Journal: bioRxiv

Article Title: RNA methylation influences TDP43 binding and disease pathogenesis in models of amyotrophic lateral sclerosis and frontotemporal dementia

doi: 10.1101/2022.04.03.486880

Figure Lengend Snippet: Density of UG nucleotide sequences 100bp upstream and downstream of m6A modifications identified by cross-linking induced mutation sites (CIMS; A ) or cross-linking induced truncation sites (CITS; B ) in relation to random sequences (red line). Grey shading represents 95% confidence regions. ( C ) Schematic of HaloTag immunoprecipitation and dot blot procedure. ( D ) Dot blot for total RNA (detected by methylene blue) or m6A-modified RNA (detected by anti-m6A antibody) isolated by immunoaffinity purification of HaloTag-labeled proteins in HEK293T cells overexpressing HaloTag, TDP43-HaloTag or YTHDF2-HaloTag from 3 biological replicates. ( E ) Diagram illustrating insertion of the HaloTag open reading frame into the endogenous TARDBP locus immediately 5’ to the TDP43 start codon, resulting in a fusion of HaloTag to the N-terminus of TDP43. ( F ) Halo-TDP43 HEK293T cells labeled live with JF646 Halo dye (red), then fixed, permeabilized, and immunostained with anti-TDP43 antibody (green) prior to imaging. DAPI (blue) marks the nucleus of each cell. Scale bar = 10µm. ( G ) Dot blot for total RNA (detected by methylene blue) or m6A-modified RNA (detected by anti-m6A antibody) isolated by immunoaffinity purification of endogenous HaloTag-TDP43 or exogenous HaloTag. Additional replicates shown in Sup. Fig. 1.

Article Snippet: Human spinal cord samples were homogenized in Trizol and RNA was extracted using phenol-chloroform extraction for the m6A mRNA&lncRNA Epitranscriptomic microarray (8×60K, Arraystar, Rockville, MD, USA).

Techniques: Mutagenesis, Immunoprecipitation, Dot Blot, Modification, Isolation, Immunoaffinity Purification, Labeling, Imaging

Journal: bioRxiv

Article Title: RNA methylation influences TDP43 binding and disease pathogenesis in models of amyotrophic lateral sclerosis and frontotemporal dementia

doi: 10.1101/2022.04.03.486880

Figure Lengend Snippet: ( A ) HaloTag-TDP43 immunoprecipitation was followed by DART-seq to delineate m6A sites within TDP43 target RNAs. HaloTag-TDP43 HEK293T cells were transfected with APOBEC1-YTH or APOBEC1-YTHmut and crosslinked before immunoaffinity purification of HaloTag-labeled proteins. Immunoprecipitated RNAs were then sequenced and C-T transitions were identified in the context of DRACH motifs (red shaded box, D=A/G/T, R=A/G, H=A/C/T). Absolute counts ( B ) and relative frequency ( C ) of base pair transitions observed by RNA-seq in each condition. Shaded boxes represent transition types expected from APOBEC1 activity. ( D ) Example m6A sites identified by DART-seq in RPL10A . C-T transitions are highlighted in red, and DRACH motifs in pink. Green arrow, transcription start site; red hexagon, transcription stop site; thick blue bars, coding exons; thin blue bars, untranslated region. ( E ) Absolute count and relative distribution ( F ) of DART-seq reads in cells expressing APOBEC1-YTH and APOBEC1-YTHmut. UTR, untranslated region; CDS, coding sequence. ( G ) Scatter plot of TDP43 targets, determined by fold enrichment in precipitated RNA from HaloTag-TDP43 cells (expressing APOBEC1-YTH and APOBEC10YTHmut) compared to cells transfected with HaloTag. Red dots signify transcripts showing > 2-fold enrichment in both APOBEC1-YTH and APOBEC1-YTHmut expressing cells. TARDBP , yellow dot, identified as high confidence target. ( H ) Stacked bar graph showing percentage of m6A modified RNA in TDP43 targets (red) and non-targets (black). ( I ) Cumulative distribution of RNA methylation in TDP43 targets (red) and non-targets (black). p = 1.87×10 −55 by Kolmogorov Smirnov test. ( J ) Euler diagram depicting overlap between TDP43 targets identified in this study, and those identified by TDP43 cross linking and immunoprecipitation followed by RNA-sequencing (CLIP-seq) in HEK293T cells (Hallegger et al ., 2021) . **p=1.5×10 −117 , hypergeometric test. ( K ) Pie charts demonstrating the percentage of methylated RNA among TDP43 targets (pink) and non-targets (grey). **p<1×10 −5 chi-square test.

