Oral Presentation Australian & New Zealand Society of Magnetic Resonance Conference 2017

Disentangling the many RNA interactions of FUS using NMR Spectroscopy- ways and means (#7)

Fionna Loughlin 1 2 , S Campagne 1 , T Kazeeva 1 , M D Ruepp 3 , P J Lukavsky 1 , F. H-T Allain 1
  1. Institute of Molecular Biology and Biophysics, ETH, Zurich, Switzerland
  2. Current address: Biochemistry and Molecular Biology, Monash University, Melbourne, Victoria, Australia
  3. Chemistry and Molecular Biology, University of Bern, Bern, Switzerland

FUsed in Sarcoma (FUS) is a multifunctional hnRNP protein which regulates transcription, pre-mRNA splicing, miRNA biogenesis, DNA damage and can drive subcellular phase transitions. Two neurodegenerative diseases Amyotrophic Lateral Sclerosis (ALS) and Fronto-Temporal Lobar Degeneration (FTLD) show neuropathological protein aggregates containing FUS and it is hypothesized that mis-regulation of RNA processing could play a major role in these diseases1. We are investigating the molecular mechanisms by which FUS recognizes RNA. FUS consists of a C-terminal prion-like region and an N-terminal RNA/DNA binding region including an RNA Recognition Motif (RRM) and a zinc-finger (ZnF) domain interspersed between methylated RGG repeats. FUS associates with a large variety of RNAs2 including pre-mRNA, pri-miRNA, lncRNA from CCND1 promoter and TERRA. Differing results from in vivo (CLIP) and in vitro (SELEX, binding assays) suggest that RNA binding by FUS is complex potentially combining structural, sequence and nonspecific interactions with additional influences from post-translational modifications and protein partners. Despite intense interest, to date there is not structural information on how FUS interacts with RNA. We present the solution structures of FUS RRM and ZnF domains bound to RNA and discuss the particular challenges associated with the structural analysis of protein-RNA interactions and the strategies that can be used to maximize data quality required for de-novo structure determination. The RRM domain bound to a stem-loop RNA3 shows an unusual binding mode in which the RRM binds in the 3’ loop region using the β-sheet and C-terminal tail with limited sequence specificity and an α1-β2 extension unique to FUS and two of its family members EWS and TAF15, contacts the stem-loop junction. The ZnF shows a sequence specific interaction with an NGGU motif and this interaction likely accounts for the preference for GU rich motif detected in CLIP based experiments. Additional studies show that the RRM and ZnF can coordinate in binding a bi-partite RNA site with contributions from RGG repeat motifs and that both domains contribute to minor intron splicing events. These results give the first insights into the structural basis of RNA recognition by FUS.

  1. 1. Shang Y. and Huang E.J. (2016) Brain Res. 1647:65-78 2. Schwartz J.C. (2015) Annu Rev Biochem 84:355-379 3. Hoell, J.I. et al. (2011) Nat Struct Mol Biol 18:1428-31.