMJ24019 ; gmr-GAL4 (YH3)/+, gmr-GAL4 + TDP-43 + lof. Nsun4 : TDP-43 (37M)/ cn[1] l(2)10685[10685]; gmr-GAL4 (YH3)/+ , gmr-GAL4 + TDP-43 + si. Nsun5 : TDP-43 (37M)/+; gmr-GAL4 (YH3)/si.Nsun5 TRiP.HMS00438 , gmr-GAL4 + TDP-43 + si. Nsun6 : TDP-43 (37M)/si. Nsun6 TRiP.HMC04118 ; gmr- GAL4 (YH3)/+ , and gmr-GAL4 + TDP-43 + si. Mt2 : TDP-43 (37M)/si.Mt2 TRiP. HMS01667 ; gmr-gal4 (YH3)/+ .

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) Expression of human TDP-43 leads to degeneration of the external eye (area in white hatched line, upper panel) and reduced retinal width (white arrow, lower panel) compared with the normal control. Reduction in Nsun1 , Nsun2 or Nsun6 reduced TDP-43–induced degeneration of the external eye (upper panel), whereas only reduction in Nsun1 mitigates TDP-43–induced degeneration of the internal retina (white arrow lower panel). Scale bars: upper panel is 25 μm, lower panel is 35 μm. (B) Reduction in Nsun1 , Nsun2 , Nsun5 , and Nsun6 by siRNA significantly reduces TDP-43–induced degeneration in the external eye. Each datapoint represents one animal and spans three independent biological repeats. Graph is the mean ± SD, one-way ANOVA ( P < 0.0001), and a Tukey’s test; ns, not significant. (C) Reduction Nsun1 significantly mitigates TDP-43–induced reduction in retinal depth. Each datapoint represents one animal from three independent repeats. Graph is the mean ± SD, one-way ANOVA ( P < 0.0001), and a Tukey’s test; ns, not significant. (D) Nsun1 expression levels are unalerted by TDP-43 expression. Expression of the siRNA to Nsun1 is reduced in the eye. TDP-43 was expressed in the Drosophila eye with gmr-GAL4 with either a control (si.mCherry) or a siRNA to Nsun1 (si. Nsun1 ) and both were compared with the normal control (si.mCherry alone). All data are relative to Tubulin. RNA was isolated from the entire head; thus, not all cells will express the siRNA to Nsun1 , and therefore, not all cells will have reduced Nsun1 levels. Graph is the mean of five biological repeats, one-way ANOVA, and a Tukey’s test; ns, not significant. Each data point is a technical replicate, three from each biological repeat. Genotypes: 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)/+ , gmr-GAL4 + TDP-43 + si. Nsun2 : TDP-43 (37M)/si. Nsun2 TRiP.HMJ24019 ; gmr-GAL4 (YH3)/+, gmr-GAL4 + TDP-43 + lof. Nsun4 : TDP-43 (37M)/ cn[1] l(2)10685[10685]; gmr-GAL4 (YH3)/+ , gmr-GAL4 + TDP-43 + si. Nsun5 : TDP-43 (37M)/+; gmr-GAL4 (YH3)/si.Nsun5 TRiP.HMS00438 , gmr-GAL4 + TDP-43 + si. Nsun6 : TDP-43 (37M)/si. Nsun6 TRiP.HMC04118 ; gmr- GAL4 (YH3)/+ , and gmr-GAL4 + TDP-43 + si. Mt2 : TDP-43 (37M)/si.Mt2 TRiP. HMS01667 ; gmr-gal4 (YH3)/+ .