Article Snippet: Human spinal cord samples were homogenized in Trizol and RNA was extracted using phenol-chloroform extraction for the m6A mRNA&lncRNA Epitranscriptomic microarray (8×60K, Arraystar, Rockville, MD, USA).

Techniques: Immunoprecipitation, Transfection, Immunoaffinity Purification, Labeling, RNA Sequencing, Activity Assay, Expressing, Sequencing, Modification, Methylation

Journal: bioRxiv

Article Title: RNA methylation influences TDP43 binding and disease pathogenesis in models of amyotrophic lateral sclerosis and frontotemporal dementia

doi: 10.1101/2022.04.03.486880

Figure Lengend Snippet: ( A ) TARDBP gene map, illustrating TDP43 binding region (TBR), the location of the DRACH motif (pink square), and the C-T transition (red box) identified by DART-seq within this domain, representing an m6A site. ( B ) Schematic of the TARDBP minigene reporter, consisting of the mCherry ORF upstream of TARDBP exon 6 and 3.4 Kb of the TARDBP 3’ UTR. The A residue adjacent to the detected C-T transition via DART-seq in the WT reporter (mCherry-TBR) was mutated to a G, precluding methylation the mutant reporter (mCherry-mTBR). Red, methylated residue; blue line, DRACH motif; dagger, C-T transition from DART-seq. ( C ) HaloTag-TDP43 was isolated by immunoaffinity purification from HaloTag-TDP43 HEK293T cells expressing mCherry-TBR or mCherry-mTBR, and reporter RNA detected in elution fractions by qRT-PCR. ( D ) Outline of TDP43 autoregulation assay. Excess TDP43 binds to the reporter, triggering reporter splicing, destabilization, and reduced mCherry fluorescence. ( E ) Primary rodent neurons were transfected with WT (mCherry-TBR) or mutant (mCherry-mTBR) reporters, together with EGFP or TDP43-EGFP. After 7d, mCherry expression was assessed by fluorescence microscopy. Scale bar= 20 µm. Normalized RFP (mCherry) intensity in primary neurons expressing WT mCherry-TBR reporter ( F ) or mutant mCherry-mTBR ( G ) reporter together with EGFP or TDP43(WT)-EGFP. Cherry-TBR+GFP n= 160, Cherry-TBR+TDP43(WT)-GFP n= 58, Cherry-mTBR+GFP n= 105, Cherry-mTBR+TDP43(WT)-GFP n= 44. Data in C plotted as mean ± SD, collected from 3 biological replicates. ns= not significant, *p< 0.05, **p< 0.01; one-way ANOVA with Tukey’s test. Data in F and G plotted as mean ± SD, color coded by biological replicate. ns = not significant, *p < 0.05; Welch’s t-test.

Article Snippet: Human spinal cord samples were homogenized in Trizol and RNA was extracted using phenol-chloroform extraction for the m6A mRNA&lncRNA Epitranscriptomic microarray (8×60K, Arraystar, Rockville, MD, USA).

Techniques: Binding Assay, Residue, Methylation, Mutagenesis, Isolation, Immunoaffinity Purification, Expressing, Quantitative RT-PCR, Fluorescence, Transfection, Microscopy

Journal: bioRxiv

Article Title: RNA methylation influences TDP43 binding and disease pathogenesis in models of amyotrophic lateral sclerosis and frontotemporal dementia

doi: 10.1101/2022.04.03.486880

Figure Lengend Snippet: ( A ) Genome-wide analysis of RNA methylation via epitranscriptomic array. RNA was extracted from control (n= 3) and sporadic ALS (sALS) patient (n= 4) spinal cord samples, prior to m6A RNA immunoprecipitation. The resulting samples were separated into methylated and non-methylated RNA, then labeled with distinct fluorescent dyes (red and green stars) prior to hybridization, allowing relative quantification of methylation at each annotated locus. ( B ) Principal component analysis (PCA) plot comparing methylation levels of control (grey) and ALS (red) patient samples. ( C ) Hierarchical clustering of mRNA methylation profiles from control and ALS mRNA samples. ( D ) Volcano plot depicting fold change in mRNA methylation levels in ALS compared to control spinal cord. ( E ) Hierarchical clustering of lncRNA methylation profiles from control ALS lncRNA samples. ( F ) Volcano plot showing fold change in lncRNA methylation levels in ALS compared to control spinal cord. In D and F , grey horizontal vertical lines represent p= 0.05 and fold change (FC)= 2. ( G ) Euler diagram demonstrating overlap (n= 322, p= 5.09×10 −119 , hypergeometric test) among TDP43 substrates and methylated transcripts identified in HEK293T cells, in additional to hypermethylated transcripts determined via m6A array in sALS spinal cord. Comparisons were limited to the subset of transcripts expressed in both HEK293T cells and human spinal cord (nTPM>2). ( H ) Based on comparisons with the GEO transcription factor loss-of-function database via Enrichr , there was strong enrichment for TDP43-regulated genes not only among the set of 2034 transcripts hypermethylated in sALS spinal cord, but also among the 322 TDP43 targets that were also hypermethylated in sALS (A1 in G ). Combined score = (log 10 p * Z-score). ( I ) Immunohistochemical staining for m6A in control and sALS spinal cord sections. Scale bars= 50 µm. ( J ) Quantification of m6A antibody reactivity in spinal cord neurons from control (n= 110 neurons) and sALS (n= 277 neurons) sections. Plot shows mean +/- SD, color coded by patient. ****p< 0.0001 via Mann-Whitney test.

