Journal: Nature Communications

Article Title: Transcriptional and physiological adaptations in nucleus accumbens somatostatin interneurons that regulate behavioral responses to cocaine

doi: 10.1038/s41467-018-05657-9

Figure Lengend Snippet: Cocaine induces 1100 DETs in NAc somatostatin interneurons. a Coronal section of anterior forebrain showing EGFP-F/SST overlap in NAc. Scale in a = 200 µm; Scale in inset = 25 µm. b Representative FACS gating used to isolate EGFP(+) nuclei from NAc of SST-TLG498 mice. We isolated ~5000 nuclei per mouse. c Differential expression analysis identifies 1100 cocaine-regulated transcripts in NAc somatostatin interneurons ( p < 0.05). 10 mice were injected with saline and 10 with cocaine every day for 7 days. Mice were analyzed 1 h after the final injection and tissue was flash frozen and stored before nuclear FACS. On average, ~1–2 ng of RNA were collected per mouse as determined by analysis on RNA Pico Bioanaylzer chips (Agilent). 0.5–2 ng were then used to generate indexed libraries for sequencing using Clontech SMARTer Stranded Total RNA-seq library preparation kits. All libraries were analyzed for mean insert size and molar concentration prior to sequencing with V4 chemistry (Illumina) at Beckman Coulter Genomics (Now: GeneWiz). Samples were pooled in groups of 8 and all pools were sequenced across multiple lanes. We performed multiplexed Illumina HiSeq to obtain > 2 × 10 7 paired reads per sample with read length of 125 × 2 bp. Individual transcripts are depicted as individual squares. Transcripts above the x axis are upregulated, while transcripts below the x axis are downregulated. Transcripts are color coded by transcript type and transcript types are quantified for relative abundance in top right inset pie chart. d DETs are enriched for gene ontology terms related to multiple neuronal compartments demonstrating different ways in which genes can regulate cellular function (Bonferoni p < 0.05). Several of the genes implicated in regulating these cellular compartments ( Ankr , Map2 , NrCam ) are implicated in regulating neuronal structural and functional plasticity. e Cell type-specific expression of GABAergic neuron and other interneuron subtype transcriptional markers. Npy neuropeptide Y, Nos1 nitrous oxide synthase 1, Lhx6 Lim-containing homeobox 6, Cck cholecystekinin, Pv parvalbumin, Chat choline acetyltransferase, Vip vasoactive intestinal peptide

Article Snippet: 0.5–2 ng were then used to generate indexed libraries for sequencing using Clontech SMARTer Stranded Total RNA-seq library preparation kits.

Techniques: Isolation, Expressing, Injection, Sequencing, RNA Sequencing Assay, Concentration Assay, Cell Function Assay, Functional Assay

Journal: Nature Communications

Article Title: Transcriptional and physiological adaptations in nucleus accumbens somatostatin interneurons that regulate behavioral responses to cocaine

