Journal: Cell

Article Title: Regulation of the RNAPII Pool Is Integral to the DNA Damage Response

doi: 10.1016/j.cell.2020.02.009

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Article Snippet: KAPA mRNA HyperPrep kit , Kapabiosystems , KK8581.

Techniques: Virus, Recombinant, Protease Inhibitor, Membrane, SYBR Green Assay, Reverse Transcription, Genome Wide, Knock-In, Mutagenesis, Selection, Software

Journal: Nature Communications

Article Title: m6a methylation orchestrates IMP1 regulation of microtubules during human neuronal differentiation

doi: 10.1038/s41467-024-49139-7

Figure Lengend Snippet: a IMP1 iCLIP crosslinks (counts per millions) and RNAseq data (sequencing reads) are mapped onto genomic coordinates in NPCs and neurons for 4 exemplar target mRNAs. iCLIP replicates are shown in blue for neurons and in green for NPCs, while the merged signal is in red. 3’ untranslated region (3ʹUTR) and last exon are marked by a blue box and separated by an arrow, while line arrows indicate other intronic boundaries. In many of the RNA targets, the increase of IMP1 recruitment is not correlated with mRNA level. b Volcano plot of IMP1-bound peaks normalized by gene expression changes in NPCs vs neurons. Peaks with significantly higher signal in NPCs vs neurons, and vice versa are shown respectively in pink and orange. P values were calculated using a two-sided likelihood-ratio test (LRT). A threshold of 1 1, while the horizontal dashed line marks an adjusted p value < 0.05. c Relative expression of IMP1 over GAPDH at NPC and neuronal stages by RT-qPCR (RNA level, left) and WB (protein abundance, right). Data presented as boxplots, where the centre line is the median, limits are the interquartile range and whiskers correspond to 1.5 times the interquartile range. Outliers are not displayed for clarity. For the qPCR experiment n = 4 data points were obtained from independent iPSC lines (biological replicates), while for the WB experiment n = 6 data points (6 replicates, including 4 biological replicates (independent iPSC lines) in 2 experimental blocks). Values are normalized by the relative expression in the NPCs of the corresponding clones. P values were calculated using a two-sided Mann–Whitney U test and reported on the plot. d Left—representative image of βIII-tubulin (top) and skeletonization (bottom) used for the branching analysis. Approximated scale bar is 20 μm. Middle and right—number of triple and quadruple branches in neuronal processes normalized against branch length, in IMP1 siRNA (IMP1 KD) vs non-targeting siRNA control (CTRL). Data presented as boxplots where the centre line is the median, limits are the interquartile range and whiskers correspond to 1.5 times the interquartile range. Outliers are not displayed, for clarity. Data points represent different fields of view, n = 3 independent iPSC lines (biological replicates) in 3 independent experiments. P values were calculated using two-sided Mann–Whitney U test and reported on the plot. Source data are provided as a Source Data file.

Article Snippet: Poly(A)+ selected reverse stranded RNA sequencing libraries were prepared from total RNA using KAPA mRNA HyperPrep Library kit from Illumina®, with 200 ng of total RNA as input.

Techniques: Sequencing, Gene Expression, Expressing, Quantitative RT-PCR, Quantitative Proteomics, Clone Assay, MANN-WHITNEY, Control

Journal: Nature Communications

Article Title: m6a methylation orchestrates IMP1 regulation of microtubules during human neuronal differentiation

doi: 10.1038/s41467-024-49139-7

Figure Lengend Snippet: a Volcano plot showing proteins that are both differentially expressed upon IMP1 knock down in neurons and whose cognate mRNA is bound by IMP1 in our CLIP experiments. Proteins significantly up- or down-regulated in IMP1 siRNA (siIMP1) vs non-targeting control (siCTRL) are highlighted in orange and pink respectively. IMP1, CLASP1 and PEBP1 (Fig. ) are highlighted with blue dots and blue squares. Selection was based on log2 FC < −1 (down-regulated proteins) or log2 FC > 1 (upregulated proteins) and p value < 0.05. A two-sided one-sample Student’s t test was performed, for transcripts containing at least one IMP1 binding site. For MS, n = 3 independent iPSC lines for each condition (biological replicates) were used; for iCLIP, n = 3 technical replicates from 3 independent iPSC lines (biological replicates) were used. b Number of upregulated and downregulated proteins in neurons, from ( a ) and NPCs, from Supplementary Fig. , reported as a barplot. For MS on neuronal samples, replicates are as stated above. For MS on NPCs, n = 2 independent iPSC lines (biological replicates) for each condition were used; for iCLIP on NPCs, n = 2 technical replicates from 2 independent iPSC lines + 3 technical replicates from 1 independent iPSC line (biological replicates) were used. c Cumulative distribution plot of log2 FC in protein expression between IMP1 KD and control in neurons, as detected by MS, was plotted for RNA classes with 0, 1–2, 3–5 and 6 or more peaks in iCLIP experiments. For MS, n = 3 independent iPSC lines for each condition (biological replicates); for iCLIP, n = 3 technical replicates from 3 independent iPSC lines (biological replicates). The reported p values were calculated using a two-sided Kolmogorov–Smirnov test comparing 1–2 peaks, 3–5 peaks or 6 and + peaks to 0 peaks. d Number of IMP1 peaks per gene detected by iCLIP for GO categories related to microtubules, neuronal or other pathways classified by PANTHER. Number of genes in each category is in the same range (between 26 and 30). Data presented as boxplots where the centre line is the median, limits are the interquartile range and whiskers correspond to 1.5 times the interquartile range. Outliers are not displayed for clarity. Red dots represent the mean. The categories “Cytoskeleton organization”, “Microtubule-based process”, “Neuron projection development” contain many common genes. Black dots represent transcripts which are present in more than one of these categories, while orange dots represent transcripts in a single category. “Positive regulation protein kinase activity” with “Small molecule biosynthetic process” include different transcripts, represented by blue dots. Each category is represented by a number below the corresponding bar. P values were calculated using a two-sided Mann–Whitney U test and added to the plot. Comparisons were performed within each cell type. e STRING analysis of the interaction network between proteins that are downregulated in IMP1 siRNA treated neurons, where the cognate encoding transcripts are also bound by IMP1 (as identified in 4a). The analysis reports on in the GO category “microtubule-based process” (PANTHER). Network protein-protein interaction (PPI) enrichment p value < 1.0e–16. P value was calculated using a hypergeometric test. Source data are provided as a Source Data file.

