Journal: Frontiers in bioscience (Landmark edition)

Article Title: Novel insights into the role of 5-Methylcytosine RNA methylation in human abdominal aortic aneurysm.

doi: 10.52586/5016

Figure Lengend Snippet: Fig. 1. Expression of m5C RNA methylation status (A) and m5C methylation modulators (B, C, D, E, F, and G) at the mRNA level in AAA tissue samples compared with the healthy control aortas analyzed by qRT-PCR. The non-parametric Mann-Whitney U test was all applied to analyze the m5C methylation ratio (A); the m5C “writers” family, NSUN1 (B), NSUN2 (C), NSUN5 (D), NSUN6 (E), Dnmt2 (F); and “readers” family Aly/REF (G). The expression of individual m5C RNA methylation modulators related to the expression of GAPDH set as 1 (100% expression). *, p < 0.05; **, p < 0.01. m5C, 5-Methylcytosine RNA methylation; AAA, abdominal aortic aneurysm; qRT-PCR, quantitative real-time polymerase chain reaction; ns, not significant.

Article Snippet: Then sections were washed 3 times with Tris-buffered saline, and incubated with rabbit anti-NSUN2 antibody (dilution 1:100; Proteintech, Wuhan, China) overnight at 4 ◦C in a humidified chamber.

Techniques: Expressing, Methylation, Control, Quantitative RT-PCR, MANN-WHITNEY

Journal: Clinical and Translational Medicine

Article Title: NSUN2 promotes colorectal cancer progression by enhancing SKIL mRNA stabilization

doi: 10.1002/ctm2.1621

Figure Lengend Snippet: NSUN2 is upregulated in CRC, associated with patient's survival and tumourigenesis. (A) NSUN2 was highly expressed in colorectal cancer tumour tissues (T) compared with adjacent normal tissues (N) from TCGA, GSE20916 and GSE33113 databases. (B) qRT‐PCR analysis showed the NSUN2 mRNA levels in 14 paired samples of CRC tumours (T) and corresponding adjacent normal tissues (N). (C) Western blot analysis showed the NSUN2 protein levels in paired samples of CRC tumours (T) and corresponding adjacent normal tissues (N). (D) Kaplan–Meier estimates of survival time of patients in cohort ( n = 267) from the sixth Affiliated Hospital of Sun Yat‐sen University and GSE17538 ( n = 232) by different NSUN2 expression levels in tumours. (E) Schematic representation of azoxymethane (AOM)/dextran sulfate sodium (DSS). (F) Colonoscopy photos of representative examples from Nsun2 +/+ and Nsun2 ‐/‐ mice. Arrow points to tumours. (G) Representative colon tumour images of Nsun2 +/+ and Nsun2 ‐/‐ mice. Arrow points to tumours. Scale bar = 1 cm. (H) Tumour numbers and volumes of Nsun2 +/+ and Nsun2 ‐/‐ mice. (I) H&E (hematoxylin and eosin) staining of colon tumour sections from Nsun2 +/+ and Nsun2 ‐/‐ mice. Scale bar = 1 mm. * P < 0.05; ** P < 0.01; *** P < 0.001.

Article Snippet: The antibodies used included NSUN2 (1:4000, Proteintech, 20854‐1‐AP), SKIL (1:4000, Proteintech, 19218‐1‐AP), GAPDH (1:5000, Proteintech, 60004‐1‐Ig), Myc (1:5000, CST, 2276s), Flag (1:5000, CST, 8146s), TAZ (1:4000, Proteintech, 22306‐1‐AP) and YBX1 (1:4000, abcam, ab12148).

