Abstracts
Specific recognition and modulation of structured RNA decay cis elements by the immunoregulatory protein Roquin
Andreas Schlundt
Universität Greifswald, Analytische Biochemie, Friedrich-Ludwig-Jahn-Str. 17, 17489 Greifswald, Germany, andreas.schlundt@uni-greifswald.de
Cis-regulatory RNA elements in 3’-untranslated regions (UTRs) of mRNAs recruit trans-acting protein and miRNA factors for the control of transcript half-lives, a process crucial e.g. during immune response. It is assumed that sequence- and structure contexts of some folded cis elements have co-evolved together with regulatory RNA-binding proteins. While individual cis-trans pairs are often well understood, also on the structural level, the concerted recognition of cis element clusters in 3’UTRs by mRNA-regulatory proteins is challenging to study at high resolution. We have recently provided the full secondary and partial tertiary structure of the 157-nt Ox40 mRNA 3’UTR using a combination of NMR, SAXS, in-line-probing and SHAPE. Two mRNA-decaying stem-loop elements in the 3’UTR are bound by the 4-domain RNA-binding immune-regulatory 125-kDa protein Roquin for selective transcript suppression. The data suggest contributions from all three RNA-binding domains (RBDs), but their concerted action for selective Ox40 transcript recognition remains unresolved. We hypothesize a particular role for the mRNA cis-elements’ native context, which is likely not covered by the available structures. In line with that: How a cell guarantees the correct folding of complete 3’UTRs for gene regulation remains unknown.
Here, combining single-molecule force spectroscopy with NMR spectroscopy and biochemistry, we provide nucleotide-resolved insight into structure and folding pathways of the Ox40 mRNA 3’UTR and cis element modulation by the inter-connected Roquin RBDs. Using an integrated microfluidics setup, we extract binding kinetics to Roquin in real time in correlation with the folding states of regulatory RNA elements. Our work suggests previously unknown RNA-engaging mechanisms and functions of the RBDs and revises our understanding of target RNA recognition by Roquin. We also suggest that a central mRNA-decaying cis element has co-evolved with Roquin into a powerful cis-trans pair in immune regulation.