Trim-NHL proteins are defined by RING, B-Box and Coiled-coil protein motifs (referred to collectively as the Trim domain) coupled to an NHL domain. The C. elegans, D. melanogaster, mouse and human Trim-NHL proteins are potential and in several cases confirmed, E3 ubiquitin ligases. Current research is focused on identifying targets and pathways for Trim-NHL-mediated ubiquitination and in assessing the contribution of the NHL protein-protein interaction domain for function and specificity. Several Trim-NHL proteins were discovered in screens for developmental genes in model organisms; mutations in one of the family members, Trim32, cause developmental disturbances in humans. In most instances mutations that alter protein function map to the NHL domain. The NHL domain is a scaffold for the assembly of a translational repressor complex by the Brat proto-oncogene, a well-studied family member in Drosophila. The link to translational control is common to at least four Trim-NHLs that associate with miRNA pathway proteins. So far, two have been shown to repress (Mei-P26 and Lin41) and two to promote (NHL-2, Trim32) miRNA-mediated gene silencing. In this chapter we will describe structure-function relations for each of the proteins and then focus on the lessons being learned from these proteins about miRNA functions in development and in stem cell biology.

Introduction

The year 2000 was an auspicious one for the miRNA field. In a series of three papers, the let-7 miRNA was first identified in a screen for mutations affecting developmental timing in C. elegans.¹ Second, let-7 was quickly demonstrated to be highly conserved and expressed in a wide range of bilaterally symmetric animals,² unlike the original miRNA gene, lin-4, that was thought to be restricted to C. elegans.³ Third, let-7 was shown to act as a repressor for a conserved gene, lin-41,^1,4 within the developmental timing pathway. Analysis of predicted protein domains in LIN-41 led to the description of a novel class of so-called Ring, B-Box, Coiled-coil (RBCC) domain proteins.^4,5 Later, the acronym Trim (Tripartite motif) was adopted for this sequence of protein domains. Current annotations recognize over 70 Trim domain proteins in mammalian genomes that can be subdivided into distinct classes based on the presence of additional C-terminal domains (reviewed in refs. 6,7). The C-terminus of LIN-41 was found to contain six copies of a 44 amino acid repeat sequence shared with two other known proteins: HT2A and NCL-1.^4,5 These were the founding members of the Trim-NHL (NCL-1, HT2A, LIN-41) family. As seen in Figure 1, there are four Trim-NHL proteins encoded in the D. melanogaster, M. musculus and H. sapiens genomes and five in C. elegans. Several of these proteins, including the mouse ortholog of LIN-41, have recently been identified as regulators of miRNA pathway activity, the subject of this review.

Figure 1

Overview of the Trim-NHL protein family. The mammalian NHLrc1-3 proteins are not considered due to space constraints and because of the lack of Trim motifs in these proteins (with the exception of the RING in NHLrc1). Protein motifs were identified using (more...)

There are several reviews of Trim domain proteins, including their structural characterization, disease phenotypes and functions in ubiquitin-mediated protein degradation.^6-8 As of this writing, however, there is no comprehensive review of Trim-NHL family members and their roles in development and miRNA regulation. For this reason, we will begin with a brief outline of the common structural features of this protein family, followed by a review of earlier work on the functional characterization of each of the members. We will then turn to recent evidence that at least some of the Trim-NHL proteins act as post-transcriptional regulators of miRNA activity in their capacity as E3 ubiquitin ligases.

The Trim-NHL Family of Developmental Regulators

Although there have been a number of important studies on Trim-NHL proteins in chicken, zebrafish or rat, the bulk of the work has concerned the proteins from C. elegans, Drosophila, mouse and humans presented in Figure 1. The phylogenetic tree presented in Figure 1 is based on alignment of the C-terminal NHL sequences. Both the phylogenetic relationships and the individual domain structures of these proteins have been more rigorously treated elsewhere.^6,7,9-11 As detailed below, the Trim-NHL sequence of motifs was first recognized in LIN-41 from C. elegans and consists of a RING-type (Really Interesting New Gene) Zinc-finger in close proximity to the N-terminus. One or two copies of a distinct Zinc-finger motif referred to as the B-Box follow the RING. The triad is completed by a hydrophobic heptad repeat termed the coiled-coil. This linear arrangement is quite constant, suggesting cooperative functional interactions between the three motifs. Less conserved is the Ig-filamin domain situated between the Trim motif and the NHL domain in several of the proteins. The paradigm NHL domain contains six copies of a 44 residue repetitive motif but the number of readily identified repeats varies from 2 to 6.

