Browsing by Subject "Oxidative stress"

Post-transcriptional modifications (PTMs) in RNA are present in all known RNA species and conserved in all kingdoms of life. Transfer RNA (tRNA) has been shown to have numerous conserved modifications, which exemplifies the importance of modifications having impact on the structure of the tRNA and its function as carrier of the amino acids. Ribosomal RNAs (rRNA) are universally modified as well, and modifications are situated at functionally important spots of the ribosome. Given the fact that types and sites of modifications are conserved, it is likely that these modifications have been selected for and that they optimize the ribosomal structure and functions. Stress, such as temperature or infection by a pathogen, is known to change the presence or abundance of modifications in RNA molecules and thereby affect translation efficacy. In line with that, this master’s thesis project sought to gain insight into the dynamics of PTMs in tRNA and rRNA upon oxidative stress, with the goal of utilizing recently optimized UPLC/MS method for identifying modified ribonucleosides. As the specific aim of the thesis was to estimate the change in PTMs in tRNA and rRNA in response to oxidative stress with 0.5 mM and 2 mM hydrogen peroxide H2O2, 3 immediate goals were: (i) to isolate total tRNA from yeast grown in stress conditions, (ii) to isolate rRNA from yeast 80S ribosomes, and (iii) to identify present modifications using mass spectrometry. Yeast was cultured in presence of H2O2 as a stressor in mentioned concentrations, and both treatments considered showed a difference in survival when compared to the control. Rough cell concentration estimates (OD600) did not show the effect of the stressor on cell survival clearly, but when number of viable cells per mL was estimated, it was clear that growth of the stressed yeast cultures was hindered 2 hours after exposure to H2O2 but recovered during the 24 hours. Firstly, using UPLC/MS analysis, 29 modifications were identified in tRNA from control and H2O2 treated yeast. Most identified modifications showed no change in abundance in treatments, which is to be verified with additional replicates. However, distinct dynamics of stress-related change was found for several modifications, revealing additional modifications that may play a role in stress related modificome reprogramming to the previously known signature modifications of oxidative stress. It was expected that recovery of culture growth after 24 hours may be accompanied with modification level recovery. However, that was not demonstrated here as downregulation at 2 hours followed by upregulation at 24 hours was seen for 2-methylthio-N6-methyladenosine, N4-acetylcytidine and 5-methoxycarbonylmethyl-2-thiouridine, and the reverse was shown for N4-methylcytidine. Upregulation in both time points was also shown here for some modifications. Taken together, these results confirm a complex and dynamic control of tRNA modifications in cellular survival responses. Modifications found to be affected by oxidative stress are most frequently located on the wobble position 34 and anticodon loop position 37, so it is expected that changes in their modification levels could directly affect the tRNA function in translation, making them a specific target for future research. Secondly, modifications in rRNA from control yeast cultures were identified, such as expected methylations of all 4 canonical nucleosides. However, further analysis will be needed to confirm the other identified modifications, due to the potential mRNA and tRNA contamination. Optimizing the method for rRNA modifications identifications by acquiring more modified nucleosides specific for the rRNA to use as standards in the analysis, analyzing rRNA types separately and using tandem mass spectrometry would enable getting a deeper understanding of which modifications are present and where they are positioned. Finally, it would enable reliable identification of the signals of novel modifications present in rRNA, such as the tRNA modification 5-carbamoylmethyluridine signal found here. In conclusion, this thesis work lays the foundation to study the evolutionary conserved function of PTM changes during stress as modulators of translation, using the methodological approaches discussed in-depth within the thesis, primarily to confirm the intriguing results found here.

Transfer RNA (tRNA) is one of the most extensively modified RNA molecules. The role of tRNA modifications become apparent during physiological condition such as oxidative stress, where it serves as an adaptive response to the changing environment. These modifications are upregulated mainly at the wobble position of the tRNA to enhance the translational efficiency of the stress response genes through enhanced decoding rate and tRNA–mRNA interaction. Hence, tRNA modification has a crucial role in regulating translational fidelity, and such modifications can be utilized to fine-tune the translation for improved production of heterologous protein. Therefore, this study aimed to analyze the tRNA modification changes in two laboratory-significant E. coli strains (BL21 (DE3) and K12) during oxidative stress and utilize these modifications to enhance the production of heterologous protein using a defined cell-free protein synthesis system. Ultra-performance liquid chromatography-mass spectrometry was used to detect and quantify the tRNA modification changes in the hydrogen peroxide-treated E. coli cells. The results showed unique tRNA modification patterns and intensities between the two bacterial strains in response to oxidative stress. Modifications such as ac4c and m2,2G were upregulated in E. coli BL21 (DE3) following hydrogen peroxide treatment, whereas k2C and chm5U were increased in E. coli K12. Further analysis of the dataset revealed that most of the upregulated ribonucleoside modifications were predominant at the anticodon loop of the tRNAs, indicating the potentiality of these tRNA pools to impact on translation. Likewise, I optimized an E. coli-based cell-free protein synthesis system to investigate the effect of modified tRNA pools on translation. Hence, this study serves as a stepping stone to understand the tRNA modification landscape of E. coli and provides a platform to depict the function of post-transcriptional tRNA modifications in translation with the CFPS system.