The endonuclease Regnase-1

Roquin-Introduction 47

targeted genes. Upon stimulation of the TCR with cognate antigen and co-stimulation of CD28 by APCs, the paracaspase MALT1 gets activated and cleaves both Roquin paralogs. Roquin-1 bears two cleavage sites, whereas for Roquin-2, one cleavage site has been described so far (Figure 3). T cells treated with the MALT1 inhibitor Mepazine or MALT-1 deficient T cells as well as T cells with an inactive form of MALT1 are incapable of cleaving Roquin proteins (Gewies et al., 2014; Jeltsch et al., 2014). However, whether the residing cleavage products (1-510 or 1-579 aa) that still own the essential ROQ-domain, are functional, has not been addressed so far. Additionally, in human Jurkat T cells autoregulation of Roquin through its own 3′-UTR has been proposed (Cui et al., 2017). Furthermore, Roquin-1 and Roquin-2-encoding mRNAs were enriched in pulldown experiments with Roquin-1, whereas both mRNAs were predicted to contain CDE elements (Leppek et al., 2013). Interestingly, the endonuclease Regnase-1 is targeted for degradation in a very comparable way and will therefore be discussed in more detail in the subsequent section.

1.3.2.1 Regnase-1 mouse models

To study the function of Regnase-1, Zc3h12a^-/- mice were generated independently by two research groups. Loss of Regnase-1 resulted in a strong autoimmune phenotype characterized by splenomegaly and lymphadenopathy, multiorgan infiltration of plasma cells and the production of antibodies of all immunoglobulin subtypes as well as anti-double-stranded DNA antibodies. On cellular level, Regnase-1-deficient mice develop class-switched B cells and accumulate highly activated splenic T cells. Macrophages lacking Regnase-1 and stimulated with LPS revealed increased expression of the cytokines IL-6 and IL-12b (Liang et al., 2010;

Matsushita et al., 2009). In addition, all lymphoid organs are drastically disorganized including spleen, lymph nodes and thymus (Miao et al., 2013). However, combining Zc3h12a deficiency with a knockout of either Il6 or Il12p40, encoding for IL-12b, did not rescue the phenotype.

Generating mice with conditionally deleted Regnase-1 in T cells, revealed that the autoimmune phenotype in the systemic knockout mice is T-cell intrinsic. Indeed, Zc3h12a^fl/fl; Cd4-cre mice have profound hyperactivated T cells, accumulate plasma cells and secrete elevated levels of all immunoglobulin subtypes and anti-nuclear autoantibodies (Uehata et al., 2013).

1.3.2.2 Domain organization and function of Regnase family members Next to Regnase-1, the Regnase protein family comprises three additional members:

Regnase-2, Regnase-3 and Regnase-4 encoded by Zc3h12b, Zc3h12c and Zc3h12d, respectively. By comparing the structural domains of these family members (Figure 5), it becomes evident that they all share a CCCH-type zinc finger similar to 1 and Roquin-2 (Figure 3) (Liang et al., Roquin-2008). In addition, all Regnase proteins bear a PilT N-terminal (PIN)-like RNase domain (NYN domain) that, in Regnase-1, comprises endoribonuclease activity which is facilitated by a catalytic center composed of magnesium ion complexed by acidic residues (Asp141, Asp225, Asp226, Asp244 and Asp248) (Xu et al., 2012). This surface is probably forming an RNA-interaction platform, however the co-crystal structure of Regnase-1 in association with RNA has not been solved, yet (Anantharaman and Aravind, 2006;

Matsushita et al., 2009; Mino et al., 2015).

The physiological role of Regnase-2 is hardly understood whereby cells of the immune system exhibit only low expression thereof (Liang et al., 2008).

Regnase-3 is involved in vascular inflammation by counteracting inflammatory responses due to inhibiting the NF-kB pathway and pro-inflammatory gene expression (Liu et al., 2013a). Very recently, the knowledge about Regnase-3 has been expanded by a study that demonstrates a role as endonuclease in regulating mRNA degradation of transcripts involved in the IFN-g pathway and immune homeostasis which is functional complement to Regnase-1. Here the authors showed that systemic Regnase-3 deficiency causes strong IFN signaling thereby

Introduction 49

suppressing GC formation. Interestingly, Regnase-3 exhibits endonucleolytic function thereby degrading mRNAs, such as Zc3h12a, localized to endosomes (von Gamm et al., 2019).

Figure 5 ç Domain organization of Regnase family proteins.

Schematic representation of the domain organization of Regnase-1, Regnase-2, Regnase-3 and Regnase-4. All proteins share a PilT N-terminal (PIN)-like RNase domain and a CCCH-type zinc finger (ZnF), whereas Regnase-1 harbors a C-terminal proline-rich region (PRR) and S435 as well as S439 are sites for phosphorylation (P). Amino acid (aa) positions of the domains are indicated. Black Scissor symbol represents proven and grey scissor symbols indicate predicted MALT1 cleavage sites.

Structures are adapted from Jeltsch & Heissmeyer, 2016 and Takeuchi, 2017.

Regnase-4 like Regnase-1 exhibits endonucleolytic function thereby regulating gene expression of Il6, Il1b and Tnf via their 3′-UTRs. In addition, interactions between Regnase-1 and -4 have been suggested (Huang et al., 2012; Wawro et al., 2017; Zhang et al., 2015).

However, Regnase-4-deficient mice appear healthy, but stimulation of T cells ex vivo induces hyperactivation, suggesting a role for Regnase-4 in controlling T cell effector functions (Minagawa et al., 2014).