Each of these motifs will be discussed in turn, beginning with the RING domain. Although referred to as a RING finger, the actual structure is not fingerlike. Instead, the RING sequence contains four pairs of Zinc binding residues (numbered 1-4). The peptide chain loops back upon itself to juxtapose two non-adjacent pairs of Zinc binding residues (pairs 1 and 3) to form the first Zinc-coordination site. The peptide chain then reverses direction again to align the two remaining pairs (pairs 2 and 4). The result is a rigid, self-reinforced globular structure referred to as a "cross-brace" (reviewed in refs. 12,13; see NCBI Conserved Domain Database 00162). RING motifs are found in a large number of proteins and were originally thought to mediate DNA binding and/or protein-protein interactions. After several RING proteins were linked to ubiquitin-mediated protein degradation, the RING domain of the Cbl (Casitas B-lineage lymphoma) proto-oncogene was shown to actively participate in protein ubiquitination,^14,15 a finding then extended to many additional family members¹⁶ (reviewed in refs. 13,17). The RING domain was later shown to possess intrinsic E3 ubiquitin ligase activity and to directly recruit and activate specific E2 ubiquitin conjugating enzymes.^18,19 A brief description of the ubiquitin pathway will be provided in the next section (see Fig. 2). However, it seems certain that E3 activity is a general feature of the RING domain. Trim32 was the first Trim-NHL protein shown to be an E3 ubiquitin ligase,^20,21 followed by Trim2²² and mouse LIN-41 (referred to as either Trim71, mLin41, or Mlin41).²³

Figure 2

Basic principles of ubiquitination. a) A ubiquitin monomer is covalently linked to an E1 activating enzyme. Interaction of the E1 with an E2 conjugating enzyme results in transfer of the primed ubiquitin to an E2 conjugating enzyme. For RING domain E3 (more...)

Less is known about the B-Box motifs, which are found either singly or in tandem. When in tandem, the upstream B-Box conforms to a distinct consensus sequence. B-Boxes are most commonly associated with a RING domain, but as seen in Brat and Dappled/Wech this is not always the case (Fig. 1). Mutations in the B-Box for the nonNHL Trim proteins Trim5a and Trim18 (Midline-1) disrupt protein function (reviewed in ref. 6). As discussed below, mutations in the B-Box of human Trim32 lead to a distinct developmental disorder (Bardet-Biedl Syndrome)²⁴ compared to mutations in the NHL domain (Limb Girdle Muscular Dystrophy).^21,25 However, in these and in other cases the precise role of the B-Box remains elusive and may entail protein turnover, oligomerization or intracellular localization in addition to specific protein-protein interactions. The solution structure of the individual and the tandem B-Boxes of Trim18 is an important advance, revealing that both adopt a cross-brace conformation quite similar to the RING domain^26-28 (see also NCBI Conserved Domain Database cl00034). This raises the interesting possibility that the B-Box may directly interact with the RING and participate in E3 activity, perhaps by serving as an accessory binding surface for E2 conjugating enzymes.

Of the three Trim motifs, the coiled-coil is the most widespread outside the Trim superfamily and represents a basic building block of protein structure (reviewed in refs. 18,29). In principle, coiled-coils can form higher order homo and heteromeric structures with a high degree of plasticity.²⁹ Less is known about their structural or functional contribution to Trim domain function, although there is experimental evidence that they support homomeric interactions.⁹ The same study showed that multimerization mediated by the coiled-coil was required for correct localization to intracellular compartments. Trim2, Trim3 and Trim32 eGFP fusions were found to be cytoplasmic with a filamentous, diffuse or "speckled" distribution, respectively.⁹

When present, the Filamin homology domain, also referred to as an Ig-filamin repeat (see NCBI Conserved Domain Database cl02665), lies between the Trim motifs and the NHL repeats. No function has been assigned to the Filamin domain in the context of Trim-NHL family members. In Filamin proteins, the Ig-Filamin repeats are present in up to twenty-four copies and adopt a rod-like β-barrel conformation that interacts with actin and many other partners (see ref. 30 for a recent review).