Article Snippet: Poly(A)+ selected reverse stranded RNA sequencing libraries were prepared from total RNA using KAPA mRNA HyperPrep Library kit from Illumina®, with 200 ng of total RNA as input.

Techniques: Knockdown, Control, Selection, Binding Assay, Expressing, Activity Assay, MANN-WHITNEY

Journal: Nature Communications

Article Title: m6a methylation orchestrates IMP1 regulation of microtubules during human neuronal differentiation

doi: 10.1038/s41467-024-49139-7

Figure Lengend Snippet: a Representative LI-COR scanning visualization of poly(A) + RNA crosslinked to an m6A antibody or IgG in neurons, immobilized on a nitrocellulose membrane. RNA is visualized via the ligated infrared adaptor. The portion of the membrane excised and used to generate miCLIP libraries is framed in dashed white lines. For both neurons and NPCs, n = 2 technical replicates from 4 independent iPSC lines (biological replicates). NPC = neural precursor cells; Neur = neurons. b Metagene plot showing m6A site distribution in neurons. The motif with the most significant p value according to the HOMER analysis is displayed. P value was calculated within the HOMER package using a binomial distribution test. c Venn diagram displaying the number of m6A sites in neurons and/or NPCs. d m6A sites detected by miCLIP (top) and RNAseq (sequencing reads, bottom) are displayed on the 3’end of three example genes in NPCs and neurons, using Integrative Genomics Viewer (IGV) software. 3’ untranslated region (3ʹUTR) and last exon are marked by a blue box and separated by an arrow. NPC = neural precursor cells; Neur = neurons. e Cumulative distribution plot of the log2 FC in protein expression between IMP1 knockdown and control neurons as detected by MS, was plotted for corresponding RNA classified by the number of IMP1-m6A peaks detected by iCLIP and miCLIP. For MS, n = 3 independent iPSC lines for each condition (biological replicates); for iCLIP, n = 3 technical replicates from 3 independent iPSC lines (biological replicates); for miCLIP, n = 2 technical replicates from 3 independent iPSC lines + n = 1 technical replicate from 1 independent iPSC line (biological replicates). P values were calculated using two-sided Kolmogorov–Smirnov test comparing 1–2 peaks or 3–5 peaks or 6 and + peaks to 0 peaks. The corresponding P values are reported on the plot. f Number of IMP1 peaks on m6A sites (IMP1-m6A peaks) calculated by superimposing the results of the IMP1 iCLIP and miCLIP analyses in different GO categories determined by the PANTHER analysis. The three microtubule/cytoskeleton/neuronal terms and two cytoskeleton-unrelated terms displayed are determined by the PANTHER analysis shown in Fig. Data presented as boxplots where the centre line is the median, limits are the interquartile range and whiskers correspond to 1.5 times the interquartile range. Outliers are not displayed for clarity. Red dots represent the mean. The categories “Cytoskeleton organization”, “Microtubule-based process”, “Neuron projection development” contain many common genes. Black dots represent transcripts which are present in more than one of these categories, while orange dots represent transcripts in a single category. “Positive regulation protein kinase activity” with “Small molecule biosynthetic process” include different transcripts, represented by blue dots. g Barplot showing the percentage of proteins belonging to the “Microtubules-based process” terms as defined by the PANTHER GO classification for each of the following categories: downregulated proteins as defined in Fig. , downregulated proteins where the encoding mRNA is directly bound by IMP1 as defined in Fig. , downregulated proteins where the encoding mRNA is directly bound by IMP1 as defined in Fig. where IMP1 binds to an m6A site as determined by miCLIP. Source data are provided as a Source Data file.

Article Snippet: Poly(A)+ selected reverse stranded RNA sequencing libraries were prepared from total RNA using KAPA mRNA HyperPrep Library kit from Illumina®, with 200 ng of total RNA as input.

Techniques: Membrane, Sequencing, Software, Expressing, Knockdown, Control, Activity Assay

Journal: Molecular Cell

Article Title: Elongation Factor TFIIS Prevents Transcription Stress and R-Loop Accumulation to Maintain Genome Stability

doi: 10.1016/j.molcel.2019.07.037

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Article Snippet: KAPA RNA Hyper prep , Illumina , KR1350.

Techniques: Ubiquitin Proteomics, Virus, Recombinant, Reverse Transcription, Single Cell Gel Electrophoresis, Sequencing, Plasmid Preparation, Software, Cell Analysis, Protease Inhibitor, Membrane, SYBR Green Assay