It is still unknown whether Regnase family members are functionally redundant, but differentially expressed, or if they have evolved to fulfill entirely different functions. Sites for MALT1-targeted cleavage have been predicted for Regnase-2, 3 and 4, but have not been proven, yet. Therefore, more investigations are required to understand the function of the Regnase family members 2, 3 and 4 in the immune response.

1.3.2.3 Molecular functions of Regnase-1

In the last years, Regnase-1 has been intensively studied and a variety of molecular functions have been identified. The PIN-like domain of Regnase-1 allows deubiquitylation by association with USP10, which in turn negatively regulates NF-kB by removing polyubiquitylation chains of the IkB kinase subunit IKKg or NEMO. Moreover, Regnase-1 removes ubiquitin moieties

from TRAF proteins by involving the CCCH ZnF thereby negatively regulating JNK and NF-kB activity. Yet, it is unsolved how this contributes to the function of Regnase-1 (Liang et al., 2010;

Niu et al., 2013). It was additionally suggested that the PIN domain of Regnase-1 has the capacity to form head-to-tail oligomers, which is mandatory for bona fide RNase activity in vitro (Yokogawa et al., 2016). The before-mentioned CCCH-zinc finger is highly conserved among different species and Regnase family member genes and the ZnF of Regnase-1 is engaged in direct RNA interaction but is not essential for mRNA degradation (Matsushita et al., 2009;

Yokogawa et al., 2016). The C-terminal PRR in Regnase-1 could serve as a protein-protein-interaction surface as it was reported for other PRR-containing proteins before (Yokogawa et al., 2016). In accordance, the carboxy terminus in human Regnase-1 enables oligomerization of multiple Regnase-1 proteins therefore facilitating cleavage of pre-miRNAs and antagonizing Dicer function (Suzuki et al., 2011). However, a later study claimed that miRNAs are not targeted for Regnase-1-mediated degradation since Regnase-1 deficient MEF cells did not show elevated abundance of several miRNAs in comparison to WT MEF cells (Mino et al., 2015).

As mentioned before, Regnase-1 is capable of recognizing stem loop structures mainly localized in 3′-UTRs and thus facilitating degradation of its target mRNAs. These target mRNAs encode for co-stimulatory receptors like Ox40, Icos and Ctla-4, cytokines like 2, 6 or IL-12b or transcription factors such as c-Rel, Irf4 or IkBz and IkBNS. RNA regulation requires the RNase catalytic center in the PIN-domain of Regnase-1, since a point mutation of Asp141 to Asn (D141N) abrogates the mRNA degradation capacity (Behrens et al., 2018; Jeltsch et al., 2014; Mino et al., 2015; Uehata et al., 2013). The degradation of target mRNAs was reported to be independent of the CCCH ZnF, but NMR studies showed that the ZnF can indeed bind Il6 mRNA (Yokogawa et al., 2016). Additionally, Regnase-1 directly cleaves circular and linear Il6 mRNA, whereas the recognition of a stem loop is mandatory for degradation, indicating that Regnase-1 acts as an endonuclease (Mino et al., 2015).

On the one hand Regnase-1 facilitates the degradation of its target mRNAs by direct cleavage dependent on its RNase catalytic center (Lipert et al., 2017; Matsushita et al., 2009). On the other hand, Regnase-1 engages with other post-transcriptional gene regulators. The helicase UPF1, which is essential for the induction of NMD, directly interacts with Regnase-1 and its helicase activity is critical for Regnase-1-mediated transcript degradation (Mino et al., 2019;

Mino et al., 2015). Not only mRNA degradation, but also inhibition of translation has been attributed to Regnase-1. A conserved translational silencing element (TSE) in the 3’-UTR of human Nfkbiz is targeted for degradation by Regnase-1. Here, two highly conserved SL (SL 4 and 5) structures are targeted by Regnase-1 to induce mRNA decay, whereas three tandem SL structures (SL1-3) are required in addition to SL4 and 5 for translational silencing by Regnase-1 (Behrens et al., 2018). Interestingly, SL4 and SL5 share sequence and structure

Introduction 51

similarities with the Roquin-targeted CDE element. These findings indicate that Regnase-1 serves a broad spectrum of molecular functions to guarantee post-transcriptional gene regulation during an acute immune response.

1.3.2.4 Control of Regnase-1 protein expression

Since the endonuclease Regnase-1 plays important roles in controlling immune responses, its expression needs to be under tight control to facilitate expression of target genes. In macrophages, Regnase-1 is phosphorylated at the canonical DSGXXS (Ser435, Ser439) sequence motif by the inhibitor of transcription factor NF-kB kinase (IKK) complex upon signaling of TLRs or IL-1R stimulation resulting in ubiquitination and degradation of 1 (Figure 5). Interestingly, self-regulation of 1 has been reported whereby Regnase-1 targets its own transcript for degradation via a stem loop structure in its 3′-UTR (Iwasaki et al., 2011; Mino et al., 2015). These mechanisms facilitate a tightly coordinated expression of Regnase-1 in macrophages and therefore a release of Regnase-1 target genes like Il6 from mRNA degradation. In T cells, TCR stimulation and co-stimulation triggers cleavage of Regnase-1 by the paracaspase MALT1 at amino acid 111 (Gewies et al., 2014; Jeltsch et al., 2014; Uehata et al., 2013). Intriguingly, the degradation kinetic is analogous to the cleavage of Roquin proteins depending on TCR signal strength (Figure 5).

1.3.3 The overlapping functions of Roquin and Regnase-1 in the