Of course, the defining domain of the Trim-NHL family is the NHL repeat (Fig. 1). The similarity of the NHL repeats to a previously characterized motif, the WD-40 β-propeller, was significant enough to suggest a similar secondary structure.⁵ Furthermore, many of the genetically isolated mutations in LIN-41 and Brat mapped to the NHL domain.^4,31 The NHL domain was necessary and sufficient for rescue of brat embryos during embryonic patterning,³² demonstrating that the isolated domain has activity as a translational repressor (see below for details). The crystal structure of the isolated NHL from Brat has been solved and revealed that each of the NHL repeats forms one "blade" of a six-bladed propeller, with the exception of the first blade. β-sheet elements derived from the first and sixth NHL repeat contribute to the first blade. This brings both ends of the structure together in a circular, or doughnut-like arrangement.³³ Each blade is composed of four β-sheets connected by exposed loops. Structural modeling suggests that the RNA-binding protein Pumilio interacts with one face of the propeller via the looped out residues. The properties of this potential interaction surface are predicted to vary substantially among the individual Trim-NHL family members such as Dappled/Wech.³³

The Trim Domain as E3 Ubiquitin Ligase

As noted above, E3 ubiquitin ligase activity has been documented for an increasing number of Trim proteins, making it the potentially largest class of E3 enzymes. In the case of Trim-NHL proteins, a role in protein ubiquitination could account for the diversity of their known functions in processes ranging from developmental timing (LIN-41, NHL-2), patterning (Brat), cell growth, division and proliferation (Brat, Mei-P26, NCL-1), endosomal trafficking (Trim3), muscle integrity (Trim32, Wech) and germ line (LIN-41, Mei-P26) or nervous system development and function (Brat, Mei-P26, mLin41, Trim2, Trim32). Ubiquitination is a post-translational modification rivaling phosphorylation in extent and consequence. The ubiquitin literature is far too vast to summarize here and there are many excellent reviews.^13,34,35 Basic features relevant to this review are summarized in Figure 2. Covalent attachment of ubiquitin to substrate proteins involves a tag team of three enzymatic activities (E1-E3). First, in an ATP-consuming reaction ubiquitin is activated by attachment to an E1 activating enzyme. Interaction with an E2 conjugating enzyme leads to transfer of ubiquitin from the E1 to the active site of the E2. E3 ligases belong to one of two large classes, HECT or RING. In the case of RING domain E3s, the E3 serves as a bridge between the E2 and the substrate protein with primary responsibility for substrate specificity. Trim E3s are believed to stimulate catalysis by inducing proximity between the E2 and the substrate.¹³ A total of six known E1s (only two utilize ubiquitin, the others employ ubiquitin-like proteins such as SUMO—Small Ubiquitin-like MOdifier) can chose among ~40 E2s. A given E3 will productively interact with only one or a few E2s and can recognize multiple substrate proteins. Substrate ubiquitination can take many forms, including monoubiquitination, oligoubiquitination and various types of polyubiquitin chains (Fig. 2). To date, for the Trim-NHLs there is experimental evidence for polyubiquitination activity using Lys48 linkages, one of seven lysines available in ubiquitin for chain elongation.^21-23,36,37 This is the classical signal for recognition and degradation by the 26S proteasome. Alternative linkages are nonproteolytic and alter substrate function, for example in directing protein traffic to vesicles (monoubiquitination) or by acting as a scaffold for protein-protein interactions (Lys63 polyubiquitination). Since linkage specificity is primarily a function of the E2, an E3 can have dual capacity and there is experimental support for substrate monoubiquitination via Trim32.³⁸

Functional Analysis of Individual Trim-NHL Family Members

Brat

The Drosophila protein Brat (Brain tumor) is perhaps the best studied of the Trim-NHL proteins, due to its dual role as an embryonic patterning gene and as a tumor suppressor in the larval brain. brat mutants also have defects in male and female fertility, cuticle formation and occasionally form melanotic tumors,⁶⁴ a constellation reminiscent of lin-41 and dappled, respectively. During early embryonic development Brat is required for the establishment of an anterior-posterior gradient in the translation of hunchback (hb) mRNA, a maternal effect gene and Zinc finger transcription factor.³² In this role as translational regulator, Brat participates in complex formation with the translational regulator Pumilio and the sequence specific RNA-binding protein Nanos. Despite detailed information regarding the structural features of the regulatory complex, less is known about the mechanistic contribution of Brat to translational control.³³ Sonoda and Wharton have suggested that Brat may assemble on multiple mRNAs by engaging in combinatorial interactions with RNA-binding proteins other than Nanos,³² but this has not yet been confirmed experimentally. This suggestion may, however, prove to be correct in light of the discovery that Brat interacts with the miRISC effector protein Ago1,⁶⁵ as discussed in more detail in the section on Mei-P26.

