Journal: Nucleic Acids Research

Article Title: Single-turnover kinetic analysis of non-long terminal repeat retrotransposition defines the pathway and rate constants leading to second-strand synthesis

doi: 10.1093/nar/gkag223

Figure Lengend Snippet: R2Bm mechanism cartoon, gene structure, and 28S DNA target. ( A ) Cartoon of the R2 retrotransposition pathway. R2 protein, bound to its mRNA, binds the 28S DNA locus and nicks the first strand . This is followed by transfer of the nicked DNA from the endonuclease active site (EN) to the polymerase active site (RT) . TPRT then occurs using the R2 mRNA as a template and the nicked lower strand as a primer . The second strand is then nicked (using the 5′RNA portion of the RNA shown in B), and the new 3′ end is used as a primer to copy the cDNA from the TPRT reaction (, , and ). Finally, host factors seal the nicks, leaving an R2 element integrated in the host genome . For simplicity in drawing, the diagram contains one R2 enzyme, but the reaction could involve a dimer. Only the polymerase and endonuclease domain of the protein is drawn. Sequence-specific DNA-binding domains are also present but are not shown due to ambiguity of their position in the second half of the reaction. ( B ) R2Bm gene structure and RNAs used in this study. The two regions of the elements whose RNA sequence is known to be recognized by the R2 enzyme correspond to the 5′RNA segment , and the 3′UTR. RNAs used in this study are diagrammed below, along with their length. The truncated ORF, containing the 5′RNA region, is 582 nt compared to a full R2Bm ORF of 3342 nt. ( C ) 28S DNA target. Regions of the 28S DNA target that are recognized by the R2Bm protein are annotated based on findings by Deng et al. . A 3′ phosphate blocking group was added to both strands to prevent non-specific addition to the 3′ ends of the starting DNA substrate. Top and bottom strands are 5′ labelled with 6-FAM and Cy3, respectively, and the 3′ ends are phosphorylated to prevent extensions by the R2 polymerase. Created in BioRender. Dangerfield, T. (2026) https://BioRender.com/e1lrntp ..

Article Snippet: In vitro transcription reactions were set up in a volume of 320 μl with 8 μg of purified PCR product, 5 mM DTT, 2 mM NTPs, and 1600 Units of T7 RNA polymerase in T7 RNA Polymerase reaction buffer (NEB).

Techniques: Sequencing, Binding Assay, Blocking Assay

Journal: Nucleic Acids Research

Article Title: 2′- O -Methylation of the second transcribed nucleotide within the mRNA 5′ cap impacts the protein production level in a cell-specific manner and contributes to RNA immune evasion

doi: 10.1093/nar/gkac722

Figure Lengend Snippet: Methylation of the first transcribed nucleotides in mRNA only partially prevents protein level decrease under IFN-induced stress. Relative protein production levels 72 h after a 5 h IFNα pre-treatment in ( A and B ) A549 and ( C and D ) JAWS II cells. Data were analysed for three independent replicates (each experiment consisted of three technical replicates). Bars for each transcript represent the mean value ± SEM normalized to protein production in mock-treated cells. Statistical significance: * P <0.05, ** P <0.01, *** P <0.001, **** P <0.0001 (one-way ANOVA with Turkey's multiple comparisons test) (data presenting changes in relative protein production with increasing concentration of IFNα are shown in for A549 and JAWS II cells, respectively). Co-purification of endogenous proteins from lysates of IFNα-treated ( E and F ) A549 (IFNα concentration was 500 U/ml) and ( G and H ) JAWS II (IFNα concentration was 5000 U/ml) cells with biotinylated RNA bearing cap0, cap1, cap2 or cap2-1 with ( E and G ) A or ( F and H ) m 6 A as the first transcribed nucleotide. Human eIF4E, IFIT1 and NCBP1 or murine counterparts (Ifit1b: lower band) were detected in precipitates by western blotting. A 1/15th and 1/21st volume of lysate used for each incubation with beads was loaded as input sample for ( E–G ) and ( H ), respectively; a 1/3rd volume of each eluate was loaded; shorter exposure is presented for input samples than for eluates; *non-specific band.

Article Snippet: The crude mRNAs were purified using NucleoSpin RNA Clean-up XS (740903, Macherey-Nagel). mRNAs were HPLC-purified using an RNASep Prep RNA Purification Column (ADS Biotec) at 55°C, a linear gradient of buffer B (0.1 M triethylammonium acetate pH 7.0 and 50% acetonitrile) from 17.5% to 25.8% in buffer A (0.1 M triethylammonium acetate pH 7.0) over 20 min and a flow rate of 0.9 ml/min. mRNAs from collected fractions were recovered by isopropanol precipitation.

Techniques: Methylation, Concentration Assay, Copurification, Western Blot, Incubation