Techniques: Quantitative RT-PCR, Western Blot, Expressing, Staining

Journal: Clinical and Translational Medicine

Article Title: NSUN2 promotes colorectal cancer progression by enhancing SKIL mRNA stabilization

doi: 10.1002/ctm2.1621

Figure Lengend Snippet: NSUN2 promotes malignant phenotypes of CRC cells both in vitro and in vivo. (A) After infection of DLD1 and HCT116 cells with shRNAs, cell growth curves were determined by Incucyte assays. The expression of NSUN2 was analyzed by Western blot. (B) Overexpression NSUN2 promoted CRC cell growth determined by Incucyte assays. (C) Overexpression of NSUN2 restored the cell growth inhibition caused by NSUN2 depletion. (D) Effects of NSUN2 depletion on the clonogenic ability of DLD1 and HCT116 cells. (E) Overexpression of NSUN2 promoted CRC cell colony formation. (F) Overexpression NSUN2 restored the reduced colony numbers caused by NSUN2 silencing. (G) After infection of DLD1 and HCT116 cells with shRNAs, cell migration was determined by transwell assays. Scale bar = 100 µm. (H) Overexpression of NSUN2 promoted CRC cell migration. (I) Overexpression NSUN2 restored the cell‐migration inhibition caused by NSUN2 silencing. (J) A xenograft mouse model was constructed by subcutaneously injecting nude mice with DLD1 cells ( n = 6 per group). Representative images of xenografts were shown. (K, L) Decreased tumour volume (K) and weight (L) were observed in the NSUN2 knockdown group, which was restored by NSUN2 overexpression. ** P < 0.01; *** P < 0.001.

Article Snippet: The antibodies used included NSUN2 (1:4000, Proteintech, 20854‐1‐AP), SKIL (1:4000, Proteintech, 19218‐1‐AP), GAPDH (1:5000, Proteintech, 60004‐1‐Ig), Myc (1:5000, CST, 2276s), Flag (1:5000, CST, 8146s), TAZ (1:4000, Proteintech, 22306‐1‐AP) and YBX1 (1:4000, abcam, ab12148).

Techniques: In Vitro, In Vivo, Infection, Expressing, Western Blot, Over Expression, Inhibition, Migration, Construct, Knockdown

Journal: Clinical and Translational Medicine

Article Title: NSUN2 promotes colorectal cancer progression by enhancing SKIL mRNA stabilization

doi: 10.1002/ctm2.1621

Figure Lengend Snippet: SKIL expression is positively regulated by NSUN2. (A, B) Immunofluorescence staining was performed to detect the NSUN2 and m5C levels with or without NSUN2 knockdown in DLD1 cells (A), or overexpression in HCT116 cells (B). Scale bar = 20 µm. (C) Original data of RNA array was normalized using Affy package in R software, and then the differential gene analysis between samples was carried out and screened by fold‐change and P value. Next, the differential genes in RNA array were overlapped with those modified by m5C methylation from Bis‐seq. ³⁰ (D) SKIL mRNA and protein levels were analyzed after NSUN2 depletion in DLD1 and HCT116 cells. (E) NSUN2 and SKIL immunohistochemistry of colon tumour sections from Nsun2+/+ and Nsun2‐/‐ mice. Scale bar = 100 µm. (F) SKIL was highly expressed in colorectal cancer tumour tissues (T) compared with adjacent normal tissues (N) from TCGA, GSE33113 and GSE20916 databases. (G) Kaplan–Meier estimates of survival time of patients in cohort ( n = 267) from the sixth Affiliated Hospital of Sun Yat‐sen University by different SKIL expression levels in tumours. (H) NSUN2 were positively correlated with the expression of SKIL at mRNA levels in TCGA and pairs of fresh tissues. (I) Adjacent and tumour tissues of colorectal cancer patients were collected and lysates were analyzed by immunoblotting with indicated antibodies. (J) Representative IHC staining for NSUN2 and SKIL in human CRC tissue microarray (TMA). Case 1 is representative of a patient with NSUN2‐high colon cancer. Case 2 is representative of a patient with NSUN2‐low colon cancer. Scale bar = 100 µm. (K) Quantification of staining intensities of indicated protein from sections in (J). NSUN2 and SKIL show a positive correlation. * P < 0.05; *** P < 0.001.

Article Snippet: The antibodies used included NSUN2 (1:4000, Proteintech, 20854‐1‐AP), SKIL (1:4000, Proteintech, 19218‐1‐AP), GAPDH (1:5000, Proteintech, 60004‐1‐Ig), Myc (1:5000, CST, 2276s), Flag (1:5000, CST, 8146s), TAZ (1:4000, Proteintech, 22306‐1‐AP) and YBX1 (1:4000, abcam, ab12148).