The most striking feature of brat mutants is the growth of larval tumors in the optic centers that can attain ten times the normal size.⁶⁴ Frank et al found that ectopic Brat inhibits RNA synthesis and cell proliferation while increasing cell size in several organ contexts.⁶⁶ However, Brat is primarily expressed in the embryonic PNS, the ventral nerve cord and the developing brain.³¹ Even within the CNS, the tumor origin was later shown to be confined to a subset of neural progenitors⁶⁷ with the characteristics of transit amplifying cells.⁶⁸ A recent review of the relevant issues in the larval nervous system is highly recommended⁶⁹ (see also ref. 70) and can only be briefly treated here. In the basic scheme, the intrinsic apical-basal polarity of neuroblasts is used as a guide to asymmetrically sort cell fate determinants to daughter cells (Fig. 3a). Different regions of the fly brain execute specific programs of proliferation and specification, but the basic scenario of asymmetric division is that the apical daughter retains neuroblast character while the basal daughter, called a ganglion mother cell (GMC), is smaller and destined for cell cycle exit and terminal differentiation. Segregation of cell fate determinants requires the action of apical proteins in the mother cell, including Par-3, Par-6 and atypical Protein Kinase C (reviewed in refs. 69,70)(Fig 3). These proteins are required for basal accumulation of a second set of proteins, which drive GMC specification. Brat is a recent entry into this group of specification factors, which includes Numb, an inhibitor of the Notch pathway and Prospero, a transcription factor that suppresses self-renewal and promotes neural differentiation.^67,71,72 Brat was shown to bind Miranda,^71,72 a scaffold protein that accumulates in a crescent at the basal pole of the mother cell destined for inheritance by the GMC (or basal neuroblast, see below). After division, Brat suppresses neuroblast markers and promotes cell cycle exit in the GMC.^67,71,72 One proposed regulatory target for Brat is the Drosophila Myc protein, a neuroblast marker that is dysregulated in brat mutants.⁷¹ A new wrinkle in this basic model comes from the discovery that the tumor lineage in brat mutants does not immediately produce a GMC but instead a secondary neuroblast⁶⁸ (Fig. 3b). This is a transit-amplifying cell that produces GMCs after further division. In this scenario, loss of Brat leads to tumors because the secondary neuroblast fails to mature. How Brat controls proliferation and maturation of these cells remains unclear, but it appears to work cooperatively and in parallel to Numb.⁶⁸ Recent evidence derived from studies of Mei-P26⁶⁵ and other Trim-NHLs to be discussed next points to a role for Brat as regulator of the miRNA pathway, but this has yet to be demonstrated.

Figure 3

Models for Trim-NHL proteins in stem cell niches. a) In the Drosophila CNS, neuroblasts (NB) divide asymmetrically to produce a smaller committed daughter termed ganglion mother cell (GMC) and a new neuroblast. An apical (A.) protein complex (or Par complex) (more...)

Trim-NHL Proteins as Regulators of the miRNA Pathway

mLin41

Based on mouse and human genetics, as discussed above, Trim2 and Trim32 are developmental regulators of the mammalian CNS. Mouse LIN-41 (mLin41) is the third mammalian Trim-NHL protein shown by genetic means to be involved in CNS morphogenesis.⁹⁷ Homozygous disruption of mLin41 in gene-trap lines revealed that mLin41 is required for embryonic viability, with a striking but uncharacterized failure of neural tube closure. Using the gene-trap as a reporter for mLin41 promoter activity in heterozygotes, strong expression was detected in the brain, dorsal root ganglia, eyes and branchial arches (and elsewhere) between E10.5 and E12.5. This corresponds closely to the period of embryonic demise in the homozygotes.⁹⁷ However, it is unclear if the craniofacial abnormalities and closure defect is specific or if it is a secondary result of growth arrest or reduced embryonic viability. Probing mLin41 expression at the protein level, we confirmed mLin41 expression in embryonic stem cells and in Oct-4⁺ cells of the E7 embryonic ectoderm.²³ After embryonic induction of let-7 and miR-125 combine to downregulate mLin41, postnatal niches of mLin41 expression were found in ciliated epithelia of the male and female reproductive tract, in male germ cells and in interfollicular epidermal stem cells. Löer et al had previously demonstrated the mLin41 protein in muscle⁵³ and, thus, the expression pattern of mLin41 seems quite analogous to that of the C. elegans protein.⁴