Techniques: Expressing, Control, Isolation

(A) Reduction in Nsun1 (si. Nsun1 ) mitigates lifespan deficits caused by expression of TDP-43 in adult neurons. Drosophila were fed 100 μl of 2 μg/ml RU486 topically on the food, >100 males per genotype were followed for each genotype, and a log-rank (Mantel-Cox) test was used for significance. (B, C) Reduction in Nsun1 (si. Nsun1 ) does not alter total TDP-43 protein levels in adult neurons. Graph is the mean (±SD) of three biological repeats and a t test. (D) Reduction in Nsun1 (si. Nsun1 ) reduces neuronal TDP-43 protein levels in the cytoplasm, increases TDP-43 protein levels in the nucleus and has no effect on insoluble TDP-43 levels. Top panel immunoblotted for TDP-43, middle panel for Lamin C and bottom panel for Tubulin. (E) TDP-43 protein levels quantified relative to the appropriate control (Lamin C for the nucleus, Tubulin for the cytoplasm and Lamin C for insoluble protein). Graph is the mean (±SD) of three biological repeats and multiple t tests; ns, not significant. (F) Micrograph of an example cryosection of an adult Drosophila head stained with Hoechst. The optic lamina is indicated as was the region selected for TDP-43 puncta quantification. (G) Micrographs of Drosophila head cryosections immunolabelled for TDP-43 and counterstained with Hoechst. Hatched white line indicates the optic lamina and region quantified. Hatched white box indicates the area magnified in inset. Expression of TDP-43 in adult neurons leads to the formation of cytoplasmic TDP-43–lablelled puncta versus the negative control ( elavGS > si.mCherry ). Co-expression of the siRNA to Nsun1 (si. Nsun1 ) reduces TDP-43 puncta. (H) The number of TDP-43–labelled puncta in the optic lamina was reduced by Nsun1 reduction. Graph is the mean (±SD) of all animals over three biological repeats with one-way ANOVA and a Tukey’s; ns is not significant. (I) TDP-43–positive puncta in the optic lamina were reduced in size by reduction in Nsun1 . Graph is the mean (±SD) of all animals over three biological repeats (left), with one-way ANOVA and a Tukey’s, ns is not significant. Genotypes are: (A, B, C, D, E) elavGS > control: w*; +/+; elavGS/si.mCherry 35785 , elavGS > si. Nsun1 : w*; +/si.Nsun1 TRiP.HMJ24019 ; elavGS/+ , elavGS > TDP-43 + control: w*; +/+; elavGS, UAS-TDP-43-52S/si.mCherry 35785 , elavGS > TDP-43 + si.Nsun1: w*; +/si.Nsun1 TRiP.HMJ24019 ; elavGS, UAS-TDP-43-52S/+ . (G, H, I) elavGS > control: w*; +/+; elavGS/si.mCherry 35785 , elavGS > TDP-43 + control: w*; UAS-TDP-43-5X/+; elavGS/si.mCherry 35785 . elavGS > 5X-TDP-43 + si. Nsun1 : w*; UAS-TDP-43-5X/si.Nsun1 TRiP.HMJ24019 ; elavGS/+ . Source data are available for this figure.

