title: Proteomic Exploration of Protein-RNA Interactions in Human Cells
creator: Trendel, Jakob
description: The interactions of proteins with RNA are ubiquitous in human cell biology and fundamental  to most if not all of life as we know it. Proteins and RNA interact for the purpose of gene  expression, where genetic information is encoded within RNA, which serves as template for  protein in messenger RNA, as scaffold for the trimming of other RNA within the spliceosome,  as a catalyst for the production of protein within the ribosome, and as component of various  other processes, many of which we are only beginning to understand. Central to our  understanding of such processes is the interplay between proteins and RNA. Protein-RNA  interactions are often disrupted by conventional extraction methods and need to be stabilized  through crosslinking in order to be appreciated by transcriptomic or proteomic methodology.  Crosslinking typically occurs through UV irradiation, which covalently connects protein bound  to RNA. However, currently there is no universal method for the extraction of proteincrosslinked  RNA available, so that insights can only be gained for certain subsets of the  transcriptome or individual RNA-binding proteins, respectively. In this thesis a new method  for the extraction of protein-crosslinked RNA from UV-crosslinked cells is described. In order  to demonstrate the comprehensive usefulness of the approach, which was termed XRNAX, it  is applied for the resolution of several proteomic and transcriptomic problems. First, XRNAX  is employed for the purification of ribonucleotide-crosslinked peptides identifying known and  unknown protein-RNA interfaces. Next, a comprehensive draft for the human RNA-binding  proteome is derived applying XRNAX to three commonly used cell lines. The same approach  is applied for the differential quantification of RNA-binding during a timeline of arseniteinduced  translational arrest in human cells. Additionally, XRNAX is combined with CLIPseq to  monitor the RNA exosome component EXOSC2 processing pre-ribosomal RNA during arsenite  stress. At last, XRNAX is used along with TMT-SILAC in order to derive protein half-lives of the  human RNA-bound proteome. Half-lives of the RNA-bound proteome are found on average  1.75 fold increased in comparison to the total proteome. For validating the stabilization of  protein-RNA complexes, ribosomal assemblies are purified from human cells using polysome  profiling and protein half-lives assessed with TMT-SILAC. Stabilization is confirmed, however,  only for ribosomal proteins in 80S ribosomes within the 80S or polysome fractions. Further  experiments interfering with ribosome biogenesis, the proteasome and autophagy are  distilled into a model for the turnover of ribosomal proteins and ribosomal assemblies.  In summary, this thesis describes new methodology and biological insight on protein-RNA  interactions in human cells.
date: 2019
type: Dissertation
type: info:eu-repo/semantics/doctoralThesis
type: NonPeerReviewed
format: application/pdf
identifier: https://archiv.ub.uni-heidelberg.de/volltextserverhttps://archiv.ub.uni-heidelberg.de/volltextserver/26436/1/Trendel_Dissertation.pdf
identifier: DOI:10.11588/heidok.00026436
identifier: urn:nbn:de:bsz:16-heidok-264362
identifier:   Trendel, Jakob  (2019) Proteomic Exploration of Protein-RNA Interactions in Human Cells.  [Dissertation]     
relation: https://archiv.ub.uni-heidelberg.de/volltextserver/26436/
rights: info:eu-repo/semantics/openAccess
rights: http://archiv.ub.uni-heidelberg.de/volltextserver/help/license_urhg.html
language: eng