The Physiological Role and Toxicological Significance of the Non

CBC-08292; No of Pages 10 Comparative Biochemistry and Physiology, Part C xxx (2017) xxx–xxx

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Comparative Biochemistry and Physiology, Part C

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The physiological role and toxicological signiﬁcance of the non-metal-selective cadmium/copper-metallothionein isoform differ between embryonic and adult helicid snails

Veronika Pedrini-Martha a, Raimund Schnegg a, Pierre-Emmanuel Baurand b, Annette deVauﬂeury b,c, Reinhard Dallinger a,⁎ a Department of Zoology, University of Innsbruck, Technikerstraße 25, 6020 Innsbruck, Austria b Chrono-Environnement, UMR 6249 University of Franche-Comté, 16 route de Gray, 25030 Besançon cedex, France c Department of Health Safety Environment, avenue des Rives du Lac, BP179, 70003 Vesoul cedex, France article info abstract

Article history: Metal regulation is essential for terrestrial gastropods to survive. In helicid snails, two metal-selective metallo- Received 16 November 2016 thionein (MT) isoforms with different functions are expressed. A cadmium-selective isoform (CdMT) plays a Received in revised form 16 February 2017 major role in Cd2+ detoxification and stress response, whereas a copper-selective MT (CuMT) is involved in Cu Accepted 16 February 2017 homeostasis and hemocyanin synthesis. A third, non-metal-selective isoform, called Cd/CuMT, was first charac- Available online xxxx terized in Cantareus aspersus. The aim of this study was to quantify the transcriptional activity of all three MT genes in unexposed and metal-exposed (Cd, Cu) embryonic Roman snails. In addition, the complete Cd/CuMT Keywords: fi Gastropoda mRNA of the Roman snail (Helix pomatia) was characterized, and its expression quanti ed in unexposed and Helix pomatia Cd-treated adult individuals. In embryos of Helix pomatia, the Cd/CuMT gene was induced upon Cu exposure. Cantareus aspersus Its transcription levels were many times higher than that of the other two MT genes, and also exceeded by far Development the Cd/CuMT mRNA concentrations of adult snails. In the hepatopancreas of adult Roman snails, no Cd/CuMT Cadmium could be detected at the protein level, irrespective of whether the snails had been exposed to Cd or not. This con- Copper trasts with the situation in the near relative, Cantareus aspersus. It appeared that the 3′-UTR of the Cd/CuMT fi Detoxi cation mRNA differed largely between Cantareus aspersus and Helix pomatia, being larger in the latter species, with a 5′ and 3′UTRs number of putative binding sites for proteins and miRNAs known to inhibit mRNA translation. We suggest this mRNA binding proteins miRNA as a possible mechanism responsible for the lack of Cd/CuMT protein expression in adult Roman snails. © 2017 The Author(s). Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

1. Introduction pigment of terrestrial gastropods (Van Holde et al., 2001). Non-essential harmful metals, on the other hand, must be inactivated. This is the case The Roman snail (Helix pomatia) is a key species of terrestrial habi- for cadmium (Cd) which can negatively affect adult and embryonic tats in central Europe. Roman snails are in close contact with the soil snails, being able to cross the eggshell (Druart et al., 2010), as well as di- substrate throughout their whole lifespan from eggs, buried a few cen- gestive epithelia of adult individuals. For example, Cd can reduce the timeters in the substrate, to mature snails thriving on the soil surface. fertility of terrestrial snails by decreasing the number of laid eggs and Due to ﬂuctuating environmental meteorological conditions, the water by delaying the egg laying cycle (Gimbert et al., 2008), by inhibiting supply and availability of metallic trace elements in the upper soil hori- the hatching of embryos (Druart et al., 2010; Baurand et al., 2014)and zon may vary according to the intensity of precipitations. Moreover, the reducing their growth rate (Baurand et al., 2014). water balance of terrestrial snails is subjected to seasonal alterations Metallothioneins (MTs) are ubiquitous metal binding proteins, in- due to intermittent periods of activity and aestivation (Pedrini-Martha volved in metal homeostasis and detoxiﬁcation (Dallinger et al., 1997; et al., 2016). Hence, the handling and regulation of metallic trace ele- Egli et al., 2006), as well as in stress response (Chabicovsky et al., ments within snail tissues becomes an important task for the species vi- 2004; Fu and Miao, 2006; Egg et al., 2009; Pedrini-Martha et al., ability. The intracellular availability of essential trace elements needs to 2016). They are characterized by a low molecular weight, a low abun- be regulated by homeostatic mechanisms. Copper (Cu) homeostasis, for dance or lack of aromatic amino acids and a high content of cysteines, example, is linked to the synthesis of hemocyanin, the respiratory arranged within the peptide chain in typical Cys-Xaa-Cys motifs. The sulfhydryl groups of the cysteine residues are essential for the formation ⁎ Corresponding author. of metal-thiolate clusters in which mono- and divalent metal ions are E-mail address: [email protected] (R. Dallinger). bound (Kojima et al., 1999). In helicid snails the development of

http://dx.doi.org/10.1016/j.cbpc.2017.02.009 1532-0456/© 2017 The Author(s). Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