Techniques: Expressing, Immunofluorescence, Staining, Knockdown, Over Expression, Software, Modification, Methylation, Immunohistochemistry, Western Blot, Microarray

Journal: Clinical and Translational Medicine

Article Title: NSUN2 promotes colorectal cancer progression by enhancing SKIL mRNA stabilization

doi: 10.1002/ctm2.1621

Figure Lengend Snippet: NSUN2‐SKIL axis promotes CRC progression both in vitro and in vivo. (A, B) Colony formation (A), and CCK8 (B) assays were conducted to evaluate the effect of the NSUN2‐SKIL axis on the growth of DLD1 and SW480 cells. (C) Trans‐well assays were conducted to evaluate the effect of the NSUN2‐SKIL axis on the migration of DLD1 and SW480 cells. Scale bar = 100 µm. (D) DLD1 cells were stably infected with the indicated lentivirus and subcutaneously injected into BALB/c‐nude mice ( n = 6 per group). About 3 weeks after injection, xenografts were removed. Representative images of xenografts were shown. (E, F) Tumour volume (E) and tumour weight (F) were determined. (G) Immunofluorescence was implemented to detect the expression levels of Ki67 using tumour tissues harvested from xenograft model mice. Scale bar = 10 µm. The green colour represents Ki67 staining; the blue colour represents DAPI staining. * P < 0.05; ** P < 0.01; *** P < 0.001.

Article Snippet: The antibodies used included NSUN2 (1:4000, Proteintech, 20854‐1‐AP), SKIL (1:4000, Proteintech, 19218‐1‐AP), GAPDH (1:5000, Proteintech, 60004‐1‐Ig), Myc (1:5000, CST, 2276s), Flag (1:5000, CST, 8146s), TAZ (1:4000, Proteintech, 22306‐1‐AP) and YBX1 (1:4000, abcam, ab12148).

Techniques: In Vitro, In Vivo, Migration, Stable Transfection, Infection, Injection, Immunofluorescence, Expressing, Staining

Journal: Clinical and Translational Medicine

Article Title: NSUN2 promotes colorectal cancer progression by enhancing SKIL mRNA stabilization

doi: 10.1002/ctm2.1621

Figure Lengend Snippet: Methylation by NSUN2 stabilizes SKIL mRNA in a YBX1‐dependent manner. (A) Effect of wild‐type or mutant NSUN2 on cell growth in NSUN2 knockdown DLD1 and HCT116 cells by incucyte assays. (B) Wild‐type but not mutant NSUN2 reversed the decrease of SKIL mRNA and protein levels caused by NSUN2 depletion. (C) Assessment of the m5C modification of SKIL mRNA by m5C‐RIP qPCR. (D) Wild‐type but not mutant NSUN2 reversed the decrease of SKIL m5C levels caused by NSUN2 depletion. The “IgG” of each group was normalized as 1. (E) RNA stability assays displayed the reduced SKIL mRNA half‐life in NSUN2 knockdown CRC cells compared with the control cells by qRT‐PCR at the indicated time points after treatment with 10 µg/mL actinomycin D. (F) Wild‐type but not mutant NSUN2 reversed the decrease of SKIL mRNA half‐life induced by NSUN2 silencing. (G) RIP assays showed that YBX1 could bind to SKIL mRNA. (H) Significant reduction of YBX1 binding to SKIL when NSUN2 was silenced. (I) YBX1 knockdown substantially decreased the RNA and protein levels of SKIL. (J) Effects of YBX1 knockdown on mRNA half‐life of SKIL by RNA stability assays. (K, L) Wild‐type NSUN2 reversed the decrease of SKIL mRNA (K) and protein (L) levels caused by NSUN2 depletion, which could be abrogated by YBX1 knockdown. * P < 0.05; ** P < 0.01; *** P < 0.001; ns, not significant.

