%0 Generic
%A Hollmann, Nele Merret
%C Heidelberg
%D 2021
%F heidok:29140
%R 10.11588/heidok.00029140
%T Structure and dynamics of Upstream of N Ras and their influence on RNA binding and translation regulation
%U https://archiv.ub.uni-heidelberg.de/volltextserver/29140/
%X 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.