2.1. Cadmium exposure and sampling of adult Helix pomatia 2.3. Characterization and quantification of Cd/CuMT mRNA from adult and embryonic Helix pomatia Adult Roman snails were obtained from a commercial dealer (Thü- ringer Weinbergschnecke, Germany) and acclimatized in transparent 2.3.1. RNA isolation octagonal plastic boxes (diameter: 12 cm; height: 6 cm) on garden For RNA isolation of adult Roman snails, hepatopancreas tissue ali- earth substrate (Bauhaus-Gartenerde, Austria, with a reported Cd con- quots were homogenized in TRIzol® Reagent (Ambion™ by Life Tech- centration of b0.3 μg/g dry weight) (information provided by the sup- nologies) using the Ultra-Turrax T25 (Janke & Kunkel IKA® plier) containing lime powder (CaCO3) under constant conditions Labortechnik, Germany). After DNase I digestion (Invitrogen) RNA was (18 °C; 12:12 h light:dark). Prior to exposure snails were fed regularly cleaned up with the RNeasy MiniElute Kit (Qiagen, Hilden, Germany). with uncontaminated lettuce (Lactuca sativa) and sprayed with water For RNA isolation of 7 day-old embryos, three eggs were pooled, where- for one week. Subsequently, control snails were fed with uncontaminat- as for 20 day-old embryos one individual was used. RNA was isolated ed lettuce and Cd-exposed snails were fed with Cd-enriched lettuce using the RNeasy® Mini Kit (Qiagen, Hilden, Germany) according to every day as described in Pedrini-Martha et al. (2016). For MT expres- the manufacturer's instructions. sion studies by means of quantitative real-time PCR, five controls and five Cd-fed snails were dissected on a cooled aluminium-plate, which 2.3.2. RACE PCR of the 5′ and 3′ UTR of the Cd/Cu-MT was cleaned with RNase AWAY (Sigma-Aldrich). Tissue aliquots from The coding region of the Cd/CuMT gene was sequenced by us previ- the hepatopancreas (approx. 1 mg fresh wt.) were transferred to ously and is already available in GenBank as two allelic variants (V1 and RNAlater™ Solution (Invitrogen by ThermoFisher Scientific, USA) and V2) under the accession numbers GU111728 (V1) and GU111729 (V2). stored at −80 °C. To obtain the full length cDNA of the Cd/CuMT isoform gene, the For MT protein purification, adult Roman snails were kept under lab- SMARTer™ Race cDNA Amplification Kit (Clontech, Canada) was ap- oratory conditions (18 °C; 12:12 h light:dark) in plastic boxes on moist- plied. Total RNA (300–700 ng) from three adult Roman snails was ened garden soil substrate. 15 animals were exposed to Cd over a period used for generation of RACE-Ready cDNA according to the of 14 days by feeding on Cd-enriched lettuce every second day prepared manufacturer's instructions. For the rapid amplification of the 5′ and as described in Dallinger et al. (2004). 15 control animals were reared the 3’UTRs, gene specific primers were employed as follows: GSP1, 5′- under the same conditions as above by feeding on uncontaminated let- ATG TGG CAA GAT TCC TGT GCG GCT GTG G-3′; and GSP2, 5′-AAC tuce. Average Cd concentration in the metal-enriched feed was about AGC AA C CCC TGC AGC TGC GGC GAC G-3′. PCR was carried out

Table 1 Primer design for qRT PCR of all three MT isoform genes of Helix pomatia. Sequences are shown in 5′–3′ direction, lengths (bp) and working concentrations (nM) of the gene speciﬁc primers are also reported. Abbreviations: bp = base pairs; nM = nanomolar; S = forward primer; AS = reverse primer.

