Structural studies of 5´ UTR of Hepatitis C viral RNA by NMR-based structural biology

verfasst von

Olga Alexandrovna Vitsyna

betreut von

Andreas Kirschning

Abstract

RNA is a single-stranded biopolymer that plays a myriad of roles in physiological and pathological processes and is the carrier of genetic information in many human pathogens. Hepatitis C virus (HCV) is one of the most impactful representatives of RNA viruses. Liver-abundant human microRNA-122 (miR-122) binds to two tandem sites within domain I of the 5´ untranslated region (5´ UTR) of HCV, ultimately resulting in upregulation of viral propagation. Despite many studies of the interaction between HCV and miR-122, the exact mechanism by which this recognition event leads to increased viral propagation is unknown. In this thesis, I have studied the 5´ UTR HCV–miR-122 interaction at different levels of structural complexity (domain I, domains I-II and the full 5´ UTR) using an integrative NMR-based structural biology approach. First, I have performed the near-complete assignment of domain I resonances and determined its secondary structure. Isolated domain I binds two copies of miR-122 with different affinities, and the binding kinetics fall into the slow-to-intermediate exchange-regime on the NMR chemical-shift timescale. Magnesium ions promote structural rearrangement of domain I, which in turn changes its interaction pattern with miR-122. Next, I have determined the secondary structures of the isolated domain II and a domain I-II construct, both in their apo (without miR-122) and holo (bound to miR-122) states. The data demonstrates that, in the domain I-II construct, domains I and II maintain independent folds; furthermore, the secondary structure of domain II remains intact upon domain I binding two copies of miR-122. However, the binding of miR-122 to the domain I-II construct does lead to a structural rearrangement that changes the relative orientation of the two domains, resulting in more open and extended conformation. Finally, I have investigated the interaction of miR-122 with the full 5´UTR. Since the differences between the low-resolution scattering data of the 5´ UTR in the apo and holo states were minimal, no major structural changes in the 5´ UTR upon miR-122 binding appear to occur. To study the local structural details of the 5´ UTR, I have explored the use of solid-state NMR. While there were clear changes in chemical shifts of the 5´ UTR upon miR-122 binding, indicating conformational changes in the 5´ UTR, acquisition of solid-state NMR data on segmentally labeled samples and isolated domain I was challenging and could not provide definitive answers at this stage. Overall, using an NMR-based integrative structural biology approach, I could show that miR-122 binding to domain I causes both widespread local rearrangements within domain I and a significant reorientation of domain I relative to domain II, while the effect of miR-122 binding on the overall structure of the full 5’ UTR was found to be minimal.

Organisationseinheit(en)

Institut für Organische Chemie

Typ

Dissertation

Anzahl der Seiten

172

Publikationsdatum

2023

Publikationsstatus

Veröffentlicht

Ziele für nachhaltige Entwicklung

SDG 3 – Gute Gesundheit und Wohlergehen

Elektronische Version(en)

https://doi.org/10.15488/14877 (Zugang: Offen)