Article Snippet: The antibodies used included NSUN2 (1:4000, Proteintech, 20854‐1‐AP), SKIL (1:4000, Proteintech, 19218‐1‐AP), GAPDH (1:5000, Proteintech, 60004‐1‐Ig), Myc (1:5000, CST, 2276s), Flag (1:5000, CST, 8146s), TAZ (1:4000, Proteintech, 22306‐1‐AP) and YBX1 (1:4000, abcam, ab12148).

Techniques: Methylation, Mutagenesis, Knockdown, Modification, Control, Quantitative RT-PCR, Binding Assay

Journal: Clinical and Translational Medicine

Article Title: NSUN2 promotes colorectal cancer progression by enhancing SKIL mRNA stabilization

doi: 10.1002/ctm2.1621

Figure Lengend Snippet: NSUN2 induces the SKIL‐TAZ axis to promote CRC tumourigenesis. (A) Western blot analysis showed that overexpression of NSUN2 led to increased TAZ protein levels. (B) NSUN2 silencing decreased protein levels of TAZ. (C) NSUN2 depletion substantially suppressed the expression of TAZ‐targeted genes. (D) DLD1 cells were infected with shNSUN2 or Scramble and then treated with cycloheximide (100 µg/mL) for indicated times. The turnover of TAZ is indicated graphically. (E) HCT116 cells were transfected with the indicated plasmids and immunoblotted with the indicated antibodies. (F) Overexpression SKIL reversed the inhibition of TAZ‐targeted gene expression caused by NSUN2 silencing. (G) Expression level of NSUN2 in indicated patient‐derived xenografts (PDXs). (H) Treatment schedule of verteporfin is indicated. (I) Impact of verteporfin on tumour growth in mice bearing indicated PDXs ( n = 8 each group). (J) Impact of verteporfin on tumour weight in mice bearing indicated PDXs ( n = 8 each group). ** P < 0.01; *** P < 0.001; ns, not significant.

Article Snippet: The antibodies used included NSUN2 (1:4000, Proteintech, 20854‐1‐AP), SKIL (1:4000, Proteintech, 19218‐1‐AP), GAPDH (1:5000, Proteintech, 60004‐1‐Ig), Myc (1:5000, CST, 2276s), Flag (1:5000, CST, 8146s), TAZ (1:4000, Proteintech, 22306‐1‐AP) and YBX1 (1:4000, abcam, ab12148).

Techniques: Western Blot, Over Expression, Expressing, Infection, Transfection, Inhibition, Targeted Gene Expression, Derivative Assay

Journal: Clinical and Translational Medicine

Article Title: NSUN2 promotes colorectal cancer progression by enhancing SKIL mRNA stabilization

doi: 10.1002/ctm2.1621

Figure Lengend Snippet: Schematic illustration shows that NSUN2 promotes CRC malignancy by facilitating SKIL mRNA stabilization, followed by TAZ activation.

Article Snippet: The antibodies used included NSUN2 (1:4000, Proteintech, 20854‐1‐AP), SKIL (1:4000, Proteintech, 19218‐1‐AP), GAPDH (1:5000, Proteintech, 60004‐1‐Ig), Myc (1:5000, CST, 2276s), Flag (1:5000, CST, 8146s), TAZ (1:4000, Proteintech, 22306‐1‐AP) and YBX1 (1:4000, abcam, ab12148).

Techniques: Activation Assay

Journal: Nucleic Acids Research

Article Title: Spatial regulation of NSUN2-mediated tRNA m 5 C installation in cognitive function