Name Sequence 5′–3′ Length [bp] Concentration [nM]

Cd/CuMT S 5′-CGG CGA CGA CTG CAA ATG-3′ 18 900 Cd/CuMT AS 5′-GGA GCA CTG GTT GCC ACA CT-3′ 20 900 CuMT S 5′-CCT TGC AGC TGT GGT AAC GA-3′ 20 900 CuMT AS 5′-CAA GAA CTG CAT CGG TCA CAA-3′ 21 300 CdMT S 5′-AAA GTG CAC CTC AGC TTG CA-3′ 20 900 CdMT AS 5′-GCA GGC GGC ACA TGT ACA G-3′ 19 300

Fig. 1. Transcription of all three MT isoform genes in 7 and 20 day-old unexposed embryos of Helix pomatia. Transcription levels are expressed as mRNA copy numbers/10 ng total RNA, represented by bar graphs (means with standard deviations) for the CdMT gene (white bars), the Cd/CuMT gene (grey bars) and the CuMT gene (black bars) in snail embryos at 7 dpf (left hand side; n =7–8) and 20 dpf (right hand side; n =6–7). Double asterisks indicate significant differences in mRNA expression between the Cd/CuMT gene and the two other genes (CdMT and CuMT). The single asterisk indicates significant differences in transcription levels between the CdMT as well as the Cd/CuMT compared to the CuMT genes. The significance level was set at P ≤ 0.05. Abbreviations: dpf = days post fertilization.

Fig. 2. Transcription of all three MT isoform genes in 7 and 20 day-old Roman snail embryos exposed to (A) Cd or (B) Bordeaux mixture. Transcription levels are expressed as mRNA copy numbers/10 ng total RNA, represented by bar graphs (means with standard deviations) in controls (Co, white bars) and Cd-exposed embryos (Cd, black bars) for the CdMT,theCuMT and the Cd/CuMT genes on the left hand side (A) and for controls (Co, white bars) and embryos exposed to the Bordeaux mixture (BM, grey bars) for the three MT genes on the right hand side (B). For each gene, results for 7 day-old embryos (controls n =7–8; exposed n =3–4) and 20 day-old embryos (controls n =6–7; exposed n =4–5) are presented. Signiﬁcant differences (P ≤ 0.05) are indicated by an asterisk. Abbreviations: dpf = days post fertilization.

Fig. 3. Sketch and sequence of the full-length Cd/CuMT cDNA of Helix pomatia. A schematic map of the mRNA shows the length of the 5′ untranslated region (5′UTR) (yellow), the coding region (green) and the 3′ untranslated region (3′UTR) (blue). In addition, the full-length cDNA and the respective amino acid sequence of the translated MT peptide (one-letter code, in bold) are shown. Single nucleotide polymorphisms (SNPs) are indicated by bold italic, underlined letters. The start and the stop codons are highlighted in bold, red letters. Ambiguous nucleotide specifications:Y=TorC;K=TorG;R=GorA. mixture (Fig. 2B). This effect disappeared, however, in 20 day-old Cu- (SNPs) at positions 224 (C/T), 244 (C/T) and 329 (G/A) (Fig. 3). Of exposed embryos (W = 7, P = 0.230) (Fig. 2B). In contrast to the these, only the first leads to an amino acid substitution in the Cd/ CdMT and Cd/CuMT genes, no transcription at all was observed for the CuMT (Val/Ala, respectively). Within the 3’UTR, a SNP was detected at CuMT gene in 7 day-old embryos. Even in 20 day-old unexposed embry- position 571 (G/A). In the presence of an A (instead of G), the total os was the transcription rate of the CuMT gene significantly lower, with length of the 3’UTR is shortened by one base triplet from 227 to an average of 2372 mRNA copy numbers/10 ng total RNA, compared to 224 bp. The Cd/CuMT protein sequence of Helix pomatia shows a high the respective CdMT (W = 2, P = 0.005) and the Cd/CuMT gene values degree of similarity with the already known Cd/CuMT of the near rela- (W = 0, P =0.001)(Fig. 1). No impact was observed on the CuMT tive species Cantareus aspersus, with a reciprocal sequence identity of mRNA transcription neither in Cd-exposed snail embryos (Fig. 2A) nor 92.19% (Fig. 4). in embryos exposed to the Bordeaux mixture (Fig. 2B). Overall, there was a clear increase of mRNA expression for all three MT isoform 3.3. MT gene upregulation and MT protein expression in the hepatopancre- genes in unexposed snail embryos with increasing development time as of adult Roman snails (Cd/CuMT (ANOVA on ranks, P = 0.001) and CdMT (ANOVA on ranks, P ≤0.001)). In order to explore the Cd-dependent upregulation and metal detoxification potential of the Cd/CuMT system of Helix pomatia,geneexpres- 3.2. Characterization of the full-length Cd/CuMT mRNA sequence from adult sion studies on the transcriptional level and MT protein induction at the Helix pomatia translational level were performed in the hepatopancreas of control and Cd-exposed adult Roman snails. These results were then compared with The sequence of the Cd/CuMT full-length mRNA was obtained by the respective induction and expression data of the already known RACE PCR. The complete mRNA is 571 bp long. It is composed of a CdMT isoform gene and protein. As shown in Fig. 5A, no significant up- 5’UTR with 149 bp followed by the coding region with 195 bp and a regulation of the Cd/CuMT gene of Helix pomatia could be detected upon 3’UTR with 227 bp (Fig. 3). In the 5′ UTR region, a variable dinucleotide Cd exposure (two tailed P = 0.084; one tailed P =0.042)(Fig. 5A). In was detected at positions 133 and 134 (combinations TT, GT, TG or -T). contrast, a 3-fold upregulation of the CdMT mRNA transcription was ob- The coding region contains three single nucleotide polymorphisms served in Cd-exposed individuals (W = 0, P =0.016)(Fig. 5B).

