Title:

Regulation of T cell signaling and autoimmunity by RNA-binding

Author information:

Katharina Jeltsch1,2, Vigo Heissmeyer1,2*

1 Institute for Immunology, Biomedical Center Munich, Ludwig- Maximilians-Universität München, Grosshaderner Str. 9, 82152 Planegg- Martinsried, Germany

2 Research Unit Molecular Immune Regulation, Helmholtz Zentrum München, Marchioninistr. 25, 81377 Munich, Germany

* corresponding author

Abstract:

Post-transcriptional regulation by RNA-binding proteins controls mRNA half-life and efficiency of translation. Recently, the RNA-binding proteins Roquin and Regnase-1 have been shown to play pivotal roles in T lymphocytes by preventing inflammatory and autoimmune disease. These factors share an overlapping set of target mRNAs and are both regulated by proteolytic cleavage through the paracaspase MALT1. This review discusses the mouse models of inactivation or deregulation and how these trans-acting factors recognize target mRNAs. Based on different affinities of cis-elements in target mRNAs and regulation of the trans-acting factors, we propose the following model: Increasing TCR signal strength will gradually inactivate Roquin and Regnase-1 causing differential target mRNA derepression that specifies cell fate decisions and effector functions of T cells.

Highlights:

• Roquin and Regnase-1 regulate shared mRNA targets via stem-loops in their 3' UTRs • Roquin and Regnase-1 are inactivated by MALT1 cleavage upon T cell stimulation • Accessibility and affinity/avidity of cis-elements determines their responsiveness • TCR strength is translated into differential expression of Roquin/Regnase- 1 targets

The challenge of understanding the molecular basis of tolerance

The breakdown of tolerance and development of autoimmunity is a multifactorial process in which environmental cues synergize with genetic predispositions. This level of complexity is reflected by genome-wide association studies with common single nucleotide polymorphisms (SNPs), which identified many autoimmune disease-associated variants with rather small susceptibility effects and limited diagnostic potential. In contrast, rare Mendelian autoimmune disorders typically have severe consequences and have provided important insights into the various processes of tolerance [1]. The large body of basic research on these monogenic causes of autoimmunity has already proven its impact on human health by fostering the development of novel potent therapies in tumor immunology [2]. With strategies of precision medicine emerging, research on individual molecule contributions in immune disorders becomes even more important for the potential treatment of cancer or autoimmune diseases.

Post-transcriptional gene regulation in lymphocyte differentiation Lymphocyte differentiation, like any other specialized cellular program, requires well-adjusted changes of gene expression. These changes are typically launched by the cooperation of transcription factors induced by antigen, costimulation and cytokine signals [3]. These then lead to the induction of subset-specifying transcription factors and, upon establishment of differential gene regulation, epigenetic mechanisms stabilize the gene expression program. Very important post-transcriptional mechanisms, often mediated by miRNAs, ensure the temporal sharpening of expression profiles and the fine-tuning of expression levels of key molecular players. Since the effects of individual miRNAs on individual target mRNAs are rather modest, stronger impacts are created by different miRNAs cooperating in the regulation of the same mRNA or by one miRNA targeting several mRNAs in the same pathway [4]. Post- transcriptional regulation of individual mRNAs by RNA-binding proteins can be very strong and can completely switch cellular responses by decreasing the levels of specific mRNAs or by blocking their translation. Many different cis- regulatory elements including binding sites for miRNAs and RNA-binding proteins can be found in long untranslated regions (UTRs) of many mRNAs. This implicates a high degree of combinatorial regulation by different post- transcriptional factors. However, such cooperative action of trans-acting factors on shared or exclusive cis-elements remains largely unexplored. Very recent findings demonstrated a critical role of the RNA-binding proteins Roquin-1, Roquin-2 and of the endonuclease Regnase-1 in T cells, and uncovered an overlapping set of target mRNAs (Figure 1) [5!!,6!!,7!!,8,9!!]. This review aims to develop a model to explain how this system controls T cell effector function. We propose that differential gene expression programs are established through potential redundant, cooperative or even compartmentalized exclusive control of the shared targets (Figure 2) by dynamic regulation of Roquin and Regnase-1 proteins. These differences then promote specific T cell fate decisions (Figure 3).

