25-30 August 2019
Henry Ford Building
Europe/Berlin timezone

Solution structure of Upstream-of-N-Ras, a 116 kDa multi-domain RNA binding protein

28 Aug 2019, 11:30
25m
Max Kade Auditorium (Henry Ford Building)

Max Kade Auditorium

Henry Ford Building

Talk Biological applications Biomolecules

Speaker

Dr Janosch Hennig (Structural and Computational Biology Unit, EMBL Heidelberg)

Description

The protein Upstream-of-N-Ras (Unr) is a highly conserved and abundant RNA binding protein with elevated expression levels in several cancer types. Here, it is supposed to bind certain mRNAs to regulate their translation. In Drosophila, Unr acts an RNA chaperone, where it binds to long non-coding RNA RoX2 to promote formation of the dosage compensation complex. In all hitherto publications concerning Unr it has been proposed that it features five cold shock domains (CSDs), which are known for binding single-stranded RNAs. These domains are presumably connected with flexible linkers.
However, our construct optimizations and NMR analysis revealed the presence of altogether nine CSDs. Interestingly, the predicted canonical CSDs are interspersed with novel non-canonical CSDs, which share the same fold with the canonical ones, but lack their otherwise conserved trademark RNA binding residues. Structure determination of several tandem and triple domain constructs by NMR and X-ray crystallography revealed that there are no flexible linkers and that the domains have indeed a fixed orientation towards each other. This indicates that Unr, although binding to single-stranded RNAs via its CSDs, achieves also RNA structure specificity due to the fixed orientations and distances of canonical CSDs.
Complementing the structures with small-angle X-ray scattering and further NMR data we were able to obtain a high-resolution structural ensemble of the full-length protein. Additional mutational analysis using biochemical, biophysical and cellular assays confirm the importance of inter-domain arrangements for RNA specificity and function.
This study presents a new paradigm of how a general single-stranded RNA binding protein achieves RNA structure specificity.

Primary authors

Ms Nele Merret Hollmann (Structural and Computational Biology Unit, EMBL Heidelberg) Dr Pravin Kumar Ankush Jagtap (Structural and Computational Biology Unit, EMBL Heidelberg) Ms Tanit Guitart (Stem Cells and Cancer Programme, Gene Regulation, Centre for Genomic Regulation (CRG) Barcelona) Dr Pawel Masiewicz (Structural and Computational Biology Unit, EMBL Heidelberg) Ms Lara Sweetapple (Structural and Computational Biology Unit, EMBL Heidelberg) Dr Bernd Simon (Structural and Computational Biology Unit, EMBL Heidelberg) Dr Fátima Gebauer (Stem Cells and Cancer Programme, Gene Regulation, Centre for Genomic Regulation (CRG) Barcelona) Dr Janosch Hennig (Structural and Computational Biology Unit, EMBL Heidelberg)

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