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) Reduction in Nsun1 (si. Nsun1 ) mitigates lifespan deficits caused by expression of TDP-43 in adult neurons. Drosophila were fed 100 μl of 2 μg/ml RU486 topically on the food, >100 males per genotype were followed for each genotype, and a log-rank (Mantel-Cox) test was used for significance. (B, C) Reduction in Nsun1 (si. Nsun1 ) does not alter total TDP-43 protein levels in adult neurons. Graph is the mean (±SD) of three biological repeats and a t test. (D) Reduction in Nsun1 (si. Nsun1 ) reduces neuronal TDP-43 protein levels in the cytoplasm, increases TDP-43 protein levels in the nucleus and has no effect on insoluble TDP-43 levels. Top panel immunoblotted for TDP-43, middle panel for Lamin C and bottom panel for Tubulin. (E) TDP-43 protein levels quantified relative to the appropriate control (Lamin C for the nucleus, Tubulin for the cytoplasm and Lamin C for insoluble protein). Graph is the mean (±SD) of three biological repeats and multiple t tests; ns, not significant. (F) Micrograph of an example cryosection of an adult Drosophila head stained with Hoechst. The optic lamina is indicated as was the region selected for TDP-43 puncta quantification. (G) Micrographs of Drosophila head cryosections immunolabelled for TDP-43 and counterstained with Hoechst. Hatched white line indicates the optic lamina and region quantified. Hatched white box indicates the area magnified in inset. Expression of TDP-43 in adult neurons leads to the formation of cytoplasmic TDP-43–lablelled puncta versus the negative control ( elavGS > si.mCherry ). Co-expression of the siRNA to Nsun1 (si. Nsun1 ) reduces TDP-43 puncta. (H) The number of TDP-43–labelled puncta in the optic lamina was reduced by Nsun1 reduction. Graph is the mean (±SD) of all animals over three biological repeats with one-way ANOVA and a Tukey’s; ns is not significant. (I) TDP-43–positive puncta in the optic lamina were reduced in size by reduction in Nsun1 . Graph is the mean (±SD) of all animals over three biological repeats (left), with one-way ANOVA and a Tukey’s, ns is not significant. Genotypes are: (A, B, C, D, E) elavGS > control: w*; +/+; elavGS/si.mCherry 35785 , elavGS > si. Nsun1 : w*; +/si.Nsun1 TRiP.HMJ24019 ; elavGS/+ , elavGS > TDP-43 + control: w*; +/+; elavGS, UAS-TDP-43-52S/si.mCherry 35785 , elavGS > TDP-43 + si.Nsun1: w*; +/si.Nsun1 TRiP.HMJ24019 ; elavGS, UAS-TDP-43-52S/+ . (G, H, I) elavGS > control: w*; +/+; elavGS/si.mCherry 35785 , elavGS > TDP-43 + control: w*; UAS-TDP-43-5X/+; elavGS/si.mCherry 35785 . elavGS > 5X-TDP-43 + si. Nsun1 : w*; UAS-TDP-43-5X/si.Nsun1 TRiP.HMJ24019 ; elavGS/+ . Source data are available for this figure.

Techniques: Expressing, Control, Staining, Negative Control

(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.

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.

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

(A) NSUN1 has a central RNA methyltransferase domain and an arginine-rich RNA-binding motif. There are four NSUN1 isoforms listed in NCBI and ensembl. These are isoform 1 (NCBI: NP_001028886.1 and ensembl: Nop2-212-i2), isoform 2 (NCBI: isoform 4, NP_001245239.1 and ensembl Nop2-218-i3), isoform 3 (NCBI: NP_001245238.1 and ensembl: Nop2-202-i4) and isoform 4 (NCBI: NP_001245239.1 and ensembl: Nop2-218-i3). The predicted molecular weight and conserved protein domains are listed. (B) The rabbit polyclonal antibody to NSUN1 detected two major NSUN1 protein bands at the predicted molecular weight in U20S protein lysate. The antigen for the rabbit polyclonal antibody was raised against the N-terminal region of NSUN1. We selected this antibody for our studies. (C) The mouse monoclonal antibody to NSUN1 detected one single high molecular weight band and several low molecular weight bands in U20S protein lysate. The antigen for the mouse monoclonal antibody to NSUN1 spans the RNA methyltransferase domain (aa 241–389). (D) Endogenous TDP-43 co-immunoprecipitated with endogenous NSUN1 in U20S cells. Top panel immunoblotted for NSUN1. Lower panel immunoblotted for TDP-43. (E) Endogenous NSUN1 co-immunoprecipitated with endogenous TDP-43 in U20S cells. Top panel immunoblotted for NSUN1. Lower panel immunoblotted for TDP-43. (F) Endogenous NSUN1 co-immunoprecipitated with endogenous TDP-43 in SH-SY5Y cells. Top panel immunoblotted for NSUN1. Lower panel immunoblotted for TDP-43. (G) Endogenous NSUN1 co-immunoprecipitated with human TDP-43-WT-YFP, human TDP-43-5F-L-YFP and human TDP-43-Q331K-YFP in U20S cells. Top panel immunoblotted for NSUN1. Lower panel immunoblotted for TDP-43. (H) Endogenous NSUN1 co-immunoprecipitated with human TDP-43-WT-YFP and human TDP-43-5F-L-YFP in SH-SY5Y cells. Top panel immunoblotted for NSUN1. Lower panel immunoblotted for TDP-43. Source data are available for this figure.