Inflammation and autoimmunity in mice with Roquin and Regnase-1 mutations

An ENU (ethylnitrosourea) mutagenesis screen discovered the sanroque mouse strain. This line harbors a single point mutation changing the codon for methionine 199 to arginine in the Roquin-1-encoding gene Rc3h1 [8]. It demonstrated an essential role of Roquin-1 in peripheral tolerance by showing that homozygous sanroque mice develop severe systemic lupus erythematosus-like autoimmunity [8]. Surprisingly, the respective knockout of the Rc3h1 gene did not reproduce systemic autoimmunity, instead revealed modest immune dysregulation [10]. Individual genetic ablation of Rc3h1 or its paralog Rc3h2, encoding the Roquin-2 (Figure 1A), showed that both are essential for postnatal survival of mice [9!!-11!]. Interestingly, the postnatal lethality coinciding with impaired neural tube closure and defective lung inflation was similarly detected in mice with a complete deletion of the Roquin-1-encoding gene and mice expressing Roquin-1 with partial deletion of only its RING finger [10,11!]. This suggests that the E3 ligase activity of Roquin contributes a vital function for the organism. Nevertheless, the combined deficiency of Rc3h1 and Rc3h2 in peripheral T cells uncovered functional redundancy of Roquin proteins [9!!]. In fact, these mice phenocopy important aspects of sanroque mice like splenomegaly, lymphadenopathy, activation of CD4+ and CD8+ T cells as well as accumulation of T follicular helper cells (TFH) and germinal center B cells [9!!]. Regnase-1 as well as its family member Regnase-4 repress IL-6 production and negatively regulate macrophage-mediated inflammation [12-15]. Regnase-3 has so far only been involved in vascular inflammation, where it counteracts the inflammatory response by inhibiting the NF-κB pathway and pro-inflammatory gene expression [16]. Very comparable to mice with mutation in Roquin-encoding genes, T cell-specific deletion of the Regnase-1- encoding gene Zc3h12a causes spontaneous activation of T cells, aberrant cytokine production, accumulation of plasma cells and hypergammaglobulinemia in mice [7!!]. To a varying degree, autoantibodies can be found in Roquin or Regnase-1 mutant mice with an abundance of anti- nuclear antibodies in sanroque and mice lacking Regnase-1 in T cells, while mice with Roquin-deficiency in T cells mostly produce tissue-directed autoantibodies recognizing certain antigens for example in the pancreas [5!!,7!!,8].

