Abstract
Roquins are a family of highly conserved RNA-binding proteins that also contain a RING-type E3 ubiquitin ligase domain. They repress constitutive decay elements containing mRNAs and play a critical role in RNA homeostasis and immunological self-tolerance. Here we present the crystal structures of the RNA-binding region of Roquin paralog RC3H2 in both apo- and RNA-bound forms. The RNA-binding region has a bipartite architecture composed of ROQ and HEPN domains, and can bind to stem-loop and double-stranded RNAs simultaneously. The two domains undergo a large orientation change to accommodate RNA duplex binding. We profiled E2 ubiquitin-conjugating enzymes that pair with Roquins and found that RC3H1 and RC3H2 interact with two sets of overlapping but not identical E2 enzymes to drive the assembly of polyubiquitin chains of different linkages. Crystal structures, small-angle X-ray scattering, and E2 profiling revealed that while the two paralogs are highly homologous, RC3H2 and RC3H1 are different in their structures and functions. We also demonstrated that RNA duplex binding to RC3H2 cross-talks with its E3 ubiquitin ligase function using an in vitro auto-ubiquitination assay.
Highlights
RC3H1 and RC3H2 both repress the mRNAs of genes that control T follicular helper cell development, such as the inducible T-cell co-stimulator (ICOS), and the activation-induced tumor necrosis receptor superfamily 4 (TNFRS4)[10,11], they are not functionally redundant
The ubiquitination activity of the MEX-3C RING domain is required for the degradation of major histocompatibility complex (MHC) mRNA15; the E3 ubiquitin ligase/phosphatase complex MID1/PP2A binds to the expanded CAG repeat of huntingtin (HTT) mRNA and the ligase activity of MID1 regulates the translation of HTT mRNA16
The crystal contained a polypeptide of approximately 17 kDa, much smaller than expected (Fig. S1A), which is likely a proteolytic product made by trace amounts of an unknown E. coli protease that survived purification
Summary
RC3H1 and RC3H2 both repress the mRNAs of genes that control T follicular helper cell development, such as the inducible T-cell co-stimulator (ICOS), and the activation-induced tumor necrosis receptor superfamily 4 (TNFRS4)[10,11], they are not functionally redundant. The ubiquitination activity of the MEX-3C RING domain is required for the degradation of major histocompatibility complex (MHC) mRNA15; the E3 ubiquitin ligase/phosphatase complex MID1/PP2A binds to the expanded CAG repeat of huntingtin (HTT) mRNA and the ligase activity of MID1 regulates the translation of HTT mRNA16. It is unknown whether the ubiquitin ligase function of Roquins cross-talks with their RNA-binding function. RLE-1 was found to induce the degradation of a FOXO family transcription factor, DAF-16, through the ubiquitin-proteasome pathway[18] These studies attest that the RING domain of Roquin proteins is functional. The cross-talk between RNA binding and ubiquitination by Roquins may be context dependent
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