Universität zu Köln

Gerbracht, Jennifer Victoria ORCID: 0000-0002-3377-4649 (2020). Analysis of post-transcriptional gene expression modulated by mRNA stability and RNA-binding proteins in human cells. PhD thesis, Universität zu Köln.

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Abstract

The output of gene products needs to be regulated in order for cells to function in development and react to signals or stress. As a first step of gene expression, genes are transcribed into RNA. Messenger RNAs (mRNAs) are produced which carry the genetic information from the DNA to ribosomes, the sites of protein biosynthesis in the cell. Also other RNA species are transcribed that perform regulatory or catalytical functions within the gene expression machinery. All RNAs in the cell undergo processing and quality control as soon as they are transcribed. Furthermore, they are bound by RNA binding proteins, forming messenger ribonucleoproteins (mRNPs). This process of post-transcriptional gene regulation ensures that the produced RNA species are stable and localize to the correct subcellular department. In this cumulative thesis three distinct aspects of post-transcriptional gene regulation are investigated. In the first part the processing of ribosomal RNAs (rRNAs) is analyzed. As the most abundant RNA species in the cell, mature rRNAs are derived from a polycistronic precursor RNA that undergoes cleavage and modification before being exported into the cytoplasm with ribosomal proteins, thus forming mature ribosomal subunits. A myriad of processing factors has been described which are involved in rRNA maturation. Here, a link between the ubiquitin E3 ligase UBR5 and the ribosomal processing machinery is identified in human embryonic stem cells. UBR5 interacts with components of the box H/ACA snoRNP which carries out pseudouridylation modifications of rRNAs. It is shown that depletion of UBR5 in stem cells impairs rRNA maturation and ribosomal biosynthesis as a whole. The second part of this thesis is focused on the exon junction complex (EJC) which is an essential RNA-binding complex in metazoan cells. The EJC is deposited during splicing upstream of exon-exon junctions of mRNAs and stays bound until it is removed during translation. During the lifetime of an mRNA, the EJC fulfills critical functions in splicing, mRNA export and translation. It is also important for the mRNA surveillance pathway nonsense-mediated mRNA decay (NMD). The assembly of the individual EJC components is intimately linked to the splicing process, however the exact dynamics and interactions at play are not fully understood. The recently published structures of the human spliceosome have given insights into the positioning of EJCs components within spliceosome intermediates. These advances are discussed in the thesis and conclusions for further studies are drawn. Next, a new function of the EJC in connection with splicing is described: after its deposition, the EJC together with associated splicing factors inhibits the usage of potential splice sites in the transcriptome, thereby maintaining splicing fidelity. Since this function is only observed for three of the four described core EJC components, the molecular function of the fourth EJC component CASC3 is investigated. Using CRISPR-Cas9-generated knockout cells it is shown that CASC3 is important for the function of the EJC in the NMD pathway. Finally, in the last part of the thesis the process of mRNA turnover is described. All mRNAs eventually undergo degradation in order to maintain dynamics in gene expression. Furthermore, faulty mRNAs are targeted by quality control pathways which leads to their degradation. Finally, mRNAs such as cytokine transcripts exist that need to be tightly controlled and are therefore unstable. The analysis of mRNA turnover is challenging, since different nucleolytic pathways can act on the degradation of transcripts. Here, a novel method is presented that employs viral structures which are resistant to the human 5′-3′ exonuclease XRN1. By using these structures in unstable reporter transcripts, the participation of exo- and endonucleolytic degradation can be determined. This approach is also described in a protocol format and can be used to monitor the degradation of mRNAs in single cells using live-cell imaging.

Item Type:	Thesis (PhD thesis)
Creators:	Creators Email ORCID ORCID Put Code Gerbracht, Jennifer Victoria jennifer.gerbracht@gmx.de orcid.org/0000-0002-3377-4649 UNSPECIFIED
URN:	urn:nbn:de:hbz:38-107179
Date:	2020
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 RNA turnover UNSPECIFIED Nonsense-mediated mRNA decay UNSPECIFIED Splicing UNSPECIFIED mRNA binding proteins UNSPECIFIED Exon junction complex UNSPECIFIED RNA biology UNSPECIFIED
Date of oral exam:	6 February 2020
Referee:	Name Academic Title Gehring, Niels Prof. Dr. Hofmann, Kay Prof. Dr. Wahle, Elmar Prof. Dr.
Refereed:	Yes
URI:	http://kups.ub.uni-koeln.de/id/eprint/10717