Molecular determinants of Roquin and Regnase-1 function

The mRNA of Roquin-1 is ubiquitously expressed [8] while higher protein levels of Roquin-1 and Roquin-2 are detected in the thymus and lymphoid organs [9!!]. Roquin-1 and Roquin-2 share an amino-terminal RING finger, a novel RNA-binding domain termed ROQ followed by a CCCH-type and a proline-rich region in the carboxy-terminus (Figure 1A) [8,17,18!,19- 22!]. The ROQ domain is embedded in adjacent sequences (HEPNN and HEPNC) that fold back together to form one HEPN domain. This domain binds to double-stranded RNA independently of the ROQ domain [21,22!]. The ROQ domain of Roquin recognizes characteristic RNA stem-loop structures of a 5-7 nucleotide long stem and a tri-loop in which a central purine base, preceded by a pyrimidine base, engages in stacking interactions with several other purine bases from the upper 3' stem [23,24!!]. The canonical stem- loop, termed the constitutive decay element (CDE), was identified first in studies of the adenine- and uridine-rich element (ARE)-regulated Tnf 3' UTR [25]. Its recognition by Roquin depends on a core winged-helix fold of the ROQ domain [18,22!] that makes multiple interactions with the phosphate backbone of the 5' stem and few contacts with bases of the loop, which recognizes the shape, rather than the sequence of the stem-loop RNA [17,18!,22!]. Interestingly, the 3' UTR from the recently described target mRNA Tnfaip3, encoding the A20 protein, contains a composite cis-element of a stem-loop structure and an ARE that are recognized in vitro by the ROQ domain and the CCCH-type zinc finger, respectively [26!]. Although the M199R sanroque mutation occurs within the ROQ domain and confers hypomorphic regulation of the ICOS target mRNA [8,27], it does not impair RNA binding [28]. Hence, the M199 residue is not contributing to RNA/protein interaction [18!,21,22!], but is rather likely to be part of an as yet to be defined protein/protein interaction surface. The paralogs of the Regnase family comprise Regnase-1, -2, -3 and -4, also known as MCPIP1, 2, 3, 4 proteins that are encoded by Zc3h12a, b, c, d genes, respectively [29]. For Regnase-1, an endonucleolytic activity with preferred cleavage of RNAs containing a stem-loop structure, as present in the Il6 3' UTR, has been demonstrated in vitro [6!!,13]. Consistent with the importance of RNA binding and processing, all Regnase paralogs show the conservation of a PIN-like (PilT N-terminus) nuclease domain as well as of a CCCH-type zinc finger (Figure 1B). The recognition of RNA by Regnase-1 has not been defined structurally. However, mapping of response elements in the 3' UTRs of Regnase-1 target mRNAs revealed several hairpins (i.e. in the Il6, Zc3h12a, c-Rel, or Il2 3' UTRs) [7!!,13,30,31]. Regnase-1 is dynamically regulated on multiple levels. The encoded gene is transcriptionally induced after TLR or IL-1R signaling [13,32]. Phosphorylation of its DSGxxS motif (Figure 1B) by IKKβ and subsequent polyubiquitination by the E3 ligase β- TrCP (β-transducin repeat–containing protein) targets Regnase-1 for proteasomal degradation [30]. At the same time, Regnase-1 negatively regulates its own transcript via a critical stem-loop structure in its 3' UTR [30,32]. Similarly, deletion of Roquin-encoding genes resulted in a strong upregulation of Regnase-1 expression [5!!].

Roquin proteins utilize exonucleolytic decay mechanisms to degrade target mRNAs. This requires recognition of the target mRNA by the ROQ domain [18!,22!] and induction of post-transcriptional repression by carboxy-terminal sequences that include the proline-rich region [33]. In fact, this region of the protein interacts with the Ccr4/Not complex and regulates deadenylation and decay of the target mRNAs [24!!,26!] (Figure 1C). Furthermore, Roquin was shown to be associated with the decapping pathway by interacting with the helicase Rck and the enhancer of decapping Edc4 through amino-terminal sequences [33]. In contrast, the intact nuclease domain in Regnase-1 is critical for post- transcriptional repression in cells, suggesting endonucleolytic cleavage of target mRNAs as the major molecular mechanism [6!!,7!!,13,30]. In addition, a region amino-terminal to the nuclease domain engages in an interaction with the helicase Upf1, which is required for post-transcriptional repression of target mRNAs by Regnase-1 [6!!] (Figure 1D). In addition, reporter experiments with pharmacologic inhibition of translation revealed that Regnase-1-mediated repression requires active translation of the target mRNA [6!!].

Functional interplay- redundancy or cooperation?

