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: bioRxiv

Article Title: 2-Thiouridine is a broad-spectrum antiviral nucleoside analogue against positive-strand RNA viruses

doi: 10.1101/2022.12.14.520006

Figure Lengend Snippet: a , Chemical structure of 2-thiouridine (s2U). b–l , Dose-response inhibition of DENV2 ( b ), ZIKV ( c ), YFV ( d ), JEV ( e ), WNV ( f ), CHIKV ( g ), HCoV-229E ( h ), HCoV-OC43 ( i ), SARS-CoV ( j ), MERS-CoV ( k ) and several SARS-CoV-2 variants ( l ) by s2U. Cell lysates were collected for viral RNA determination, and viral RNA levels were determined relative to ACTB or 18S rRNA transcripts. m , Dose-response inhibition of viral protein expression in the DENV2-, CHIKV-, HCoV-OC43- and SARS-CoV-2-infected cells. Cells were stained with viral-specific antibodies (green) and counterstained with Hoechst 33342 nuclear dye (blue). Scale bars indicate 200 μm. n–o , s2U did not inhibit RABV ( n ) and RVFV ( o ) virus replication. Cell lysates were collected for viral RNA determination; viral RNA levels were determined relative to ACTB or 18S rRNA transcripts. p , s2U did not inhibit HSV-1 virus replication. Cell lysates were collected for viral DNA determination; viral DNA levels were determined relative to ACTB transcripts. Data are presented as mean values of biological triplicates from one of the experiments, and error bars indicate standard deviation (SD). Statistically significant differences were determined using a one-way ANOVA followed by Dunnett’s multiple comparisons test to compare with non-treated cells; * p < 0.01, ** p < 0.005, *** p < 0.0005 and **** p < 0.0001.

Article Snippet: Ribavirin, Favipiravir, GS-5734 (Remdesivir), GS-441524, 2-thiouridine and Chloramphenicol were purchased from Sigma-Aldrich, PharmaBlock Sciences, Inc., MedChemExpress, Carbosynth Limited, Cayman Chemical Company and Calbiochem, respectively.

Techniques: Inhibition, Expressing, Infection, Staining, Virus, Standard Deviation