Journal: Nature Communications

Article Title: In vivo PAR-CLIP (viP-CLIP) of liver TIAL1 unveils targets regulating cholesterol synthesis and secretion

doi: 10.1038/s41467-023-39135-8

Figure Lengend Snippet: a Schematic representation of in vivo PAR-CLIP (viP-CLIP) applied in mice. Mice are injected 6 times with 4SU over the time course of 12.5 h and sacrificed 15 h after the first dose. Intact organs are flash-frozen, grinded, and cross-linked with UV light (365 nm) under liquid nitrogen cooling. After cell lysis of the tissue powder, the RNA-RBP complexes are immunoprecipitated in presence of RNase, radiolabeled and the recovered RNA in the size of 19–35 nt is ligated to sequencing adapter, and used for cDNA library preparation and sequencing. b 4SU incorporation rates in tissues ( n = 3) after injection regime displayed in ( a ). HEK293 ( n = 3) and primary hepatocytes ( n = 3) were cultured in the presence of 100 mM 4SU for 16 h and subjected together with the tissue samples to HPLC analysis of 4SU incorporation rates. Values are mean +/– S.D. c Phosphorimage of urea-PAGE transferred to a nitrocellulose membrane that resolved 32 P-labeled RNA-TIAL1 complexes after immunoprecipitation of TIAL1 in several tissues. d Immunoblot showing tissue distribution of TIAL1 in mouse organs. e Autoradiograph of recovered RNAs as displayed in ( c ), after proteinase K treatment and 15% urea gel electrophoresis. 5’ radiolabeled synthetic RNAs of 19 and 35 nt length served as size markers. c , d , e Representative images of three independent replicates. f Graphic presentation of viP-CLIP cDNA library composition by RNA categories from recovered RNA from ( e ). Source data are provided as a Source Data file.

Article Snippet: HPLC purified ( ≥ 98%) 4-thiouridine (4SU) was purchased from Tocris (Cat No. 7005), protected against light, and dissolved fresh before use.

Techniques: In Vivo, Injection, Lysis, Immunoprecipitation, Sequencing, cDNA Library Assay, Cell Culture, Membrane, Labeling, Western Blot, Autoradiography, Nucleic Acid Electrophoresis

Journal: Nature Communications

Article Title: Nanodynamo quantifies subcellular RNA dynamics revealing extensive coupling between steps of the RNA life cycle

doi: 10.1038/s41467-024-51917-2

Figure Lengend Snippet: A Typical polysome trace for untreated SUM159 cells. B Schematic representation of the Nanodynamo framework, from input data to kinetic rates interpretation, for the gene GSTP1 . Left, RNA species expression levels (bars and dots report mean and single replicate values respectively). Centre, kinetic rates coloured according to their relative values; the synthesis rate was reported in grey because of its different unit of measurement. Right, cartoon depicting the different efficiencies of the RNA life cycle steps. C Kinetic rates distributions in log 10 scale. D Heatmap reporting for each modelled gene (rows) RNA species abundance and kinetic rates magnitude (columns). For each column, values are saturated between the 1 st and 99 th percentiles and normalised against the latter. The left bar indicates groups of genes identified by k-means clustering according to normalised abundances and kinetic rates. E Gene-level median polyA tail length distributions across RNA pools. F RNA species and kinetic rates for the simplified models (rows) implemented in Nanodynamo, compared to the complete model (1 st row); input data required for each model are indicated in blue (not required in yellow) while the provided kinetic rates are indicated in grey (missing rates in red). In the figure, Ch, N, C, and P denote chromatin associated, nucleoplasmic, cytoplasmic, and polysomal RNA, respectively, while p and m additionally specify premature or mature forms. Analyses in C , D were performed on 1914 genes processed with the complete model while the analysis in E was performed on all the genes with at least one read with profiled polyA tail after replicates pooling (11774, 11957, 11760, 12739 genes for the chromatin, nucleoplasmic, cytoplasmic, and polysomal fractions respectively). For boxplots, the horizontal line represents the median value, the box edges represent the 25 th (Q1) and 75 th (Q3) percentiles, and the whiskers show the range of data excluding outliers (observations lower that Q1 − 1.5 * interquartile range or larger than Q3 + 1.5 * interquartile range). Source data are provided as a Source Data file.

Article Snippet: SUM159 cells were labelled with 500uM of 4-Thiouridine (Santa Cruz CAS 13957-31-8) for 20 min and treated with cycloheximide 100 μg/ml (Merk C4859) for 10 min before being lysed and washed twice with ice-cold PBS containing cycloheximide 10 μg/ml.

Techniques: Expressing

Journal: bioRxiv

Article Title: Direct Nanopore Sequencing of Individual Full Length tRNA Strands

doi: 10.1101/2021.04.26.441285

Figure Lengend Snippet: Panel A, tRNA fMet ; panel B tRNA Phe ; Panel C tRNA Lys . In each panel ( i ) is base coverage along the reference sequence at each position (coverage plot), and ( ii ) is a randomly selected subset of individual aligned Nanopore reads. The total number of aligned reads are shown to the left of the coverage plots. The positions of expected modifications on biological tRNA are indicated above the coverage plots and are abbreviated: 4= 4-thiouridine; D= Dihydrouridine; B= 2′-O-methylcytidine; 7= 7-methylguanosine; T= 5-methyluridine; P= Pseudouridine; X= 3-(3-amino-3-carboxypropyl)uridine; * = 2-methylthio-N6-isopentenyladenosine; S= 5-methyl-aminomethyl-2-thiouridine; 6= N6-threonylcarbamoyl-adenosine. Grey columns in the coverage plots indicate positions along the reference where 80% or more of the quality weighted reads are the expected canonical nucleotide. At positions where the value is under the 80% threshold, the proportion of each nucleotide call is shown in color where U(T) = red, A = green, C = blue, and G = gold. Similarly, the rows of individual aligned reads (A-C, ii ) are grey at positions matching the reference and colored (using the previously mentioned convention) at positions with mismatches. The black horizontal bars in the aligned reads indicate a deletion, and purple bars indicate an insertion. The rows of aligned reads are presented as they were displayed on IGV .

Article Snippet: Standards included 5-methyluridine (TCI America), B -pseudouridine (TRC Canada), uridine (Sigma), 4-thiouridine (MP Biomedicals), 2′-O-methylcytidine (Alfa Aesar), 7-methylguanosine (Sigma).

Techniques: Sequencing

Journal: bioRxiv

Article Title: Direct Nanopore Sequencing of Individual Full Length tRNA Strands

doi: 10.1101/2021.04.26.441285

Figure Lengend Snippet: Systematic base miscalls in purified biological and canonical tRNA fMet , tRNA Lys and tRNA Phe . The coverage plots (A-C) for biological and canonical synthetic tRNAs were generated from alignments using marginAlign. The number of aligned reads for each tRNA is shown under each coverage plot. Boxes surround positions of systematic miscalls (posterior probability > 30%). No systematic miscalls were identified in the synthetic canonical tRNAs. Colored bars are at positions where less that 80% of the quality weighted alignments match the reference. At these positions, the proportion of individual bases called are shown in color (U(T) =red, A = green, C = blue, and G = gold). The known modifications for the biological tRNAs are indicated above the coverage plots. Modified nucleotides are indicated above the reference sequence with abbreviations (4= 4-thiouridine, D= Dihydrouridine, B= 2′-O-methylcytidine, 7= 7-methylguanosine, T= 5-methyluridine, P = Pseudouridine, X= 3-(3-amino-3-carboxy-propyl)uridine, * = 2-methylthio-N6-isopentenyladenosine, S= 5-methylaminomethyl-2-thiouridine, 6= N6-threonylcarbamoyl-adenosine).

Article Snippet: Standards included 5-methyluridine (TCI America), B -pseudouridine (TRC Canada), uridine (Sigma), 4-thiouridine (MP Biomedicals), 2′-O-methylcytidine (Alfa Aesar), 7-methylguanosine (Sigma).

Techniques: Purification, Generated, Modification, Sequencing