Article Snippet: Human spinal cord samples were homogenized in Trizol and RNA was extracted using phenol-chloroform extraction for the m6A mRNA&lncRNA Epitranscriptomic microarray (8×60K, Arraystar, Rockville, MD, USA).

Techniques: Genome Wide, Methylation, Control, RNA Immunoprecipitation, Labeling, Hybridization, Quantitative Proteomics, Immunohistochemical staining, Staining, MANN-WHITNEY

Journal: bioRxiv

Article Title: RNA methylation influences TDP43 binding and disease pathogenesis in models of amyotrophic lateral sclerosis and frontotemporal dementia

doi: 10.1101/2022.04.03.486880

Figure Lengend Snippet: ( A ) Representative images of rodent primary neurons transfected with plasmids expressing Cas9-2A-EGFP and sgRNA targeting the neuronal protein NeuN or negative control (LacZ). 5d after transfection, neurons were fixed and immunostained for NeuN (red). White dashed circles indicate nucleus stained with Hoechst (blue). ( B ) NeuN antibody reactivity measured in EGFP-positive neurons expressing sgLacZ (n= 565) or sgNeuN (n= 654), ****p < 0.0001 by Mann-Whitney. ( C ) Schematic depicting m6A writers (green), erasers (red), and readers (orange) targeted by CRISPR/Cas9. ( D ) Primary neurons expressing EGFP and TDP43-mApple were assessed at regular 24h intervals by fluorescence microscopy, and their survival assessed by automated image analysis. Individual neurons are assigned unique identifiers (yellow number) and tracked until their time of death (red), indicated by cellular dissolution, blebbing, or neurite retraction. Scale bar= 20µm. ( E ) Cumulative hazard plot depicting risk of death for neurons expressing TDP43(WT) + non-targeting (NT) (red line), mApple + NT (grey line), or TDP43(WT) + Atxn2 sgRNA (purple line). †p<2.0 ×10 −16 , Hazard ratio (HR)= 3.45; ***p= 5.81 ×10 −4 , HR= 0.80). ( F ) Forest plot showing HR for TDP43-overexpressing neurons upon knockdown of m6A writers (green), erasers (dark red), and readers (orange), in comparison to nontargeting (NT) control. Dashed line indicates HR= 1, representing the survival of the reference condition, neurons expressing TDP43-mApple and NT sgRNA. Values >1 indicate increased toxicity, whereas values <1 denote relative protection. Error bars represent 95% CI. ( G ) Alkbh5 knockout significantly increases TDP43 associated toxicity. †p=3.11 ×10 −5 , HR= 1.59; ***p= 2.65×10 −11 , HR= 2.03. ( H ) Ythdf2 knockout significantly extends survival in TDP43-expressing neurons. ***p <2.0 ×10 −16 , HR= 1.69; †p= 6.2 ×10 −6 , HR= 0.71. ( I ) YTHDF2 overexpression is toxic to neurons. ***p= 3.07×10 −5 , HR= 1.30. ( J ) METTL3/14 overexpression enhances TDP43-dependent toxicity in neurons. †p = 5.53 ×10 −4 , HR= 1.32; ***p =4.16 ×10 −6 , HR= 1.31. p values in E, G-J determined via Cox proportional hazards analysis, with a minimum 3 of biological replicates.

Article Snippet: Human spinal cord samples were homogenized in Trizol and RNA was extracted using phenol-chloroform extraction for the m6A mRNA&lncRNA Epitranscriptomic microarray (8×60K, Arraystar, Rockville, MD, USA).

