Inhaltszusammenfassung

For less than a decade, high-throughput sequencing became a very powerful, sensitive and precise technique for the study of ribonucleic acids. During my PhD thesis, I have used this technology for in-depth characterization of the extracellular RNA (exRNA) content of human plasma. exRNA in plasma exists either in a “soluble state” as a component of ribonucleoprotein (RNP) complexes or encapsulated into extracellular vesicles (EV) of diverse origins (exosomes, microvesicles, …). In this project...For less than a decade, high-throughput sequencing became a very powerful, sensitive and precise technique for the study of ribonucleic acids. During my PhD thesis, I have used this technology for in-depth characterization of the extracellular RNA (exRNA) content of human plasma. exRNA in plasma exists either in a “soluble state” as a component of ribonucleoprotein (RNP) complexes or encapsulated into extracellular vesicles (EV) of diverse origins (exosomes, microvesicles, …). In this project, I have demonstrated that whole human plasma contains mostly micro RNAs and the fragment of RNA hY4, as well as degraded ribosomal RNA. Moreover, using a rigorous strategy via size exclusion chromatography or consecutive proteinase K/RNase A treatments, highly purified EVs can be obtained. miRNAs and RNA hY4 fragments were nor present in majority of samples, demonstrating a huge difference between soluble exRNA and exRNA from purified EVs. The RNA content of these EVs mainly reflects RNA composition of human microbiota. In addition, I have also performed a comparative analysis of commercially available “exosome-enrichment” kits which supposed to purify human exosomes by precipitation. RNA composition of such fractions was found to be almost identical to human plasma, showing strong uncontrolled contamination by soluble RNPs. Based on this study, we were able to propose a protocol for studies in exRNA in the field of liquid biopsies with clinical sample in order to discover new diagnostic biomarkers. Apart from the characterization of RNA, high-throughput sequencing can be used for detection and quantification of RNA post-transcriptional modifications. During my PhD thesis I have applied deep sequencing for analysis of transfer RNA (tRNA) 2’-O-methylations in model bacteria (E. coli) using RiboMethSeq. Under several stress conditions, such as starvation and non-lethal antibiotics concentrations, some 2’-O-methylated nucleotides show an adaptive response. While over than half of Gm18 show a global increase under all investigated stress conditions, ribomethylated residues at position 34 show an opposite effect for some antibiotic treatments: mostly for chloramphenicol and streptomycin. Each of these dynamic profiles can be linked to cell regulation in response to stress. Change at the tRNA wobble base (position 34) could be a way to regulate translation by modifying the codon usage. Concerning Gm18, its role in the escape from the human innate immune system during host invasion is currently elucidated.» weiterlesen» einklappen

Autoren

Klassifikation

DDC Sachgruppe:
Biowissenschaften, Biologie