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) NSUN1 has a central RNA methyltransferase domain and an arginine-rich RNA-binding motif. There are four NSUN1 isoforms listed in NCBI and ensembl. These are isoform 1 (NCBI: NP_001028886.1 and ensembl: Nop2-212-i2), isoform 2 (NCBI: isoform 4, NP_001245239.1 and ensembl Nop2-218-i3), isoform 3 (NCBI: NP_001245238.1 and ensembl: Nop2-202-i4) and isoform 4 (NCBI: NP_001245239.1 and ensembl: Nop2-218-i3). The predicted molecular weight and conserved protein domains are listed. (B) The rabbit polyclonal antibody to NSUN1 detected two major NSUN1 protein bands at the predicted molecular weight in U20S protein lysate. The antigen for the rabbit polyclonal antibody was raised against the N-terminal region of NSUN1. We selected this antibody for our studies. (C) The mouse monoclonal antibody to NSUN1 detected one single high molecular weight band and several low molecular weight bands in U20S protein lysate. The antigen for the mouse monoclonal antibody to NSUN1 spans the RNA methyltransferase domain (aa 241–389). (D) Endogenous TDP-43 co-immunoprecipitated with endogenous NSUN1 in U20S cells. Top panel immunoblotted for NSUN1. Lower panel immunoblotted for TDP-43. (E) Endogenous NSUN1 co-immunoprecipitated with endogenous TDP-43 in U20S cells. Top panel immunoblotted for NSUN1. Lower panel immunoblotted for TDP-43. (F) Endogenous NSUN1 co-immunoprecipitated with endogenous TDP-43 in SH-SY5Y cells. Top panel immunoblotted for NSUN1. Lower panel immunoblotted for TDP-43. (G) Endogenous NSUN1 co-immunoprecipitated with human TDP-43-WT-YFP, human TDP-43-5F-L-YFP and human TDP-43-Q331K-YFP in U20S cells. Top panel immunoblotted for NSUN1. Lower panel immunoblotted for TDP-43. (H) Endogenous NSUN1 co-immunoprecipitated with human TDP-43-WT-YFP and human TDP-43-5F-L-YFP in SH-SY5Y cells. Top panel immunoblotted for NSUN1. Lower panel immunoblotted for TDP-43. Source data are available for this figure.

Techniques: RNA Binding Assay, Molecular Weight, High Molecular Weight, Immunoprecipitation

(A) NSUN1 is enriched in the nucleolus in human U20S cells. Cells are co-labelled with Fibrillarin (FBL) and Nucleophosmin (NPM1) and counterstained with Hoechst. Upper scale bar is 30 μm lower scale bar is 9 μm. (B) Quantification of NSUN1 co-labelling with Fibrillarin (FBL) and Nucleophosmin (NPM1). The NSUN1 signal extends beyond Fibrillarin and completely overlaps with Nucleophosmin. Pearson’s co-efficient of NSUN1 co-labelling with FBL and Nucleophosmin. Graph is mean (±sem) and a t test. (C) Schematic of the three nucleolar compartments and localisation of NSUN1, FBL and NPM1. The nucleolus is composed of three distinct compartments, the fibrillar centre, the dense fibrillar component, and the granular component, where rRNA transcription, rRNA processing and pre-ribosome assembly takes place, respectively. FBL localises to the dense fibrillar component , whereas Nucleophosmin is a granular component protein ( ; ), our data indicate that NSUN1 is in the granular component where it may function in rRNA processing and pre-ribosome assembly. (D, E) Treatment with 1 μg/ml actinomycin D (act D) for 3 h causes nucleolar breakdown, an accumulation of NSUN1 in the nucleoplasm, and cytoplasmic accumulation of TDP-43. Cells were co-labelled for TDP-43 and NSUN1 and counterstained with Hoechst. (D, E) Micrographs are wide filed images (D) and confocal images (E), respectively. (D, E) Scale bar in (D) is 25 μm, in (E) upper scale bar is 30 μm and lower scale bar is 44 μm. ( F, G, H) Treatment with 1 μg/ml actinomycin D (act D) causes NSUN1 nucleoli to decrease in number, size and intensity. Graphs are the mean (±sem) and a Dunnet’s test. (I) Treatment with 1 μg/ml actinomycin D (act D) causes NSUN1 levels in the nucleoplasm to increase. Graph is the mean (±sem) and a Dunnet’s test. (J) Actinomycin D (act D) treatment (1 μg/ml) causes a decrease in TDP-43 in the nucleus. Graph is the mean (±sem) and a Dunnet’s test. (K) The intensity of TDP-43 n the nucleolus is unchanged by 1 μg/ml actinomycin D (act D). Graphs are the mean (±sem) and a Dunnet’s test. (L) The ratio of nucleolar to nuclear TDP-43 increases by 1 μg/ml actinomycin D (act D) likely because of the overall decrease in nuclear TDP-43. Graphs are the mean (±sem) and a Dunnet’s test. (M) The ratio of nuclear to nucleolar TDP-43 decreases by 1 μg/ml actinomycin D (act D) likely because of the overall decrease in nuclear TDP-43 (see (K)). Graphs are the mean (±sem), one-way ANOVA, and a Dunnet’s test.

