Characterisation of human ribosome assembly factors

by Merle Klein Helmkamp
Doctoral thesis
Date of Examination:2023-11-22
Date of issue:2023-11-09
Advisor:Prof. Dr. Markus Bohnsack
Referee:Prof. Dr. Dörthe Katschinski
Referee:Prof. Dr. Margarete Schön
Persistent Address: http://dx.doi.org/10.53846/goediss-10187

 

 

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Abstract

English

Ribosomes are highly conserved ribonucleoprotein complexes for translating the genetic code encoded in mRNAs into functional proteins. In consist of a small and large subunit containing ribosomal RNAs as well as ribosomal proteins. Their biogenesis pathway which is best studied in yeast is an energy-consuming process linked to cell stress, cell proliferation and nutrient availability. During assembly, a vast number of RNA processing and remodelling events as well as incorporation of ribosomal proteins are taking place, mostly dependent on transacting biogenesis factors. Some of these factors have enzymatic activity such as kinases, RNA helicases, ATPases, nucleases or GTPases. Nog1, Nug1 and Nug2 are GTPases known to be involved in ribosome biogenesis in yeast with largely uncharacterised human homologues: GTPBP4, GNL3L and GNL2. Additionally, a new candidate potentially involved in ribosome biogenesis based on its nucleolar localisation but without a yeast homologue was found: GNL3. This work provides evidence for involvement in human ribosome biogenesis not only for the proteins with known yeast homologues but also for GNL3. Sucrose density centrifugation confirmed co-migration of all of these proteins with (pre-)ribosomal particles, in addition to RNA-binding ability shown for GTPBP4 and GNL3L. Pre-rRNA processing defects of the LSU as well as SSU were detected for these proteins using RNAi-mediated depletion. For GTPBP4, GNL3 and GNL2 depletion not only led to processing defects of rRNA precursors but to a lack of mature 28S rRNA and in the case of GNL3 and GNL2, to an additional decrease in synthesis of mature 18S rRNA. This study also involved the recombinant expression of these human proteins in E. coli and their purification. GTPBP4 was chosen as a representative GTPase and employed for establishing in vitro GTPase assays to characterise enzymatic activity. Together, this study defines GTPBP4, GNL2, GNL3 and GNL3L as human ribosome biogenesis factors and provides hints to their possible functions in this pathway that can be the basis of future work.
Keywords: Ribosome; GTPase; Ribosome assembly