doi: 10.1093/nar/gkae1169

Figure Lengend Snippet: G679R-NSUN2 is located in the nucleoplasm and leads to diminished m 5 C in tRNAs. ( A ) Wild type NSUN2 installs m 5 C on cytoplasmic tRNAs at positions C48, C49, C50, C40 and C34. ( B ) Western blots with anti-NSUN2 and anti-GAPDH antibodies showing NSUN2 expression in NSUN2 knockout clones ( NSUN2 KO). GAPDH serves as a loading control. ( C ) LC-MS/MS quantification of m 5 C in small RNAs (<200 nt) isolated from parental (wild type) or NSUN2 KO HEK293T clones. Three biological replicates represent every sample, each was measured in two technical replicates. The two-tailored t -test is used to calculate the P -value ( P < 0.0001). Error bars represent mean ± S.D. ( D ) Representative immunofluorescence images with anti-NSUN2 antibody of WT- and G679R-NSUN2 which have been transiently transfected in NSUN2 KO HEK293T cells. Fibrillarin serves as a nucleolus marker. Nuclei is stained with DAPI. Scale bars, 10 μm. ( E ) Representative western blots with anti-NSUN2 and anti-GAPDH antibodies showing the expression levels of exogenous WT-NSUN2 and G679R-NSUN2, which have been transiently transfected in NSUN2 KO HEK293T cells. ( F ) LC-MS/MS quantification of m 5 C in <200 nt RNAs isolated from NSUN2 KO cells transiently expressing empty vector, WT-NSUN2 and G679R-NSUN2. Three biological replicates represent every sample, each was measured in two technical replicates. The two-tailored t -test was used to calculate the P -value ( P < 0.0001). Error bars represent mean ± S.D. ( G ) Structural model of human NSUN2 predicted using AlphaFold3. The G679R mutation site, SAM-binding pocket, and IDRs are indicated.

Article Snippet: Comparable NSUN2 concentration in the nuclear extracts was confirmed using Western blotting, where Lamin B1 (Proteintech, 660 095) served as a loading control.

Techniques: Western Blot, Expressing, Knock-Out, Clone Assay, Control, Liquid Chromatography with Mass Spectroscopy, Isolation, Immunofluorescence, Transfection, Marker, Staining, Plasmid Preparation, Mutagenesis, Binding Assay

Journal: Nucleic Acids Research

Article Title: Spatial regulation of NSUN2-mediated tRNA m 5 C installation in cognitive function

doi: 10.1093/nar/gkae1169

Figure Lengend Snippet: ΔIDR-NSUN2 truncation variant is located in the nucleoplasm and partially rescues the m 5 C levels in tRNAs. ( A ) Structural prediction of NSUN2 using PONDR (natural disordered regions). The largest predicted IDRs are indicated. ( B ) Representative immunofluorescence images with anti-NSUN2 antibody of ΔIDR-NSUN2, which has been transiently transfected in NSUN2 KO HEK293T cells. Fibrillarin serves as a nucleolus marker. Nuclei is stained with DAPI. Scale bars, 10 μm. ( C ) Representative western blots with anti-NSUN2 and anti-GAPDH antibodies showing the expression of exogenous WT-NSUN2, and ΔIDR-NSUN2, which have been transiently transfected in NSUN2 KO HEK293T cells. ( D ) LC-MS/MS quantification of m 5 C in <200 nt RNAs isolated of NSUN2 KO cells transiently expressing empty vector, WT-NSUN2 and ΔIDR-NSUN2. Every sample is represented by three biological replicates; each was measured in two technical replicates. The two-tailored t -test was used to calculate the P -value ( P < 0.0001). Error bars represent mean ± S.D.

Article Snippet: Comparable NSUN2 concentration in the nuclear extracts was confirmed using Western blotting, where Lamin B1 (Proteintech, 660 095) served as a loading control.

Techniques: Variant Assay, Structural Proteomics, Immunofluorescence, Transfection, Marker, Staining, Western Blot, Expressing, Liquid Chromatography with Mass Spectroscopy, Isolation, Plasmid Preparation

Journal: Nucleic Acids Research

Article Title: Spatial regulation of NSUN2-mediated tRNA m 5 C installation in cognitive function

