Universität zu Köln

Kückelmann, Sabrina ORCID: 0000-0001-8070-8823 (2024). SMG proteins license and execute mRNA degradation via nonsense-mediated mRNA decay in human cells. PhD thesis, Universität zu Köln.

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Abstract

The cell undergoes various processes such as proliferation, differentiation, and intracellular adjustments due to changes of external conditions. For the adaption to these states precise regulation of gene expression is necessary. Nonsense-mediated mRNA decay (NMD) is a translation-dependent quality control mechanism in eukaryotes. Although NMD is best known for degrading transcripts harboring premature termination codons (PTC), NMD was additionally found to regulate gene expression via targeting physiological transcripts. Phosphorylation of the central NMD factors UPF1 via the SMG1:SMG8:SMG9 complex is a key step during NMD initiation. The kinase SMG1, responsible for UPF1 phosphorylation, is proposed to be negatively regulated by the SMG8 C-terminus and SMG9. Upon phosphorylation, the N- and C-terminal tails of UPF1 function as binding platforms for the decay-inducing factors SMG5:SMG7 and SMG6. Subsequently, the SMG7 C-terminus initiates deadenylation via the recruitment of the CCR4-NOT complex, and the endonuclease SMG6 cleaves the transcript in the vicinity of the PTC. While extensive studies were conducted over the last years, the exact biological mechanism remains undisclosed. Therefore, the aim of this thesis was to examine the regulation of the SMG1:SMG8:SMG9 complex and to investigate the connection of the SMG5:SMG7 and SMG6 pathways in human cultured cells. The regulatory function of SMG8 for SMG1 kinase activity was tested via the deletion of the SMG8 C-terminus. However, cells lacking the SMG8 C-terminus resulted in unchanged NMD activity. Furthermore, the depletion of SMG8 and SMG9 led to minor NMD inhibition and unchanged UPF1 phosphorylation, questioning their regulatory role for SMG1. Nevertheless, treatment with the SMG1 inhibitor SMG1i revealed hypersensitivity of SMG8- or SMG9-deleted cells compared to WT cells: Transcriptome-wide analysis showed an enrichment of NMD-annotated transcripts demonstrating that SMG8 and SMG9 contribute to the robustness of the NMD machinery. Degradation via the SMG5:SMG7 and SMG6 pathway was proposed to be redundant and independent. However, the loss of the SMG5:SMG7 led to the inactivation of SMG6, revealing a functional dependence between both pathways. Transcriptome-wide analysis of SMG5:SMG7-depleted cells revealed severe NMD impairment. Interaction studies demonstrated the intact SMG6-UPF1 binding under these conditions, showing that SMG5:SMG7 are crucial for SMG6 activation. Furthermore, complete NMD abolishment resulted in an accumulation of stalled NMD complexes, suggesting that endonucleolytic cleavage is required for the dissociation of the NMD machinery. Taken together, these data provide comprehensive insights into the SMG1:SMG8:SMG9 complex and the interplay between SMG5:SMG7 and SMG6. Furthermore, these data support an improved model, which consists of two consecutive authentication steps to active SMG6 endonucleolytic activity including UPF1 phosphorylation via SMG1:SMG8:SMG9 and recruitment of SMG5:SMG7.

Item Type:	Thesis (PhD thesis)
Creators:	Creators Email ORCID ORCID Put Code Kückelmann, Sabrina s.kueckelmann@gmx.de orcid.org/0000-0001-8070-8823 UNSPECIFIED
URN:	urn:nbn:de:hbz:38-732868
Date:	2024
Language:	English
Faculty:	Faculty of Mathematics and Natural Sciences
Divisions:	Faculty of Mathematics and Natural Sciences > Department of Biology > Institute for Genetics
Subjects:	Life sciences
Uncontrolled Keywords:	Keywords Language Nonsense-mediated mRNA decay; RNA decay; SMG1; SMG5; SMG6; SMG7; SMG8; SMG9; UPF1 English
Date of oral exam:	18 July 2024
Referee:	Name Academic Title Gehring, Niels H. Prof. Dr. Hofmann, Kay Prof. Dr.
Refereed:	Yes
URI:	http://kups.ub.uni-koeln.de/id/eprint/73286