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) NSUN1 is enriched in the nucleolus in human U20S cells. Cells are co-labelled with Fibrillarin (FBL) and Nucleophosmin (NPM1) and counterstained with Hoechst. Upper scale bar is 30 μm lower scale bar is 9 μm. (B) Quantification of NSUN1 co-labelling with Fibrillarin (FBL) and Nucleophosmin (NPM1). The NSUN1 signal extends beyond Fibrillarin and completely overlaps with Nucleophosmin. Pearson’s co-efficient of NSUN1 co-labelling with FBL and Nucleophosmin. Graph is mean (±sem) and a t test. (C) Schematic of the three nucleolar compartments and localisation of NSUN1, FBL and NPM1. The nucleolus is composed of three distinct compartments, the fibrillar centre, the dense fibrillar component, and the granular component, where rRNA transcription, rRNA processing and pre-ribosome assembly takes place, respectively. FBL localises to the dense fibrillar component , whereas Nucleophosmin is a granular component protein ( ; ), our data indicate that NSUN1 is in the granular component where it may function in rRNA processing and pre-ribosome assembly. (D, E) Treatment with 1 μg/ml actinomycin D (act D) for 3 h causes nucleolar breakdown, an accumulation of NSUN1 in the nucleoplasm, and cytoplasmic accumulation of TDP-43. Cells were co-labelled for TDP-43 and NSUN1 and counterstained with Hoechst. (D, E) Micrographs are wide filed images (D) and confocal images (E), respectively. (D, E) Scale bar in (D) is 25 μm, in (E) upper scale bar is 30 μm and lower scale bar is 44 μm. ( F, G, H) Treatment with 1 μg/ml actinomycin D (act D) causes NSUN1 nucleoli to decrease in number, size and intensity. Graphs are the mean (±sem) and a Dunnet’s test. (I) Treatment with 1 μg/ml actinomycin D (act D) causes NSUN1 levels in the nucleoplasm to increase. Graph is the mean (±sem) and a Dunnet’s test. (J) Actinomycin D (act D) treatment (1 μg/ml) causes a decrease in TDP-43 in the nucleus. Graph is the mean (±sem) and a Dunnet’s test. (K) The intensity of TDP-43 n the nucleolus is unchanged by 1 μg/ml actinomycin D (act D). Graphs are the mean (±sem) and a Dunnet’s test. (L) The ratio of nucleolar to nuclear TDP-43 increases by 1 μg/ml actinomycin D (act D) likely because of the overall decrease in nuclear TDP-43. Graphs are the mean (±sem) and a Dunnet’s test. (M) The ratio of nuclear to nucleolar TDP-43 decreases by 1 μg/ml actinomycin D (act D) likely because of the overall decrease in nuclear TDP-43 (see (K)). Graphs are the mean (±sem), one-way ANOVA, and a Dunnet’s test.

