RNA in motion : NMR insights into ribosomal RNA dynamics

<p dir="ltr">RNAs perform many different tasks in biology. The ribosome is an RNA-protein complex which synthesises proteins based on the message from DNA. It consists of two subunits which associate during translation initiation and form non- covalent interactions, known as intersubunit bridges. rRNA helix 44 (h44) participates in several of the intersubunit interactions, some of which are purely rRNA-mediated.</p><p dir="ltr">The ribosome is a common antibiotic target. The rise of antibiotic resistance, along with side effects of existing antibiotics, highlights the need for research to explore alternative structural layers within rRNA as targets, and to compare them across species. During the COVID-19 pandemic, multiple ways to target the virus were studied, with the viral genome being one of them.</p><p dir="ltr">The first chapter introduces RNA structure and conformational dynamics, as well as the ribosome, which is the system under study in Paper I. The theory of NMR spectroscopy and its application to studying conformational dynamics of RNA is also introduced. The SARS-CoV-2 (SCoV2) viral RNA genome is introduced to contextualise Papers III and IV.</p><p dir="ltr">In Paper I, the work to characterise the dynamics at the intersubunit bridge B3 of the human cytosolic 18S rRNA h44 is described. We show a transient protonation of an adenine, as a result of a structurally altered pKa, and a guanine nucleobase flip about the glycosidic bond. As B3 is located at the pivot point of intersubunit rotation during translation and is important for subunit association, protonation at B3 may play a role in translation initiation. In Paper II, one of the workflows employed within Paper I is described. Paper II also provides a Python script pipeline to extract and visualise chemical shifts from different base pairs to aid in inferring excited-state structures from chemical shifts. In Paper III, we determine the secondary structure of conserved stem-loop elements of the SCoV2 genome by NMR spectroscopy and show the validity of studying RNA stem-loops in isolation by NMR. In Paper IV, we provide near-complete NMR resonance assignments of stem-loop 5a of the 5'-UTR, as a basis for further structure elucidation.</p><h3 dir="ltr">List of scientific papers</h3><p dir="ltr">I. <b>Riad, M.</b>, Marušič, M., Albers, M., Halbeisen, M., Montserrat-Canals, M., Steinmetzger, C. & Petzold, K. Visualizing a transiently protonated RNA state at intersubunit bridge B3 of human ribosomal RNA helix 44 by relaxation dispersion NMR spectroscopy. [Manuscript]</p><p dir="ltr">II. <b>Riad, M.</b>, Hopkins, N., Baronti, L., Karlsson, H., Schlagnitweit, J. & Petzold, K. Mutate-and-chemical-shift-fingerprint (MCSF) to characterize excited states in RNA using NMR spectroscopy. Nat Protoc. 16, 5146-5170 (2021) <a href="https://doi.org/10.1038/s41596-021-00606-1" rel="noreferrer" target="_blank">https://doi.org/10.1038/s41596-021-00606-1</a></p><p dir="ltr">III. Wacker, A., Weigand, J. E., Akabayov, S. R., Altincekic, N., Bains, J. K., Banijamali, E., Binas, O., Castillo-Martinez, J., Cetiner, E., Ceylan, B., Chiu, L .- Y., Davila-Calderon, J., Dhamotharan, K., Duchardt-Ferner, E., Ferner, J., Frydman, L., Fürtig, B., Gallego, J., Grün, J. T., Hacker, C., Haddad, C., Hähnke, M., Hengesbach, M., Hiller, F., Hohmann, K. F., Hymon, D., de Jesus, V., Jonker, H., Keller, H., Knezic, B., Landgraf, T., Löhr, F., Luo, L., Mertinkus, K. R., Muhs, C., Novakovic, M., Oxenfarth, A., Palomino-Schätzlein, M., Petzold, K., Peter, S. A., Pyper, D. J., Qureshi, N. S., <b>Riad, M.</b>, Richter, C., Saxena, K., Schamber, T., Scherf, T., Schlagnitweit, J., Schlundt, A., Schnieders, R., Schwalbe, H., Simba- Lahuasi, A., Sreeramulu, S., Stirnal, E., Sudakov, A., Tants, J .- N., Tolbert, B. S., Vögele, J., Weiß, L., Wirmer-Bartoschek, J., Wirtz Martin, M. A., Wöhnert, J. & Zetzsche, H. Secondary structure determination of conserved SARS-CoV-2 RNA elements by NMR spectroscopy. Nucleic Acids Res. 48, 12415-12435 (2020) <a href="https://doi.org/10.1093/nar/gkaa1013" rel="noreferrer" target="_blank">https://doi.org/10.1093/nar/gkaa1013</a></p><p dir="ltr">IV. Schnieders, R., Peter, S. A., Banijamali, E., <b>Riad, M.</b>, Altincekic, N., Bains, J. K., Ceylan, B., Fürtig, B., Grün, J. T., Hengesbach, M., Hohmann, K. F., Hymon, D., Knezic, B., Oxenfarth, A., Petzold, K., Qureshi, N. S., Richter, C., Schlagnitweit, J., Schlundt, A., Schwalbe, H., Stirnal, E., Sudakov, A., Vögele, J., Wacker, A., Weigand, J. E., Wirmer-Bartoschek, J. & Wöhnert, J. 1H, 13C and 15N chemical shift assignment of the stem- loop 5a from the 5'-UTR of SARS-COV-2. Biomol Nmr Assigm. 15, 203-211 (2021) <a href="https://doi.org/10.1007/s12104-021-10007-w" rel="noreferrer" target="_blank">https://doi.org/10.1007/s12104-021-10007-w</a></p>