doi: 10.1038/s41467-018-05657-9

Figure Lengend Snippet: JunD expression in NAc somatostatin interneurons regulates locomotor behavior. a There is a positive correlation in our RNA-sequencing data between JunD expression levels and locomotor activity across saline- and cocaine-treated mice on the last day of testing (Pearson Correlation: r = 0.46, n = 20, p = 0.043). Green circles represent JunD expression levels from individual mice. b Representative images used for quantification of JUND corrected total cell fluorescence (CTCF) in EGFP(+) cells. Each image is a single plane in a z-stack to ensure true fluorescence signal (not the sum of multiple planes). Scale bar = 25 µm. c JUND protein expression is increased in EGFP(+) cells of cocaine-treated mice (1 h after last dose) compared to saline controls (Student's t -test with Welch’s Correction: *** p < 0.0001; Saline mean CTCF = 1807 ± 138.9 SEM ( n = 20 cells/3 mice) vs Cocaine mean CTCF = 2920 ± 207.7 SEM ( n = 23 cells/3 mice). Welch’s correction: t = 4.54, df = 37.77). d Stereotaxic targeting of HSV-LSL-JUND + HSV-LSL-mCHERRY or HSV-LSL-ΔJUND + HSV-LSL-mCHERRY in NAc of Sst-Cre mice. e qPCR quantification of transgene expression in NAc of Sst-Cre mice infected with HSV-LSL-JUND or HSV-LSL-mCHERRY. ∆ JunD is a truncated form of JunD that lacks the N-terminus, making it a dominant-negative protein. (Student’s t -test with Welch’s correction: * p < 0.05; HSV-LSL-JUND mean = 0.01859 ± 0.008903 SEM ( n = 8) vs HSV-LSL-ΔJUND mean = 0.0007039 ± 0.0004589 SEM ( n = 10) Welch’s Correction: t = 2.259, df = 16). f JunD overexpression in NAc somatostatin interneurons increases locomotor activity in an open field (Students t -test: * p = 0.051; mCherry mean = 303.4 ± 26.25 SEM vs + JunD mean = 395.5 ± 35.62 SEM ( n = 11,11)). g Dominant-negative ΔJunD expression in NAc somatostatin interneurons decreases locomotor responses to cocaine (Repeated Measures Two-way ANOVA: Significant effect of Virus F (1,20) = 4.937. p < 0.04; n = 10,10). Data are represented as ± SEM

Article Snippet: 0.5–2 ng were then used to generate indexed libraries for sequencing using Clontech SMARTer Stranded Total RNA-seq library preparation kits.

Techniques: Expressing, RNA Sequencing Assay, Activity Assay, Fluorescence, Infection, Dominant Negative Mutation, Over Expression

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: CXCR6 + ST2 + memory T helper 2 cells induced the expression of major basic protein in eosinophils to reduce the fecundity of helminth

doi: 10.1073/pnas.1714731115

Figure Lengend Snippet: Nb-induced ST2− and ST2+ mTh cells exhibit distinct gene expression profiles. (A) Experimental protocols for the infection of wild-type mice with Nb. #Assay: RNA sequencing. (B) The expression of the genes with a difference in expression of more than fivefold in ST2+ mTh cells compared with the other two groups obtained by RNA sequence analysis. (C and D) MA plots comparing the different expression of transcription factors (C) as well as cytokines and chemokines (D) between ST2+ and ST2− mTh cells. Horizontal line denotes log2[(ST2+ mTh)/(ST2− mTh)], while vertical lines denote (1/2) × log2[(ST2− mTh) × (ST2+ mTh)]. (E) A qRT-PCR analysis of the indicated genes in ST2+ and ST2− mTh cells. The minimum data values were set as one. The mean values (four data per group) are shown with the SEM. n.s., not significant. **P < 0.01; ***P < 0.001. (F) MA plots comparing the differential expression of surface markers between ST2+ and ST2− mTh cells. (G) Staining profiles of indicated markers on ST2+ and ST2− mTh cells are shown with the mean fluorescence intensity of cells in histograms. At least two independent experiments were performed and showed similar results. The mean of FPKM was used for analyses in B–D and F. We set positive/negative gates using staining with isotype controls in G.

Article Snippet: For cDNA library construction, we used SMARTer Stranded Total RNA-Seq Kit-Pico Input Mammalian (634412; Clontech) according to the manufacturer’s protocol.

