The Effects of Ultraviolet Radiation on Nucleoside Modifications in RNA

Ultraviolet radiation (UVR) is a known genotoxic agent. Although its effects on DNA have been well-documented, its impact on RNA and RNA modifications is less studied. By using <i>Escherichia coli</i> tRNA (tRNA) as a model system, we identify the UVA (370 nm) susceptible chemical groups and bonds in a large variety of modified nucleosides. We use liquid chromatography tandem mass spectrometry to identify specific nucleoside photoproducts under <i>in vitro</i> and <i>in vivo</i> conditions, which were then verified by employing stable-isotope labeled tRNAs. These studies suggest that the -amino or -oxy groups of modified nucleosides, in addition to sulfur, are labile in the oxidative environment generated by UVA exposure. Further, these studies document a range of RNA photoproducts and post-transcriptional modifications that arise because of UVR-induced cellular stress

Directed Evolution of Heterologous tRNAs Leads to Reduced Dependence on Post-transcriptional Modifications

Heterologous tRNA:aminoacyl tRNA synthetase pairs are often employed for noncanonical amino acid incorporation in the quest for an expanded genetic code. In this work, we investigated one possible mechanism by which directed evolution can improve orthogonal behavior for a suite of <i>Methanocaldococcus jannaschii</i> (<i>Mj</i>) tRNA^Tyr-derived amber suppressor tRNAs. Northern blotting demonstrated that reduced expression of heterologous tRNA variants correlated with improved orthogonality. We suspected that reduced expression likely minimized nonorthogonal interactions with host cell machinery. Despite the known abundance of post-transcriptional modifications in tRNAs across all domains of life, few studies have investigated how host enzymes may affect behavior of heterologous tRNAs. Therefore, we measured tRNA orthogonality using a fluorescent reporter assay in several modification-deficient strains, demonstrating that heterologous tRNAs with high expression are strongly affected by some native <i>E. coli</i> RNA-modifying enzymes, whereas low abundance evolved heterologous tRNAs are less affected by these same enzymes. We employed mass spectrometry to map ms²i⁶A37 and Ψ39 in the anticodon arm of two high abundance tRNAs (Nap1 and tRNA^Opt_CUA), which provides (to our knowledge) the first direct evidence that MiaA and TruA post-transcriptionally modify evolved heterologous amber suppressor tRNAs. Changes in total tRNA modification profiles were observed by mass spectrometry in cells hosting these and other evolved suppressor tRNAs, suggesting that the demonstrated interactions with host enzymes might disturb native tRNA modification networks. Together, these results suggest that heterologous tRNAs engineered for specialized amber suppression can evolve highly efficient suppression capacity within the native post-transcriptional modification landscape of host RNA processing machinery

Normalized expression data for the NASC Arabidopsis biotic stress series (Additional file ) were extracted and plotted as shown

The legends indicate the correspondence between the plots and the respective Arabidopsis gene identification designation. The numerical key for each array experiment is given along the X-axis. While the full list of the agents can be found in Additional file , here is a brief list: 1–16, control and infection; 17–22, control and infection; 23–36, control and elicitors treatment; 37–52, dark and different light treatment.<p><b>Copyright information:</b></p><p>Taken from "Arabidopsis mRNA polyadenylation machinery: comprehensive analysis of protein-protein interactions and gene expression profiling"</p><p>http://www.biomedcentral.com/1471-2164/9/220</p><p>BMC Genomics 2008;9():220-220.</p><p>Published online 14 May 2008</p><p>PMCID:PMC2391170.</p><p></p

Normalized expression data for the NASC Arabidopsis developmental series (Additional file ) were extracted and plotted as shown

The set of genes listed in Table 1 were split into three groups; the grouping was done according to historical views of the polyadenylation complex. Thus, genes encoding CPSF and CSTF subunits are shown in the top panel, PAPS and PABN genes in the middle, and the remaining genes in the lower panel. This grouping also applies for the plots shown in Figures 3–5. The legends indicate the correspondence between the plots and the respective Arabidopsis gene identification designation. The numerical key for each array experiment is given along the X-axis. The full list of the keys can be found in the Additional file . Here is a brief description of these samples, including wt and some mutants: 1–7, root 7–21 days; 8–10, stem 7–21 days; 11–27, leaf 7–35 days; 28–38, whole plant 7–23 days; 39–49, shoot apex 7–21 days; 50–71, flowers and floral organs 21+ day; 72–79, 8 week seeds and siliques. The arrows point to the positions for mature pollen.<p><b>Copyright information:</b></p><p>Taken from "Arabidopsis mRNA polyadenylation machinery: comprehensive analysis of protein-protein interactions and gene expression profiling"</p><p>http://www.biomedcentral.com/1471-2164/9/220</p><p>BMC Genomics 2008;9():220-220.</p><p>Published online 14 May 2008</p><p>PMCID:PMC2391170.</p><p></p

Normalized expression data for the NASC Arabidopsis chemical/hormone series (Additional file ) were extracted and plotted as shown

The legends indicate the correspondence between the plots and the respective Arabidopsis gene identification designation. The numerical key for each array experiment is given along the X-axis, and the detail can be found in Additional file . The single arrows indicate the position for cycloheximide; double arrows for GA mutants; empty arrows for imbibition and ABA treatment.<p><b>Copyright information:</b></p><p>Taken from "Arabidopsis mRNA polyadenylation machinery: comprehensive analysis of protein-protein interactions and gene expression profiling"</p><p>http://www.biomedcentral.com/1471-2164/9/220</p><p>BMC Genomics 2008;9():220-220.</p><p>Published online 14 May 2008</p><p>PMCID:PMC2391170.</p><p></p

The values for each gene in the array analysis of mature pollen were plotted as shown

<p><b>Copyright information:</b></p><p>Taken from "Arabidopsis mRNA polyadenylation machinery: comprehensive analysis of protein-protein interactions and gene expression profiling"</p><p>http://www.biomedcentral.com/1471-2164/9/220</p><p>BMC Genomics 2008;9():220-220.</p><p>Published online 14 May 2008</p><p>PMCID:PMC2391170.</p><p></p

Normalized expression data for the NASC Arabidopsis abiotic stress series (Additional file ) were extracted and plotted as shown

The legends indicate the correspondence between the plots and the respective Arabidopsis gene identification designation. The numerical key for each array experiment is given along the X-axis and the detail can be found in Additional file . Here is a brief list of the stress treatments: 1–18, control; 19–30, cold; 31–42, osmotic; 43–54, salt; 55–68, drought; 69–80, genotoxic; 81–92, oxidative; 93–106, UV-B; 107–120, wound; 121–136, heat; 137–141, cell culture control; 142–149, cell culture + heat.<p><b>Copyright information:</b></p><p>Taken from "Arabidopsis mRNA polyadenylation machinery: comprehensive analysis of protein-protein interactions and gene expression profiling"</p><p>http://www.biomedcentral.com/1471-2164/9/220</p><p>BMC Genomics 2008;9():220-220.</p><p>Published online 14 May 2008</p><p>PMCID:PMC2391170.</p><p></p