In line with the obvious overlap of mouse mutant phenotypes, the 3' UTRs of the bona-fide target mRNAs of Roquin, including Icos, Irf4, Nfkbid and Nfkbiz, and those of Regnase-1, including Il6, Ctla4, cRel and Zc3h12a itself, exhibited regulation by either Roquin-1 or Regnase-1 in reporter assays [5!!]. Combined deletion of Roquin-1 and -2 encoding genes or knockdown of + Regnase-1 in naive CD4 T cells similarly promoted TH17 differentiation, whereas inactivation of their shared targets Nfkbiz or Nfkbid inhibited this program [5!!,34-36]. Similarly, Regnase-4-deficient mice were more susceptible to experimental autoimmune encephalomyelitis (EAE) in correlation with an increased TH17 presence in the CNS [14]. The sequencing of mRNAs co-immunoprecipitated with Roquin-1 or Regnase- 1 confirmed and extended an overlapping target set [6!!]. Therefore, both factors are, at least in reporter studies, able to repress targets redundantly, but they may also control some targets involved in T cell differentiation in a cooperative manner (Figure 2). Mechanistic experiments with a reporter construct of a minimal response element from the Tnf 3' UTR containing the CDE demonstrated full repression by overexpressed Roquin-1 only in the presence of endogenous Regnase-1 and vice versa [5!!]. Consistently, the functional interdependence was overcome when the RNA-binding ROQ domain of Roquin-1 was artificially fused to the open reading frame of Regnase-1 [5!!]. Therefore, both trans-acting factors can work cooperatively by likely binding to the same, but potentially also to several cis-elements in the 3' UTR of one mRNA (Figure 2). However, more recent work questioned a general cooperation of both factors [6!!]. The data include migration of Regnase-1 but not Roquin in polysomal fractions of sucrose gradients and localization of Regnase-1 to the endoplasmatic reticulum but not to P bodies [6!!]. This is different from previous reports showing colocalization of overexpressed GFP-tagged Regnase-1 with GW182 [37] similar to the localization of Roquin in P bodies [33]. Moreover, Regnase-1 exhibited effects on selective target mRNAs earlier during LPS stimulation of macrophages and, different from Roquin, required the helicase activity of Upf1 [6!!]. The currently available data do not rule out that Roquin and Regnase-1 work cooperatively, but they argue for the existence of compartmentalized and exclusive functions at least in myeloid cells (Figure 2). Overall, many differences may be explained by both factors being able to employ multiple unique as well as shared mechanisms of post-transcriptional gene regulation, especially upon overexpression. In search for more target mRNAs and cis-elements, the first genome-wide crosslinking and immunoprecipitation studies (i.e. PAR-CLIP and HITS-CLIP) for Roquin and Regnase-1 have been reported [6!!,26!]. The overexpression and crosslinking of Roquin-1 to mRNA in HEK293 cells resulted in an enrichment of U-rich loops with 3, 4 and 5 nucleotides within hairpin structures [26!]. However, these did not resemble the consensus CDE stem-loop [26!]. It had been evident before that Roquin also interacts physically and functionally with mRNAs devoid of high-affinity CDEs [18!,24!!]. In fact, individual and combined point mutations of the ROQ domain in Roquin-1 interfered with repression of the extremely long 3' UTR of ICOS or the extremely short 3' UTR of Tnfrsf4 (Ox40) much more than repression of the short response element of the 3' UTR of Tnf that contains the high affinity CDE [18!]. This indicates a much lower accessibility, affinity or avidity of binding sites in ICOS and Tnfrsf4 (Ox40) compared to the canonical CDE in cells (Figure 2). Consistently, overexpression of Roquin was able to rescue the regulation of low-affinity CDE-mutants that appeared derepressed at endogenous Roquin expression levels [18!,23]. The regulation through low and high affinity binding sites (Figure 2) is also supported by our own recent work that provides evidence for Roquin interaction with and regulation of a novel hexa-loop structure in the 3' UTR of Tnfrsf4 (Ox40), which functions as an alternative decay element (ADE) (Janowski et al., under revision). Therefore, the crosslinked U-rich stem-loop structures may represent unexplored ADEs as well as crosslinking of a variety of lower affinity interactions that are formed at higher expression levels of Roquin. Surprisingly, a much better match with high-affinity Roquin consensus stem- loops was obtained by crosslinking of overexpressed Regnase-1, which exhibited a preference for tri-loops with purine-pyrimidine-purine loop structures on a 5-7 nucleotides long stem [6!!]. Whether this interaction with cellular RNA occurs unchanged in the absence of endogenous Roquin has not been determined yet.

