Molecular mechanisms underlying the prevention of autoimmunity by Roquin revealed

[melihtas]

Only abstract is available for free, full text is behind paywall and there is a good article about it on phys.org

http://www.nature.com/nsmb/journal/vaop/ncurrent/full/nsmb.2855.html

Structural basis for RNA recognition in roquin-mediated post-transcriptional gene regulation
Abstract
Roquin function in T cells is essential for the prevention of autoimmune disease. Roquin interacts with the 3′ untranslated regions (UTRs) of co-stimulatory receptors and controls T-cell activation and differentiation. Here we show that the N-terminal ROQ domain from mouse roquin adopts an extended winged-helix (WH) fold, which is sufficient for binding to the constitutive decay element (CDE) in the Tnf 3′ UTR. The crystal structure of the ROQ domain in complex with a prototypical CDE RNA stem-loop reveals tight recognition of the RNA stem and its triloop. Surprisingly, roquin uses mainly non-sequence-specific contacts to the RNA, thus suggesting a relaxed CDE consensus and implicating a broader spectrum of target mRNAs than previously anticipated. Consistently with this, NMR and binding experiments with CDE-like stem-loops together with cell-based assays confirm roquin-dependent regulation of relaxed CDE consensus motifs in natural 3′ UTRs.

The post-transcriptional control of gene regulation is essential for a range of cellular functions and is linked to human disease. Regulation of mRNA stability and translation mediates fast changes of protein expression and involves trans-acting protein factors, which recognize specific cis elements in the UTRs of target mRNAs. The molecular requirements for targeting elements and binding factors are often poorly understood.

Roquin is an essential factor in the prevention of autoimmune disease. Both mouse and human roquin are found in two isoforms, roquin-1 and roquin-2, of high sequence identity. The Rc3h1 gene encoding the roquin-1 protein has been identified in an N-ethyl- N-nitrosourea (ENU) mouse mutagenesis screen, in which one point mutation changed Met199 to arginine. Homozygous mice of this so-called sanroque strain (Rc3h1san/san) develop high-affinity autoantibodies and a pathology that resembles the human autoimmune disease systemic lupus erythematosus. The phenotype is related to T cell intrinsic causes because the mice spontaneously develop activated T cells and accumulate follicular helper T (TFH) cells. TFH cells in sanroque mice provide inappropriate B-cell help so that high-affinity autoantibodies against self-antigens are produced. In addition, sanroque mice show increased susceptibility to autoimmune diabetes and autoantibody-induced arthritis as well as angioimmunoblastic T-cell lymphomas. Surprisingly, deletion of roquin-1 did not manifest autoimmune phenotypes. This apparent paradox was explained by the ability of roquin-2 to exert redundant functions. Roquin-2 may therefore compensate when roquin-1 is missing but not when it is present as the sanroque M199R mutant.

Regulation of T-cell activity by roquin has been correlated to its mRNA binding and downregulation of the expression of the inducible co-stimulator (ICOS) and another co-stimulatory receptor, Ox40, encoded by the Tnfrsf4 gene (here denoted Ox40). The minimal RNA-binding domain of roquin is currently unknown. More than half of the 125-kDa roquin protein is predicted to be intrinsically disordered. The N-terminal region of roquin is highly conserved and comprises a RING finger with a potential E3 ubiquitin-ligase func- tion13 and a conserved but not well-defined ROQ domain (residues 64-400) as well as a CCCH-type zinc finger that is potentially involved in RNA recognition. It has been suggested that the ROQ domain mediates binding to the ICOS mRNA. However, the ROQ domain has been predicted solely on the basis of sequence conservation, and RNA binding analysis did not exclude the involvement of additional regions. Recently, it was reported that an N-terminal fragment of roquin-1 (residues 131 to 360) mediates binding to a region in the 3′ UTR of the Tnf mRNA comprising a 23-mer CDE. However, structural details of the roquin-CDE interaction are not known, and the sequence requirements for functional decay elements are poorly understood.