doi: 10.1093/nar/gkae1169

Figure Lengend Snippet: G679R-NSUN2 presents significantly impaired tRNA-binding affinity and catalytic efficiency. ( A ) Sodiumdodecyl sulphate-polyacrylamide gel electrophoresis showing the purity of purified recombinant MBP-WT-NSUN2 and MBP-G679R-NSUN2. ( B ) LC-MS/MS quantification of m 5 C levels in an unmodified RNA probe after in vitro methylation reaction with recombinant WT-NSUN2 at different incubation time intervals. Error bars represent mean ± SEM. ( C ) LC-MS/MS quantification of m 5 C levels of RNA probe after in vitro methylation reaction with recombinant WT- and G679R-NSUN2 for 1 h. Error bars represent mean ± S.D. The two-tailored t-test is used to calculate the P -value ( P < 0.0001). ( D ) Surface electrostatic potential of human NSUN2 (structure prediction was done with AlphaFold3). ( E ) EMSA showing WT- and G679R-NSUN2 binding to in vitro transcribed tRNA . (F) Quantification of the EMSAs. n = 6. Error bars represent mean ± S.D. n.d., not determined.

Article Snippet: Comparable NSUN2 concentration in the nuclear extracts was confirmed using Western blotting, where Lamin B1 (Proteintech, 660 095) served as a loading control.

Techniques: Binding Assay, Polyacrylamide Gel Electrophoresis, Purification, Recombinant, Liquid Chromatography with Mass Spectroscopy, In Vitro, Methylation, Incubation

Journal: Nucleic Acids Research

Article Title: Spatial regulation of NSUN2-mediated tRNA m 5 C installation in cognitive function

doi: 10.1093/nar/gkae1169

Figure Lengend Snippet: Assess the function of NSUN2-mediated m 5 C installation in tRNA in Drosophila social interactions. ( A–C ) In the social space assay, Nsun2 5-2 knockout ( Nsun2 KO) flies display disrupted social behavior as revealed by their aberrant distribution in the device and the increased interdistance. ( A ) Representative images showing fly distribution in the social behavior assay. Error bars represent mean ± SEM. ( B ) Histogram of the nearest neighbor distance. ( C ) The cumulative frequency of fly interdistance in the device. Data are analyzed by two-way ANOVA followed by Sidak's test. n = 3 groups, with ∼30–40 flies per group. ( D ) LC-MS/MS quantification of m 5 C levels in small RNA (<200 nt) of wild type or Nsun2 KO flies. RNA is extracted either from the whole flies or flies’ heads. Three biological replicates represent every sample, each was measured in two technical replicates. The two-tailored t-test was used to calculate the P -value. Error bars represent mean ± S.D. ( E–H ) Rescue of social interaction defects in Nsun2 KO by pan-neural overexpression of human NSUN2 transgenes. WT-NSUN2 and ΔIDR-NSUN2 show robust and partial rescue, respectively, while G679R fails to rescue. ( E ) The cumulative frequency of fly interdistance in the device. Data are analyzed by two-way ANOVA followed by Tukey's test. ( F ) Representative images showing fly distribution in the social behavior assay. ( G ) Quantification of the distance between any of the two flies in each trial. Data are analyzed by one-way ANOVA followed by Tukey's test. Each group is compared to every other group. Compared to wild type, all other groups are significantly increased. Compared to WT-NSUN2, G679R-NSUN2 and ΔIDR-NSUN2 exhibit significantly less rescue. ( H ) Histogram of the nearest neighbor distance. n = 3–5 groups, with ∼30–40 flies per group. Error bars represent mean ± SEM. ( I ) LC-MS/MS quantification of m 5 C levels in heads’ small RNA (< 200 nt) extracted from Nsun2 KO flies after pan-neuronal expression of human WT-, G679R- and ΔIDR-NSUN2. Every sample is represented by three biological replicates; each was measured in two technical replicates. Error bars represent mean ± S.D. In all figures: ns stands for not significant. * P < 0.05, *** P < 0.001, **** P < 0.0001.

Article Snippet: Comparable NSUN2 concentration in the nuclear extracts was confirmed using Western blotting, where Lamin B1 (Proteintech, 660 095) served as a loading control.