Regulation of T cell responses by MALT1

Roquin-1, Roquin-2 and Regnase-1 share a regulatory mechanism, since they are all cleaved by the paracaspase MALT1 in response to T cell receptor (TCR) and costimulatory signaling (Figure 1A-B and Figure 3) [5!!,7!!]. The conservation of the essential P1 arginine and the surrounding amino acid sequence argues for the existence of a MALT1 cleavage site in Regnase-2, -3 and -4, but actual cleavage of these proteins has not been established yet (Figure 1B). Besides its proteolytic activity, MALT1 functions as a scaffold protein. As such it is important for transducing TCR and costimulatory receptor CD28 signals to NF-κB activation. In a complex with Bcl10 and Carma1 (CBM complex), MALT1 mediates activation of the IκB kinase (IKK) complex. Consistently, MALT1 knockout mice show defects in TCR-induced activation, proliferation and IL-2 production coupled with general immunodeficiency [38,39]. The MALT1 paracaspase cleaves a number of NF- κB-related targets, including A20, RelB, Bcl10, NIK, and CYLD, and was also shown to contribute to optimal NF-κB and T cell activation [40-44]. The observed lack of Roquin and Regnase-1 cleavage in MALT1 knockout cells [5!!,7!!] can explain some effects in MALT1 knockout mice that seemed unrelated to abrogation of NF-κB signaling. For example, Malt1-/- T cells were shown to be greatly impaired in TH17 differentiation [45], which correlates with protection from EAE [45,46]. However, analysis of mice with specific genetic inactivation of the MALT1 protease activity revealed an unforeseen lethal inflammatory syndrome [47-50], while phenocopying certain MALT1 knockout symptoms like impaired antibody responses [47,48,50], defective TH17 differentiation, insensitivity to induction of EAE [47] and impaired regulatory T cell (Treg) development [47-49]. In addition, the knock-in mice expressing the protease-deficient MALT1 protein showed weight loss, multi-organ infiltration by lymphocytes [47-50] and spontaneous neurodegeneration with ataxia in a lymphocyte- and IFN-γ-dependent manner [48]. Actually, the paracaspase activity was largely dispensable for T cell activation, since IKK activation was undisturbed by protease-insufficient MALT1 [47-49]. Hence, productive T cell signaling that relies on the scaffold function of MALT1 in the face of strongly reduced thymic Tregs and substantially diminished peripheral Tregs [47-49] could explain the disturbed T cell responses. Indeed, adoptive transfer of wildtype Tregs into newborn mice expressing protease-deficient MALT1 rescues the weight loss, T cell activation, and corrects the frequencies of IFN-γ+ or IL-4+ CD4+ T cells as well as the development of gastritis [49]. Although the reason for impaired Treg cell development is not clear, downregulation of two Roquin- and Regnase-1-controlled proteins could play a role, since loss of either c-Rel or IκBNS, have been shown to impair individual steps of thymic Treg cell development [51,52]. With the critical importance of the novel MALT1 targets Roquin and Regnase-1 and the availability of MALT1 paracaspase inhibitors [53,54], new therapeutic options to strengthen the activity of Roquin and Regnase-1 arise and could be used acutely to counteract immune disorders. However, more work is required to find out how constitutive deregulation of the different MALT1 paracaspase targets can itself be the cause of disease.

