title: Structure and dynamics of Upstream of N Ras and their influence on RNA binding and translation regulation
creator: Hollmann, Nele Merret
subject: 500
subject: 500 Natural sciences and mathematics
description: The interplay of multiple RNA binding domains (RBD) in a single RNA binding protein (RBP) to achieve RNA target specificity is far from being understood. In this thesis, a multidisciplinary study of Upstream of N-Ras (Unr) is presented. Unr is an RBP that has been predicted to contain five single-stranded RNA binding cold shock-domains (CSD). The thesis aims to unravel how Unr binds RNA targets specifically in a cellular context.   Several NMR and crystal structures of multidomain constructs were determined. As a result, four non canonical CSDs in addition to the five previously known canonical CSDs were discovered. These non-canonical CSDs play a scaffolding role between the canonical domains, but do not bind RNA independently. Using NMR relaxation and small angle X-ray scattering, it could be shown that the linker between most of the canonical and non-canonical domains is rigid, leading to a restricted flexibility of the full-length protein. Different in vitro and cellular mutational studies, including a reporter gene assay and a RIP seq experiment showed that a disruption of the fixed domain arrangement has influence on RNA recognition and the protein function.  Additionally, a crystal structure of a multidomain construct of Unr bound to poly-A RNA provides further information on the complexity of the multidomain RNA binding mechanism of Unr. Several non-canonical binding residues, some even in the non-canonical CSDs, contribute to cooperative RNA binding, suggesting that RNA binding of the full-length protein is likely to be of even higher complexity and plasticity.   Further insights into Unr-RNA binding are provided by a full-length protein model, that describes a restricted flexibility of the protein, which might play an essential role within target specificity. To expand the studies towards Unr-ribonucleotide complexes a quantitative mass spectrometry analysis was conducted, that defined several Unr interactors. The protein-protein interaction with the top hit of this result, namely pAbP, was further characterized, which paves the way towards future structure analysis of a larger translation repressor complex, as it assembles on the 3’ UTR of the msl2 mRNA.
date: 2021
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/29140/1/thesis_Nele-Hollmann.pdf
identifier: DOI:10.11588/heidok.00029140
identifier: urn:nbn:de:bsz:16-heidok-291408
identifier:   Hollmann, Nele Merret  (2021) Structure and dynamics of Upstream of N Ras and their influence on RNA binding and translation regulation.  [Dissertation]     
relation: https://archiv.ub.uni-heidelberg.de/volltextserver/29140/
rights: info:eu-repo/semantics/openAccess
rights: http://archiv.ub.uni-heidelberg.de/volltextserver/help/license_urhg.html
language: eng