Regulation of miRNA homeostasis by Roquin

Abstract

Post transcriptional and posttranslational gene regulation by transacting RNA-binding proteins is a rapid and efficient way to modify gene expression and cellular responses. Mutations in these trans-acting factors are often associated with pathology. The protein Roquin, known to bind target mRNAs and promote their decay, has been found to be crucial in the maintenance of peripheral tolerance and the prevention of autoimmunity. Mice homozygous for a point mutation in Roquin (Sanroque) behave similarly to mice that lack both Roquin and its paralogue Roquin 2; Sanroque appears to behave as a niche-filling allele. Sanroque mice develop autoimmunity due to impaired posttranscriptional regulation of T cell and macrophage target mRNAs [29; 78, 309, 31]. Recent work has identified sequence and structural requirements for the binding of Roquin to one of its targets mRNAs: a constitutive decay element (CDE) that folds into a stem loop structure in the 3'UTR of Tnf mRNA is required for Roquin-mediated mRNA decay of this transcript [5]. The mechanisms by which Roquin is known to mediate posttranscriptional repression include binding to the decapping and deadenylation complexes and at least the former activity (Roquin-facilitated decapping of target mRNAs) has been reported to be microRNA (miRNA)-independent (Glassmacher et al., 2010). miRNAs, small 20-22nt RNAs post transcriptional regulators that originate from cytoplasmic hairpin precursor intermediates, regulate their mRNA targets mainly by mRNA decay or translational inhibition. Emerging evidence has shown the involvement of microRNAs in almost every critical cellular response and their dysregulation has been shown to be associated with several autoimmune diseases. A paradoxical increase of microRNAs (miRNAs) and their target mRNAs in Sanroque mice, including key mRNAs involved in disease pathogenesis, led us to investigate the role of Roquin in miRNA homeostasis and regulation of miRNA-mediated mRNA repression. Global microRNA profiling of naive T cells from Sanroque mice showed changes in the expression of 15 mature miRNAs, compared with naive T cells from wild-type littermates. All differentially expressed miRNAs were upregulated, with miR-146a being close to 20-fold higher; this miRNA was selected for most studies investigating Roquin-mediated miRA regulation. Accumulation of miR-146a occurred in a T cell autonomous manner and after Dicer processing. In vitro processing assays excluded a direct role for Roquin in miRNA biogenesis. Investigation of Roquin binding to miR-146a using RNA-protein interaction studies and demonstrated that Roquin binds directly to this miRNA in vitro and in vivo, with Roquin-san binding with increased affinity. Finally, we showed that Roquin-san prolongs the half-life of miRNAs. An interaction - albeit weak - between Roquin and Ago2 suggests Roquin may interact with miRISC to control miRNA and mRNA homeostasis. Stabilization of specific miRNAs by Roquin to control miRNA longevity and function represents a novel and previously uncharacterized function of Roquin, and a novel unique regulatory mechanism of posttranscriptional regulation in the mammalian immune system.