A model for post-transcriptional control of T cell differentiation

From the analyses of Roquin mutant mice it has become clear that these factors potently control cell fate decisions of T cells. Specifically, hypomorphic sanroque or combined loss-of-function mutations in Roquin-encoding genes similarly lead to spontaneous activation of CD4+ and CD8+ T cells and + predispose CD4 T cells towards TFH cell differentiation [8,9!!,55,56]. In addition, effector/memory CD4+ T cells of sanroque mice abundantly produce + the TH1 cytokine IFN-γ [57]. Consistently, Roquin-deficient CD4 T cells activated under TH0 or TH1 conditions reveal a similar predisposition to become IFN-γ–producing cells [5!!]. However, different from sanroque mice, inactivation of Roquin-encoding genes in peripheral T cells enriches TH17 cells in the lung, and causes overt lung pathology with neutrophil attraction and non-allergic asthma symptoms like increased mucus production by goblet cells [5!!]. Interestingly, concurrent symptoms and pulmonary inflammation also resulted from transgenic expression of hyperactive STAT3 in T cells that were ameliorated by treatment of the mice with IL-17A-neutralizing antibodies [58]. Increased TCR signal strength has been linked with TFH, TH1 and TH17 differentiation, although the use of different model systems precludes direct comparisons [59,60]. It has been proposed that lower antigen doses induce TH2 and TFH precursors cells while intermediate doses induce TH1, and high doses induce germinal center TFH cells [59,60]. In this regard, only strong TCR stimuli induce maximum IL-21 expression in TFH cells, and in vivo TFH precursors with the highest peptide-MHCII binding capacity are selected into effector TFH cells [61]. Moreover, TH17 cell differentiation is supported by strong antigenic stimulation that prevents iTreg development [62-64]. Comparisons of different peptide concentrations or of mutant peptides with decreased affinity to a transgenic TCR showed that gradually increasing TCR signal strength is followed by increasing MALT1-dependent cleavage of Roquin and Regnase-1 [5!!]. This in turn causes increased derepression of the target transcription factors IRF4, IκBζ and IκBNS, and augments TH17 differentiation in the ex vivo activated TCR transgenic CD4+ T cells [5!!]. Based on these data and the existence of 3' UTRs with different affinities, varying lengths and numbers of cis-elements (Figure 2), we propose that different TCR signal strength is translated into differential gene expression via the MALT1/Roquin/Regnase-1 axis. Low TCR strength will only promote expression of mRNAs with low affinity binding sites, and especially those with short 3' UTRs that are not redundantly regulated will become derepressed firstly (Figure 3). In contrast, mRNAs with high affinity binding sites and particularly those with long 3' UTRs and many redundantly regulated binding sites will remain repressed (Figure 3). Presumably oversimplified and not yet tested, our model would predict affinity differences of binding sites in TH17- promoting target mRNAs (i.e. Nfkbid and Nfkbiz) and those in TH17- as well as TFH-inducing mRNAs (i.e. Irf4). There is currently no experimental evidence for MALT1 cleavage products of Roquin or Regnase-1 with functional importance. However, it appears conceivable that the rather stable amino- terminal cleavage products of Roquin proteins, which contain all domains involved in RNA recognition [5!!,9!!,33], could inhibit the activity of full- length molecules by blocking their access to physiologic binding sites on mRNAs (Figure 3).

Conclusions

Roquin and Regnase-1 proteins are key players in T cells by controlling their activation and differentiation and thus preventing autoimmunity. Recent research on these proteins has elucidated how they are regulated themselves and how they recognize and impose post-transcriptional gene regulation on target mRNAs. Bringing the available information now into testable concepts will strongly enhance our understanding of immune disorders and potentially lead to therapies for treatment of cancer or autoimmune diseases.

Figure legends

Figure 1 Domain organization of Roquin-1 and its paralog Roquin-2 (A) and of Regnase-1 and its paralogs Regnase-2, -3 and -4 (B) indicating proven (red) and potential (black) MALT1 cleavage sites. In Regnase-1, the DSGxxS motif for serine phosphorylation by IκB kinases (IKK) and subsequent ubiquitination/proteasome-mediated decay is shown (B). Mechanisms of mRNA decay mediated by Roquin and Regnase-1 proteins are depicted (C- D). RING, really interesting new gene finger; HEPN, higher eukaryotes and prokaryotes nucleotide-binding; CCCH-type zinc finger; PRR, proline-rich region; M199R, sanroque point mutation.