Techniques:

(A) PLA shows that TDP-43 and NSUN1 interact in the nucleolus and nucleoplasm and that the interaction increases upon 1 μg/ml actinomycin D (act D) treatment. Micrographs of U20S cells showing the PLA signal (magenta) counterstained with Nucleolus Bright Green and Hoechst. Scale bars are 25 μm. (B) Actinomycin D (act D) treatment (1 μg/ml) caused an increase in TDP-43-NSUN1 PLA puncta number and intensity but had no effect on TDP-43-NSUN1 PLA puncta size. Graph is mean (±sem) of five separate experiments, one-way ANOVA, and a Dunnet’s test; ns, not significant. (C) TDP-43-NSUN1 PLA puncta in the cytoplasm were unaffected by actinomycin D (act D) treatment (1 μg/ml). Graph is mean (±sem) of 5 separate experiments, one-way ANOVA, and a Dunnet’s test; ns, not significant. (D) TDP-43-NSUN1 PLA puncta form in the nucleolus and nucleoplasm and this is unaltered by actinomycin D (act D) treatment (1 μg/ml). Graphs are mean (±sem) of three separate experiments, one-way ANOVA and a Dunnet’s test; ns, not significant. (E) Endogenous Nsun1 isoform 3 co-immunoprecipitated with TDP-43-YFP in U20S cells and this is unchanged by actinomycin D (act D) treatment (1 μg/ml). Source data are available for this figure.

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) PLA shows that TDP-43 and NSUN1 interact in the nucleolus and nucleoplasm and that the interaction increases upon 1 μg/ml actinomycin D (act D) treatment. Micrographs of U20S cells showing the PLA signal (magenta) counterstained with Nucleolus Bright Green and Hoechst. Scale bars are 25 μm. (B) Actinomycin D (act D) treatment (1 μg/ml) caused an increase in TDP-43-NSUN1 PLA puncta number and intensity but had no effect on TDP-43-NSUN1 PLA puncta size. Graph is mean (±sem) of five separate experiments, one-way ANOVA, and a Dunnet’s test; ns, not significant. (C) TDP-43-NSUN1 PLA puncta in the cytoplasm were unaffected by actinomycin D (act D) treatment (1 μg/ml). Graph is mean (±sem) of 5 separate experiments, one-way ANOVA, and a Dunnet’s test; ns, not significant. (D) TDP-43-NSUN1 PLA puncta form in the nucleolus and nucleoplasm and this is unaltered by actinomycin D (act D) treatment (1 μg/ml). Graphs are mean (±sem) of three separate experiments, one-way ANOVA and a Dunnet’s test; ns, not significant. (E) Endogenous Nsun1 isoform 3 co-immunoprecipitated with TDP-43-YFP in U20S cells and this is unchanged by actinomycin D (act D) treatment (1 μg/ml). Source data are available for this figure.

Techniques: Immunoprecipitation

(A) Quantification of PLA puncta show that TDP-43 and NSUN1 interact in the nucleus versus the negative controls. Graphs are the mean (±sem) and two-way ANOVA and a Dunnet’s test. (B) PLA shows that TDP-43 and NSUN1 do not interact in the cytoplasm as the signal is no different to the negative controls. Graphs are the mean (±sem), two-way ANOVA and a Dunnet’s test. (C) Actinomycin D (act D) treatment (1 μg/ml) caused a relative increase in the number of TDP-43-NSUN1 PLA puncta in the nucleus. Graph is mean (±sem) of five individual experiments, one-way ANOVA, and a Dunnet’s test; ns, not significant. (D) Actinomycin D (act D) treatment (1 μg/ml) caused no change in the number of cytoplasmic TDP-43-NSUN1 PLA puncta. Graph is mean (±sem) of five individual experiments, one-way ANOVA, and a Dunnet’s test; ns, not significant.

