Roquin recognizes a non-canonical hexaloop structure in the 3'-UTR of Ox40.

Robert Janowski,Nina Wommelsdorf,Dierk Niessing,Andreas Schlundt,Mihaela Zavolan,Thorsten Buch,Michael Sattler,Gitta A Heinz,Andreas R Gruber,Sven Brenner,Vigo Heissmeyer,Raymund Buhmann,Michael Blank

doi:10.1038/ncomms11032

Abstract

The RNA-binding protein Roquin is required to prevent autoimmunity. Roquin controls T-helper cell activation and differentiation by limiting the induced expression of costimulatory receptors such as tumor necrosis factor receptor superfamily 4 (Tnfrs4 or Ox40). A constitutive decay element (CDE) with a characteristic triloop hairpin was previously shown to be recognized by Roquin. Here we use SELEX assays to identify a novel U-rich hexaloop motif, representing an alternative decay element (ADE). Crystal structures and NMR data show that the Roquin-1 ROQ domain recognizes hexaloops in the SELEX-derived ADE and in an ADE-like variant present in the Ox40 3′-UTR with identical binding modes. In cells, ADE-like and CDE-like motifs cooperate in the repression of Ox40 by Roquin. Our data reveal an unexpected recognition of hexaloop cis elements for the posttranscriptional regulation of target messenger RNAs by Roquin.

Highlights

The RNA-binding protein Roquin is required to prevent autoimmunity
We have recently reported structural and functional data of the Roquin-1 ROQ domain bound to a canonical constitutive decay element (CDE), a short stem loop (SL) that acts as a cis-regulatory RNA element in the 30-untranslated regions (30-UTRs) of target genes such as Tnf
Bioinformatic analysis of Next-generation sequencing (NGS) data sets derived from the starting pool and enriched selection rounds revealed that the complexity was reduced to 78.6% unique reads in 3.7 Â 106 sequences that were analysed after 3 rounds of selection and enrichment

Summary

Introduction

The RNA-binding protein Roquin is required to prevent autoimmunity. Roquin controls T-helper cell activation and differentiation by limiting the induced expression of costimulatory receptors such as tumor necrosis factor receptor superfamily 4 (Tnfrs[4] or Ox40). We have recently reported structural and functional data of the Roquin-1 ROQ domain bound to a canonical constitutive decay element (CDE), a short stem loop (SL) that acts as a cis-regulatory RNA element in the 30-untranslated regions (30-UTRs) of target genes such as Tnf (ref 11). In addition to this mechanism, the composition and binding affinity of cis-regulatory SL elements in the 30-UTRs of target mRNAs may determine the sensitivity to repression by the trans-acting factor Roquin. We present structural and functional evidence for a greatly expanded repertoire of RNA elements that are regulated by Roquin as demonstrated with a novel U-rich hexaloop SL in the 30-UTR of Ox40 bound to the Roquin-1 ROQ domain. Our X-ray crystallographic, NMR, biochemical and functional data combined with mutational analysis demonstrate that both triloop and hexaloop SL RNAs contribute to the functional activity of Roquin in T cells

Methods

Results

Conclusion