Techniques: Knock-Out, Behavioral Assay, Liquid Chromatography with Mass Spectroscopy, Over Expression, Expressing

Journal: Nucleic Acids Research

Article Title: Spatial regulation of NSUN2-mediated tRNA m 5 C installation in cognitive function

doi: 10.1093/nar/gkae1169

Figure Lengend Snippet: ΔIDR-NSUN2 restores m 5 C levels in specific tRNAs. ( A ) Western blots with anti-NSUN2 and anti-GAPDH antibodies showing the expression levels of mClover-WT-NSUN2 (WT-NSUN2) and mClover-ΔIDR-NSUN2 (ΔIDR-NSUN2), which have been transiently expressed in NSUN2 KO cells. GAPDH serves as a loading control. ( B ) LC-MS/MS quantification of m 5 C levels in small RNA (<200 nt) used as input for tRNA purification in D. RNA were extracted from parental HEK293T cells, NSUN2 KO cells and NSUN2 KO transiently expressing mClover-WT-NSUN2 (WT-NSUN2) or mClover-ΔIDR-NSUN2 (ΔIDR-NSUN2). ( C ) Representative northern blot analysis showing enrichment of the target tRNAs (tRNA Glu , tRNA Gly , tRNA Leu and tRNA Asp ) after affinity purification. 5 S rRNA serves as a negative control. ( D ) LC-MS/MS quantification of m 5 C levels of the purified tRNAs (tRNA Glu , tRNA Gly , tRNA Leu and tRNA Asp ) from parental HEK293T cells, NSUN2 KO cells and NSUN2 KO transiently expressing mClover-WT-NSUN2 (WT-NSUN2) or mClover-ΔIDR-NSUN2 (ΔIDR-NSUN2). Every sample is represented by three biological replicates, each was measured in two technical replicates. The two-tailored t-test was used to calculate the P -value (* P < 0.05, *** P < 0.001, **** P < 0.0001). Error bars represent mean ± S.D. ( E ) Heat map showing relative m 5 C level in bulk tRNA, tRNA Glu , tRNA Gly , tRNA Leu and tRNA Asp of parental HEK293T cells, NSUN2 KO cells, and NSUN2 KO transiently expressing mClover-WT-NSUN2 (WT-NSUN2) or mClover-ΔIDR-NSUN2 (ΔIDR-NSUN2). Values are normalized to the m 5 C level of parental cells, n = 6. ( F ) Schematic representation of in vitro methylation with nuclear extracts. Cells transiently expressing mClover-WT-NSUN2 or mClover-ΔIDR-NSUN2 were fractionated into cytoplasmic and nuclear fractions. Nuclear extracts were used for in vitro methylation on 5′biotynilated RNA oligo. After the reaction, biotinylated RNA probe was purified using magnetic streptavidin beads. ( G ) LC-MS/MS quantification of m 5 C levels of the purified RNA probe after in vitro methylation performed with the nuclear extracts prepared from NSUN2 KO cells (KO), and NSUN2 KO transiently expressing mClover-WT-NSUN2 (KO + WT-NSUN2) or mClover-ΔIDR-NSUN2 (KO + ΔIDR-NSUN2). Every sample is represented by three biological replicates, each was measured in two technical replicates. The two-tailored t-test was used to calculate the p -value (**** p < 0.0001). Error bars represent mean ± S.D.

Article Snippet: Comparable NSUN2 concentration in the nuclear extracts was confirmed using Western blotting, where Lamin B1 (Proteintech, 660 095) served as a loading control.

Techniques: Western Blot, Expressing, Control, Liquid Chromatography with Mass Spectroscopy, Purification, Northern Blot, Affinity Purification, Negative Control, In Vitro, Methylation

Journal: Nucleic Acids Research

Article Title: Spatial regulation of NSUN2-mediated tRNA m 5 C installation in cognitive function

doi: 10.1093/nar/gkae1169

Figure Lengend Snippet: NSUN2-mediated m 5 C installation is indispensable for proper social behavior in Drosophila . Schematic illustrating how WT-NSUN2 and NSUN2 mutants install m 5 C in tRNAs, leading to distinct impacts on social behavior in Drosophila melanogaster . Created in BioRender. Upenn, L. (2024) https://BioRender.com/s35c630 .

Article Snippet: Comparable NSUN2 concentration in the nuclear extracts was confirmed using Western blotting, where Lamin B1 (Proteintech, 660 095) served as a loading control.

Techniques: