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: Science (New York, N.Y.)

Article Title: mRNA initiation and termination are spatially coordinated

doi: 10.1126/science.ado8279

Figure Lengend Snippet: (A) Heatmaps of Pearson’s r values for the pairwise correlations between the relative usage (Ψ) of a gene’s AFEs and ALEs based on their genomic order in HEK293T-A2 cells expressing for WT RNAPII ( left , n = 49), fast elongating RNAPII ( middle , R749H mutation, n= 69) and slowly elongating RNAPII ( right , E1126C mutation, n= 62). All heatmaps show pairwise correlations for genes expressing exactly 3 AFEs and 3 AFEs. ( B ) Schematic of the 4sU-DRB-LRS protocol, which involves transcription blockage and then synchronization with DRB, followed by labeling of nascent RNA with 4sU for 10 minutes, then long-read library preparation using polyI tailing to facilitate direct RNA long-read sequencing. The bottom right schematic shows the estimation of genomic distances the reads map to, which reflects the distance that RNAPII traveled from each TSS in the labeling period. ( C ) Direct RNA-seq for HNRNPL from 4sU-DRB-LRS data in K562 cells. Shown are annotated isoforms ( top , black ) and LRS reads ( middle , introns in thin grey lines ) colored by whether they start in the first expressed AFE ( blue ) or the second expressed AFE ( yellow ). Inset shows the distributions of genomic distances across reads for each AFE. ( D ) Distribution of mean genomic distances for each gene, conditioned on reads starting in AFEs with increasing ordinal positions for no PITA ( grey ) and PITA genes ( blue ). Boxes show the median and 5–95% confidence intervals for each distribution. ( E ) Distribution of elongation velocities around upstream and downstream TSSs ( left ) and PASs ( right ) for no PITA ( grey ) and PITA ( blue ) genes. Elongation velocities are calculated using 50nt bins across +/− 5kb windows around each site and smoothed with a sliding window approach for visualization ( Methods ). Background density represents the confidence intervals across genes.

Article Snippet: During the last 5 minutes of DRB treatment, 4-thio-uridine (4sU; ThermoFisher #J60679.MD) was added to a final concentration of 1mM (as described in ( 61 )).

Techniques: Expressing, Mutagenesis, Labeling, Sequencing, RNA Sequencing

Journal: eLife

Article Title: Global donor and acceptor splicing site kinetics in human cells

doi: 10.7554/eLife.45056

Figure Lengend Snippet:

Article Snippet: Chemical compound, drug , 4-thiouracil , Carbosynth, UK , NT06186 , CAS 13957-31-8.

Techniques: Software

Journal: Molecules

Article Title: Synthesis and Antiviral Activity of a Series of 2′- C -Methyl-4′-thionucleoside Monophosphate Prodrugs

doi: 10.3390/molecules25215165

Figure Lengend Snippet: Examples of early modified nucleoside drugs ( a – c ) and the aryloxy phosphoramidate monophosphate prodrug sofosbuvir ( d ).

Article Snippet: In 2014 Idenix filed a patent claiming the triphosphates of 4′-thioguanosine, 4′-thiouridine, and 2′-β-methyl-4′-thiouridine, as well as monophosphate prodrugs of the latter two, for the treatment of HCV [ ]; they were acquired by Merck that same year, purportedly for their anti-HCV portfolio.

Techniques: Modification