Techniques: Expressing, Infection, RNA Sequencing Assay, Sequencing, Quantitative RT-PCR, Staining, Fluorescence

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: CXCR6 + ST2 + memory T helper 2 cells induced the expression of major basic protein in eosinophils to reduce the fecundity of helminth

doi: 10.1073/pnas.1714731115

Figure Lengend Snippet: Helminth-induced ST2+ mTh2 cells accumulated MBP-expressing eosinophils in the lungs, reducing the fecundity of Nb. (A) The expressions of the genes with a difference in expression of more than fivefold in the sorted eosinophils obtained from the lungs of ST2+ mTh cell-transferred mice compared with the other two groups according to an RNA sequence analysis were selected as depicted in SI Appendix, Fig. S7. The genes listed in two independent experiments are shown. (B) A qRT-PCR analysis of Prg2 and Epx in the sorted eosinophils obtained from the lung of naïve CD4+ T cell-, ST2− mTh cell-, or ST2+ mTh cell-transferred mice. The minimum data values were set as one. The mean values (six data per group) are shown with the SEM. (C) Experimental protocols for the infection of the mice with Nb. #Assay: count numbers of eggs and worms. (D) Mice were killed 6 d after infection with Nb, as described in C. The absolute numbers of parasite eggs in feces per gram and the numbers of worms are shown. The mean values (six mice per group) are shown with the SEM. At least two independent experiments were performed and showed similar results. n.s., not significant. **P < 0.01; ***P < 0.001.

Article Snippet: For cDNA library construction, we used SMARTer Stranded Total RNA-Seq Kit-Pico Input Mammalian (634412; Clontech) according to the manufacturer’s protocol.

Techniques: Expressing, Sequencing, Quantitative RT-PCR, Infection

Journal: The Journal of Biological Chemistry

Article Title: The METTL5-TRMT112 N 6 -methyladenosine methyltransferase complex regulates mRNA translation via 18S rRNA methylation

doi: 10.1016/j.jbc.2022.101590

Figure Lengend Snippet: METTL5 is an m 6 A methyltransferase stabilized by TRMT112. A , METTL5 expression in cell lines as evaluated by Western blot ( top ) with anti-GAPDH loading control ( bottom ). HEL (replicate 1) and (replicate 2) represent two different passages of HEL cells. Arrowhead signifies METTL5 band; ∗ signifies background band. B , Western blot analysis of METTL5 levels in wild-type (WT) and knockout (KO) HeLa samples, each expanded from a single isolated clone ( top ) with anti-GAPDH loading control ( bottom ). Arrowhead signifies METTL5 band; ∗ signifies background band. C , coimmunoprecipitations of TRMT112 with FLAG-METTL5. Left , anti-FLAG ( top ) and anti-TRMT112 ( middle ) Western blots in anti-FLAG immunoprecipitation samples. Input TRMT112 levels shown on bottom . Lanes from left to right are untransfected HeLa, HeLa transfected with FLAG-METTL5, and HeLa transfected with the FLAG-METTL5-3A mutant. Right , anti-FLAG ( top ) and anti-TRMT112 ( middle ) Western blots in anti-FLAG immunoprecipitation samples. Input TRMT112 levels shown on bottom . Labels: METTL5-WT, METTL5-3A, METTL5-G61D, METTL5-K191V: HeLa transfected with FLAG-METTL5-WT, FLAG-METTL5-3A, FLAG-METTL5-G61D, and FLAG-METTL5-K191Vfs∗10, respectively. D , top , Western blots for METTL5, TRMT112, and GAPDH (loading control) levels upon siRNA knockdown for METTL5 and TRMT112. Labels: Ø, untransfected HeLa cells; cont., HeLa transfected with two different nontargeting control siRNAs; METTL5, HeLa transfected with two different siRNAs targeting METTL5; TRMT112, HeLa transfected with three different siRNAs targeting TRMT112. Bottom , ratio of METTL5 to GAPDH signal intensity as quantified by Fiji software. E , top , Western blot analysis of FLAG-METTL5 and FLAG-TRMT112 expression from HeLa transfected with labeled combinations of vectors, with GAPDH loading control. Bottom , band intensity normalized to GAPDH band intensity, as quantified by Fiji software. F , LC-MS/MS analysis of d 3 m 6 A ( left ) and d 3 m 1 A ( right ) levels normalized to guanosine from in vitro methyltransferase reactions performed on total RNA isolated from METTL5-WT and METTL5-KO HeLa clones. n = 2 replicate reactions, mean and s.e.m. plotted, analyzed by one-way ANOVA, comparing all samples to HeLa WT with Dunnett’s test for multiple comparisons. ns: not significant, ∗ p <0.05. All indicated band sizes in Western blots are in kilodaltons (kDa).