In pluripotent cells and in transfection assays in heterologous cells mLin41 was found to accumulate in cytoplasmic P-bodies, as confirmed by colocalization with the P-body markers Dcp1a and Hedls as well as the miRISC proteins Ago2, Mov10 and Tnrc6b.²³ This observation dovetailed with a report that the C. elegans protein copurifies with the Dicer protein.⁹⁸ Association with Dicer was confirmed for mLin41 and extended to Ago2.²³ The interaction surface was mapped to the coiled-coil domain, suggesting the NHL domain is free to participate in additional protein-protein interactions. Although the association with Ago2 was studied in greater detail, cotransfection assays with tagged proteins confirmed interaction with Ago1 and Ago4. mLin41 is thus the sixth Trim-NHL protein shown to interact with one of the Argonautes (Mei-P26, Brat, Dappled,⁶⁵ NHL-2⁸² and Trim32³⁷). Unlike the others, however, mLin41 was shown to mediate degradative ubiquitination of Ago2.²³ First, in an autoubiquitination assay E3 ubiquitin ligase activity was confirmed, with the same E2 preference displayed by Trim2 (UbcH5a).²² Adding immunoprecipitated Ago2 to the assay led to the accumulation of polyubiquitinated Ago2. In a transfection assay, Ago2 ubiquitination was dependent on an intact RING domain and was strongly enhanced in the presence of the proteasome inhibitor MG132. In keeping with these results, depletion of endogenous mLin41 in embryocarcinoma cells decreased Ago2 ubiquitination and increased steady state Ago2 levels.²³

If mLin41 regulates Ago2 turnover and potentially other miRISC components, it should also act as a general inhibitor of miRNA-mediated silencing. Two observations support such a role. Argonautes are thought to be limiting for miRNA-mediated silencing and ectopic Ago2 expression enhances miRNA accumulation,⁹⁹ most likely by protecting small RNAs from ribonucleolytic degradation.¹⁰⁰ mLin41 was shown to block the ability of Ago2 to increase let-7 levels.²³ It will be interesting to see if a similar mechanism is responsible for the global reduction in miRNA levels observed after overexpression of Mei-P26.⁶⁵ Furthermore, mLin41 reduced silencing mediated by let-7, miR-124 or miR-128 in reporter assays. Interference with miRNA activity was dependent on an intact RING domain and could be compensated by increased expression of Ago2.²³ This finding was expanded by demonstrating that mLin41 cooperated with Lin28 in suppressing let-7 in the reporter assay, providing evidence for dual negative autoregulatory loops in the post-transcriptional control of let-7 in pluripotent cells (see Fig. 4a and 4b for a schematic view).

Figure 4

Summary of miRNA regulation during stem cell differentiation. a) In committed cells such as neural progenitors and their offspring, miRNA precursors such as prelet-7 are processed by a core complex of Dicer, Trbp and one of the Argonaute proteins (Ago). (more...)

Conclusion

Trim-NHL proteins serve as pleiotropic regulators of developmental processes and cell function. In development, a picture is emerging in which Trim-NHL proteins coordinate miRNA activity to sequentially drive transitions between self-renewal, commitment and terminal differentiation of stem cells. The picture is incomplete, in part because recent advances have come from parallel studies in C. elegans, D. melanogaster and mammals so that the issues of sequential action and redundancy have not yet been thoroughly addressed for any one developmental context. For example, in C. elegans LIN-41 and NHL-2 have opposite roles in the heterochronic pathway governing stem cell maturation in the hypodermis and vulva.⁸² Assuming that LIN-41 is an inhibitor of miRNA pathway activity, as has been shown for the mouse protein, then LIN-41 most likely blocks the acquisition of adult cell fates in the hypodermis at least in part by inhibiting full let-7 activity (the primary miRNA driver of maturation in progenitor populations). Since LIN-41 is itself a let-7 target,⁴ this is yet another example of a double negative loop enhancing the potency of a miRNA-target gene interaction (see Fig. 4). By promoting miRNA activity, NHL-2 can flip this switch and unleash the full effects of let-7 on downstream heterochronic regulators.