Figure 2 Illustration of Roquin and/or Regnase-1-targeted stem-loops in the 3' UTR of mRNAs. Hypothetical mRNAs are grouped according to having short 3' UTRs with presumably few binding sites and long 3' UTRs with many binding sites. These mRNAs can be further divided into groups containing high versus low affinity binding sites in their 3' UTRs. Finally, the different 3' UTRs are either post-transcriptionally regulated exclusively by Roquin or Regnase-1 or regulated in a cooperative or redundant manner by both factors (see text for more details).

Figure 3 A current model of Roquin- and Regnase-1-controlled T cell activation and T cell effector responses. Combined TCR activation and costimulatory signaling induces cleavage of Roquin-1 and -2 and Regnase-1 by the paracaspase MALT1. Increasing TCR signal strength gradually leads to more cleaved Roquin and Regnase-1 proteins and in this way releases their respective mRNA targets from post-transcriptional repression. mRNAs with 3' UTRs comprising low affinity binding sites and those that require cooperative binding or regulation by both Roquin and Regnase-1 become deregulated first. mRNAs containing long 3' UTRs with multiple binding sites that can be regulated by either Roquin or Regnase-1 are only derepressed upon strong TCR stimulation. According to the organization of their 3' UTRs and functional avidity of their cis-elements, different targets are differentially derepressed. These include cytokines, costimulatory receptors, signal transducers, transcriptional modulators and transcription factors that then determine the effector T cell fate. A possible dominant-negative action of the stable amino- terminal Roquin cleavage product, which still needs to be experimentally tested, is depicted by binding of the inactive molecule to mRNA. This may block access of the repressive full-length proteins to individual cis-elements.

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Acknowledgements This work was supported by Grants from the Deutsche Forschungsgemeinschaft (SFB 1054 TP-A03) and the European Commission (ERC-StG to VH). We thank Dirk Baumjohann and Dierk Niessing for critical reading of the manuscript.

A B

PLVPR111GGS DS435GIGS439 1 476 533 596 112 281 Regnase-1 Nuclease CCCH PRR

LLLPR GAS QLIPR510GTD 165 835 1 89 176 M199R 326 400 786 1130 1 533 188 344 Roquin-1 Regnase-2 RING ROQ* CCCH PRR Coiled-coil HEPN HEPN N C NSVMR240 ETS QMVPR579GSQ 1 903 264 420 Regnase-3 QLIPR509GTD 1 1187 VLIPR GCC Roquin-2 64 1 533 Hydrophobic region 92 245 Regnase-4

C D

Roquin

Interaction of the Regnase-1 5’ CAP carboxy-terminus Requirement for Interaction of AAAAAA of Roquin with the active translation Regnase-1 with of the target mRNA the helicase Upf1 CDS Ccr4/Not complex AAAAAA CDS Induced mRNA decay via deadenylation and decapping Induced mRNA decay via endonucleolytic cleavage BINDING SITES

High affinity Low affinity

Roquin Regnase-1 CDS CDS AAAAAA AAAAAA 5’ CAP 3’ UTR 5’ CAP 3’ UTR

Exclusive

Short/few POST-TRANSCRIPTIONAL CONTROL binding sites AAAAAA AAAAAA

AAAAAA AAAAAA

Cooperative 3’ UTR LENGTH 3’

AAAAAA AAAAAA

Long/many binding sites AAAAAA AAAAAA

Redundant

AAAAAA AAAAAA Induced cytokines TCR/ costimulation Induced costimulatory AAAAAA receptors

AAAAAA

MALT1 AAAAAA Induced signal mRNAs become transducer Cleavage of Roquin derepressed and Regnase-1

mRNAs remain repressed

Induced modulators AAAAAA of transcription

Induced AAAAAA transcription factors