Techniques: Transfection, Expressing, Negative Control, Staining, MANN-WHITNEY, CRISPR, Fluorescence, Microscopy, Dissolution, Knockdown, Comparison, Control, Knock-Out, Over Expression

Journal: Journal of Clinical Laboratory Analysis

Article Title: Super‐enhancer–associated long noncoding RNA RP11‐569A11.1 inhibited cell progression and metastasis by regulating IFIT2 in colorectal cancer

doi: 10.1002/jcla.23780

Figure Lengend Snippet: SE‐LncRNA microarray analysis of CRC tissues. A, The scatter plot displayed variation in SE‐LncRNA expression between group‐N (normal) and group‐T (CRC). B, The volcano plot represented the fold change values and P‐values of the microarray data. C, Hierarchical clustering revealed differentially expressed SE‐LncRNAs (fold change >2, p ‐value <0.05). Red and green colors represented upregulated and downregulated SE‐LncRNAs, respectively

Article Snippet: Given that the expression abundance of SE‐LncRNA at lower levels than that of mRNA, and RNA‐seq is not sensitive to low‐abundance SE‐LncRNAs, we conducted Arraystar human SE‐LncRNAs microarray to detect differentially expressed SE‐LncRNAs.

Techniques: Microarray, Expressing

Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

Article Title: LINC01278 Sponges miR‐500b‐5p to Regulate the Expression of ACTG2 to Control Phenotypic Switching in Human Vascular Smooth Muscle Cells During Aortic Dissection

doi: 10.1161/JAHA.120.018062

Figure Lengend Snippet: A , miRNA expression profile. The green plots are decreased and red plots are upregulated differentially expressed genes. B , Heat map of miRNA profile. C , Quantitative reverse transcription‐PCR validation of miRNA microarray. D , lncRNA expression profile. E , Heat map of lncRNA profile. F , Quantitative reverse transcription‐PCR validation of lncRNA microarray. AD indicates aortic dissection; and qPCR, quantitative reverse transcription–polymerase chain reaction. ** P <0.01.

Article Snippet: All the RNAs (6 AD and normal control samples) were extracted using TRIzol, quantified (for integrity and concentration) using NanoDrop ND‐1000, and analyzed using the Arraystar Human miRNA and lncRNA microarray.

Techniques: Expressing, Reverse Transcription, Biomarker Discovery, Microarray, Dissection, Polymerase Chain Reaction

Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

Article Title: LINC01278 Sponges miR‐500b‐5p to Regulate the Expression of ACTG2 to Control Phenotypic Switching in Human Vascular Smooth Muscle Cells During Aortic Dissection

doi: 10.1161/JAHA.120.018062

Figure Lengend Snippet: A , miRNA profile and miRNA predicts by TargetScan online tool. B , lncRNA profile and lncRNA predictions by DIANA online tool. C , The interactive network of lncRNA‐miRNA‐mRNA. There were 13 co‐expressed miRNAs and 5 mRNAs and 6 lncRNAs related in the vascular smooth muscle contraction pathway in the network. The red triangle represents mRNAs, purple diamond represents miRNAs, and blue circle represents lncRNAs. Solid lines represent relationship between 2 nodes.

Article Snippet: All the RNAs (6 AD and normal control samples) were extracted using TRIzol, quantified (for integrity and concentration) using NanoDrop ND‐1000, and analyzed using the Arraystar Human miRNA and lncRNA microarray.

Techniques:

Journal: BMC Oral Health

Article Title: Identification of key module and hub genes in pulpitis using weighted gene co-expression network analysis

doi: 10.1186/s12903-022-02638-9

Figure Lengend Snippet: Summary of two data sets of pulpitis

Article Snippet: GSE92681 including 7 pulpitis and 5 healthy pulp samples from human, using the microarray platform GPL16956 (Agilent045997 Arraystar Human LncRNA Micro-array V3).

Techniques: Quantitative Proteomics, Gene Expression

Journal: Journal of Cellular and Molecular Medicine

Article Title: A novel antisense lncRNA NT5E promotes progression by modulating the expression of SYNCRIP and predicts a poor prognosis in pancreatic cancer

doi: 10.1111/jcmm.15718

Figure Lengend Snippet: LncNT5E is up‐regulated in pancreatic cancer (PC) tissues and cell lines. A, The heat map from our previous lncRNA microarray reflected the differentially expressed lncRNAs in PC and normal tissues. T represents PC tissue, and N represents normal pancreatic tissue. ENST00000421594 indicates lncNT5E. B, Relative expression of lncNT5E in 45 paired PC and adjacent normal tissues. LncNT5E expression from all tissues was normalized to 18S expression (ΔCT) and then compared with adjacent normal tissues and converted to the fold change (2 −ΔΔCT ). C, Relative expression of lncNT5E in different cell lines. Data are shown as fold change (2 −ΔΔCT ). * P < .05, ** P < .01, *** P < .001

Article Snippet: We used microarray detection (H1602063, Arraystar Human LncRNA 8 × 60 k v3.0 1‐color) to study lncRNAs in three pairs of PC and adjacent normal tissues.

Techniques: Microarray, Expressing