Specification of mouse embryonic stem cells (ESCs) appears to proceed by a similar mechanism. In undifferentiated, pluripotent cells self-renewal is reinforced by a regulatory loop comprising a unique class of ESC-specific miRNAs and transcription factors such as Oct-4, Nanog and Myc.^91,101 In ESCs, let-7 is inhibited by the combined action of Lin28 and Lin41²³ (reviewed in ref. 102 and in Chapter 7). The importance of let-7 is threefold. First, the impact of let-7 on the proteome is amplified due to direct targeting of miRNA pathway genes.^{23,96,103-105} Second, let-7 targets cell cycle regulators, thereby directly influencing proliferation.¹⁰⁶ Third, let-7 suppresses the transcriptional program of transcription factors required for the maintenance of pluripotency and self-renewal by both direct (c-Myc, N-Myc) and indirect mechanisms (Oct-4, Nanog, Sox2, Tcf3).⁹¹ By analogy to the opposing action of LIN-41 and NHL-2 in C. elegans, it is reasonable to expect a counterpoint to mLin41 that activates the miRNA pathway during commitment. In neural lineages Trim32 fulfills two of the expected criteria: it activates a select class of miRNAs, including let-7a and suppresses c-Myc.³⁷

The dual function exerted by at least some Trim-NHLs in the ubiquitin and miRNA pathways should provide a framework for exploring their functional pleiotropy. It will be important to determine whether miRISC association is common to all family members. Within the miRISC, evidence from a variety of sources suggests that Ago2 is not the only substrate for ubiquitination.^23,107,108 The ubiquitination of additional miRISC components could certainly affect more than just the turnover rate of the individual proteins in the complex. Complex assembly, activity and intracellular compartmentalization might also be influenced. One obvious possibility is that the ability of many Trim-NHLs to associate with cytoskeletal components in a variety of cellular compartments may reflect a role in miRISC transport.^108-111 Alternatively, the association of Wech with focal adhesions or Trim3 with CART may be evidence of miRISC independent activities.

A recent study directly assayed functional redundancy and cooperation among the five Trim-NHLs in C. elegans. Mutations in four of the five (with the exception of LIN-41) were able to suppress a mutation in the embryonic polarity gene par-2, suggesting substantial functional redundancy in this pathway.¹⁰ However, other specific functions differed¹⁰ and the function of NHL-2 in the heterochronic pathway later in development appears to be independent of NCL-1, NHL-1 or NHL-3.⁸² In the mouse, loss of Lin41 cannot be compensated and leads to embryonic lethality.⁹⁷ On the other hand, no obvious defect in neurogenesis was reported after deletion of Trim32,⁴⁴ in contrast to the direct assays of neuronal differentiation reported by Schwamborn et al.³⁷ In this case Trim2 and Trim3 may functionally compensate for Trim32 deficiency. Coiled-coil domains can mediate heteromeric interactions,⁹ but there is no evidence yet for cooperativity among Trim-NHLs or between Trim-NHLs and other E3 ligases. If Trim-NHLs can form heterodimers, then the RING-less proteins Brat and Dappled/Wech might promote ubiquitination indirectly.

The discovery of ubiquitin-mediated regulation of miRNAs has revealed a nexus between two of the cells' most powerful post-transcriptional regulatory pathways. The miRNA pathway appears to make extensive use of autoregulatory feedback loops to adjust miRNA activity during development and stem cell differentiation. Several of the key molecules in these loops are involved in tumor formation (Brat in Drosophila or let-7, Lin28 and Myc in humans), underscoring their potential relevance for human disease. Two developmental disturbances have been linked to Trim32, more may be uncovered as the other human Trim-NHLs are studied. In addition to development, regulation of Trim-NHL protein synthesis or activity, for example in response to cell signaling, may allow cells to modulate miRNA efficiency to maintain cellular homeostasis.

Acknowledgements

F.G.W., E.F. and A.R. received support from the DFG Collaborative research project 665; E.C. and A.R were supported by the DFG Graduate School 1123.

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