Article Snippet: Libraries were prepared using the SMARTer Stranded Total RNA-Seq Kit v2 (Takara) per manufacturer’s instructions and sequenced on a NovaSeq6000 with 100 bp single-end reads.

Techniques: Expressing, Western Blot, Knock-Out, Isolation, Immunoprecipitation, Transfection, Mutagenesis, Software, Labeling, Liquid Chromatography with Mass Spectroscopy, In Vitro, Clone Assay

Journal: The Journal of Biological Chemistry

Article Title: The METTL5-TRMT112 N 6 -methyladenosine methyltransferase complex regulates mRNA translation via 18S rRNA methylation

doi: 10.1016/j.jbc.2022.101590

Figure Lengend Snippet: Mettl5 knockout mice have developmental and metabolic defects. A , levels of m 6 A ( left ) and m 6,6 A ( right ), normalized to G, obtained by LC-MS/MS of 40-nt probe-purified segments of 18S rRNA surrounding the m 6 A 1832 site from the brains and livers of Mettl5 +/+ , Mettl5 +/− , and Mettl5 −/− mice. n = 4, Mettl5 +/+ , n = 3, Mettl5 +/− , and n = 4, Mettl5 −/− . Analyzed by one-way ANOVA comparison with Sidak test for multiple comparisons. B , pie chart of the predicted ( left ) and actual ( right ) mice born of wild type (+/+, dark blue ), heterozygous (+/−, grey ), and knockout (−/−, red ) genotypes from heterozygous x heterozygous breeding pairs (total = 106 mice; 30 +/+, 56 +/−, 20 −/− mice). C , photograph of male littermate Mettl5 +/+ , Mettl5 +/− , and Mettl5 −/− mice at 8 weeks of age. D , body weights in grams of male ( top ) and female ( bottom ) Mettl5 +/− and Mettl5 −/− mice from 4 weeks (weaning) to 10 weeks of age. n = 6 mice for male +/−, n = 4 for male −/−, n = 5 for female +/− and −/−; unpaired t test was used to compare each time point. E , volcano plots from mouse liver ( left ) and brain ( right ) RNA-seq data displaying the negative log 10 of the adjusted p value versus the log 2 fold change comparing the transcriptomes of Mettl5 −/− and Mettl5 +/− mice (n = 3 pairs). F , dysregulated pathways based on analysis of RNA-seq data from Mettl5 −/− and Mettl5 +/− mouse livers by the software DAVID . G , effects of METTL5 knockout on translation and transcription as displayed by log 2 (fold change) of translation efficiency (TE) versus log 2 (fold change) of reads per kilobase of transcript per million mapped reads (RPKM) from ribosome profiling input and ribosome-protected fragment sequencing of mouse livers. Case = Mettl5 −/− , Control = Mettl5 +/+ . H , latency in seconds for Mettl5 +/− and Mettl5 −/− mice to fall off the rotarod in a rotarod performance test. I , total distance in centimeters travelled by Mettl5 +/− and Mettl5 −/− mice when placed in a new environment in an open field test. Unpaired t test shown. H and I , n = 11 pairs. ns: not significant, ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.005.

Article Snippet: Libraries were prepared using the SMARTer Stranded Total RNA-Seq Kit v2 (Takara) per manufacturer’s instructions and sequenced on a NovaSeq6000 with 100 bp single-end reads.

Techniques: Knock-Out, Liquid Chromatography with Mass Spectroscopy, Purification, RNA Sequencing Assay, Software, Sequencing