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) Quantification of PLA puncta show that TDP-43 and NSUN1 interact in the nucleus versus the negative controls. Graphs are the mean (±sem) and two-way ANOVA and a Dunnet’s test. (B) PLA shows that TDP-43 and NSUN1 do not interact in the cytoplasm as the signal is no different to the negative controls. Graphs are the mean (±sem), two-way ANOVA and a Dunnet’s test. (C) Actinomycin D (act D) treatment (1 μg/ml) caused a relative increase in the number of TDP-43-NSUN1 PLA puncta in the nucleus. Graph is mean (±sem) of five individual experiments, one-way ANOVA, and a Dunnet’s test; ns, not significant. (D) Actinomycin D (act D) treatment (1 μg/ml) caused no change in the number of cytoplasmic TDP-43-NSUN1 PLA puncta. Graph is mean (±sem) of five individual experiments, one-way ANOVA, and a Dunnet’s test; ns, not significant.

Techniques:

$(A) Representative micrographs of control and ALS spinal cord postmortem tissue immunolabeled for NSUN1 and counterstained with hematoxylin. Arrow indicates nucleolus in a motor neuron. Scale bar is 100 μm. (B) Schematic of the biochemical fractionation of frontal cortex tissue. (C) Protein isolated from frontal cortex tissue by sequential biochemical fractionation and immunoblotted for NSUN1 showed that in four of five control samples and four of five ALS/FTD samples NSUN1 was mostly in the soluble fractions (red boxes). LS, low-salt buffer; TX, Triton X-100 buffer; sark, sarkosyl buffer; urea, urea buffer. (D) The low-salt protein fraction isolated from control and ALS/FTD frontal cortex immunoblotted for NSUN1 and GAPDH. (E) The levels of NSUN1 isoform 3 is unaltered in ALS/FTD frontal cortex. NSUN1 is quantified relative to the GAPDH control on the same blot. Graph is the mean (±SD), t test; ns, not significant. (F) The levels of NSUN1 isoform 1/2 is significantly reduced in ALS/FTD frontal cortex. NSUN1 is quantified relative to the GAPDH control on the same blot Graph is the mean (±SD) and a t test. (G) The levels of NSUN1 isoform 4 are unaltered in ALS/FTD frontal cortex. NSUN1 is quantified relative to the GAPDH control on the same blot. Graph is the mean (±SD), t test; ns, not significant. Source data are available for this figure.$

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) Representative micrographs of control and ALS spinal cord postmortem tissue immunolabeled for NSUN1 and counterstained with hematoxylin. Arrow indicates nucleolus in a motor neuron. Scale bar is 100 μm. (B) Schematic of the biochemical fractionation of frontal cortex tissue. (C) Protein isolated from frontal cortex tissue by sequential biochemical fractionation and immunoblotted for NSUN1 showed that in four of five control samples and four of five ALS/FTD samples NSUN1 was mostly in the soluble fractions (red boxes). LS, low-salt buffer; TX, Triton X-100 buffer; sark, sarkosyl buffer; urea, urea buffer. (D) The low-salt protein fraction isolated from control and ALS/FTD frontal cortex immunoblotted for NSUN1 and GAPDH. (E) The levels of NSUN1 isoform 3 is unaltered in ALS/FTD frontal cortex. NSUN1 is quantified relative to the GAPDH control on the same blot. Graph is the mean (±SD), t test; ns, not significant. (F) The levels of NSUN1 isoform 1/2 is significantly reduced in ALS/FTD frontal cortex. NSUN1 is quantified relative to the GAPDH control on the same blot Graph is the mean (±SD) and a t test. (G) The levels of NSUN1 isoform 4 are unaltered in ALS/FTD frontal cortex. NSUN1 is quantified relative to the GAPDH control on the same blot. Graph is the mean (±SD), t test; ns, not significant. Source data are available for this figure.

Techniques: Control, Immunolabeling, Fractionation, Isolation

(A, B, C) NSUN1 isoforms quantified relative to GAPDH. Graphs are the mean (±SD) and a t test. ns, not significant. The data point with the *symbol is the patient that lacks TDP-43 pathology.

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, B, C) NSUN1 isoforms quantified relative to GAPDH. Graphs are the mean (±SD) and a t test. ns, not significant. The data point with the *symbol is the patient that lacks TDP-43 pathology.

Techniques:

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