Here, we present structural, biochemical and functional analyses of the roquin-1 RNA-binding domain and its interaction with CDEs. Our results unravel structural requirements for recognition of CDE RNA by the ROQ domain. The data indicate that CDE motifs with confined variations of the consensus sequence are functional and occur in natural targets of roquin-mediated post-transcriptional gene regulation.

http://phys.org/news/2014-07-molecular-mechanisms-underlying-autoimmunity-roquin.html

Scientists at the Helmholtz Zentrum München, the Ludwig-Maximilians University of Munich (LMU) and the Technische Universität München (TUM) have moved an important step closer to understanding molecular mechanisms of autoimmune diseases. They solved the three-dimensional structure of the Roquin protein when bound to messenger ribonucleic acid (mRNA) molecules. The results revealed that there is a much wider range of functionally important Roquin binding partners than previously assumed. The novel findings are published in the journal Nature Structural & Molecular Biology.


The Roquin protein, discovered in 2005, controls T-cell activation and differentiation by regulating the expression of certain mRNAs. In doing so, it helps to guarantee immunological tolerance and prevents immune responses against the body's own structures that can lead to autoimmune disease. Roquin is thus an immune regulator.Autoimmune diseases affect between five and ten per cent of the population. They usually occur as a result of complex environmental influences when a genetic predisposition exists. Only in rare cases the development of the disease is determined by a single mutated gene. However, a single mutation in the Roquin gene in a mouse model was shown to be responsible for the development of the autoimmune disease systemic lupus erythematosus. This mutation in the Roquin protein also led to a high susceptibility to type 1 diabetes and rheumatoid arthritis and induced angioimmunoblastic T-cell lymphoma.

Elucidation of the three-dimensional structure of the Roquin-RNA complex

An interdisciplinary team comprising the research groups led by Prof. Michael Sattler, Dr. Dierk Niessing and Prof. Vigo Heissmeyer at the Helmholtz Zentrum München, Ludwig-Maximilian University (LMU) and the Technische Universität München (TUM) has now revealed unprecedented insight into how Roquin recognizes its RNA binding partner and thereby controls T-cell functions. To this end, the scientists Dr. Andreas Schlundt, Gitta Heinz, and Dr. Robert Janowski used the X-ray crystallography platform of the Helmholtz Zentrum München to determine the spatial structure of the RNA binding domain of Roquin when bound to its RNA target. The interaction of Roquin with additional RNA binding partners was studied in solution using nuclear magnetic resonance (NMR) spectroscopy at the Bavarian NMR Center, a joint research infrastructure of the Helmholtz Zentrum München and TUM. Furthermore, the researchers could confirm the biological significance of the molecular recognition of the RNA by studying Roquin-dependent gene regulation in cellular systems.

The results obtained reveal for the first time the molecular interactions with which roquin recognizes a binding motif in a gene's mRNA. "To our surprise, these results indicate that a greater range of binding modes plays an important functional role for the gene regulation in T-cells," says Prof. Michael Sattler. "Thus, our findings suggest that Roquin regulates a larger number of genes than was previously assumed," Dr. Niessing adds. In addition to the mRNAs with optimal recognition motifs, which are tightly bound and predominantly regulated by Roquin, there is a potentially much larger number of mRNAs which are more weakly bound, but nevertheless regulated by Roquin. "On the basis of these findings we will now focus on understanding how Roquin levels are regulated in T-cells, since strong and weakly bound target mRNAs will experience a principally different regulation when the availability of the protein varies" explains Prof. Vigo Heissmeyer.

Basis for developing treatment

Defining the molecular interplay between Roquin and RNA is a prerequisite for con-trolling the function of Roquin and using its role for therapeutic strategies to treat autoimmune diseases. To this end, the scientists are now planning follow-up studies to find out how the function of Roquin can be manipulated.