Fig. 4. Sequence alignment of the Cd/CuMT peptide sequence of Helix pomatia and Cantareus aspersus. The two variants from Helix pomatia (V1: GU111728; V2: GU111729) with the already known Cd/CuMT sequence of Cantareus aspersus (EF206312) are aligned. The conserved positions of cysteine residues are highlighted in green. The putative linker region between the two protein domains is marked by a box. The polymorphic site within Helix pomatia at position 32 (valine or alanine) is highlighted in pink. Variable amino acids between the two helicid species are marked in yellow.

exposed individuals (Fig. 6B) besides a CuMT peak that was always present in the hepatopancreas tissue of both, control and Cd-fed Roman snails (Fig. 6).

4. Discussion

4.1. MT transcription patterns in Roman snail eggs suggest a role for the Cd/ CuMT gene in embryonic development

In the present study, the transcription of all three MT isoform genes (CdMT, CuMT and Cd/CuMT) was quantified for the first time in embryos of Helix pomatia. Overall, it appeared that the mRNA levels of the Cd/ CuMT gene in embryonic snails were consistently higher compared to the respective transcription levels of adult individuals of Helix pomatia (Fig. 7). This indicates that the Cd/CuMT gene may play a major role in embryonic development of Helix pomatia, whereas it may be of minor physiological importance in adult Roman snails. In the related helicid species Cantareus aspersus too, the transcription of the Cd/CuMT gene in embryonic snails was significantly elevated compared to the respec- Fig. 5. Transcriptional upregulation of the Cd/CuMT and the CdMT genes in the tive transcription levels of this gene in the hepatopancreas of adult indi- hepatopancreas of control and Cd-exposed adult Roman snails. mRNA quantification viduals (Fig. 7)(Höckner et al., 2011; Palacios et al., 2011; Baurand et al., values of the Cd/CuMT (A) and the CdMT (B) genes are shown by bar graphs (means 2015, 2016a). The expressed Cd/CuMT protein of helicid snails can bind with standard deviations) in control individuals (Co, white bars) and Cd-exposed snails both, Cu+ and Cd2+ ions simultaneously, as shown in previous studies (Cd, black bars) (n =4–5). Significant differences (P ≤ 0.05) between control and Cd- exposed snails are indicated by an asterisk. for Cantareus aspersus (Hispard et al., 2008; Höckner et al., 2011). This may be advantageous for very young embryonic snails (1 or 7 dpf), where the CuMT gene is not yet expressed at all, or is present at only Furthermore, even the basal CdMT gene expression in unexposed indi- low transcription levels (present study; see also Baurand et al., 2015). viduals was about 14-fold higher (W = 0, P = 0.008) compared to the Under such conditions, the expressed Cd/CuMT gene of embryonic snails Cd/CuMT gene transcription. At the protein level, however, no Cd/ may serve both, Cu detoxification and homeostatic Cu regulation. This CuMT protein could be detected in the hepatopancreas of adult hypothesis is supported by the fact that in the present study, the Cd/ Roman snails, neither in control nor in Cd-exposed animals (Fig. 6). In- CuMT gene of 20 day-old Roman snail embryos is upregulated upon Cu stead, a distinct induction peak of the CdMT was observed in metal- exposure. Similar findings were reported from embryos of Cantareus

Fig. 6. Reverse-phase HPLC elution proﬁles of MT isoforms in the hepatopancreas of control (A - Co) and Cd-exposed adult Roman snails (B - Cd). Absorption (A) at 254 nm and the elution gradient (% solvent B) (upper graphs) as well as Cd and Cu concentrations (lower graphs) in eluted peaks are plotted against elution time. Note that distinct elution peaks were detected, in dependence of the kind of metal treatment, only for the CdMT and CuMT isoforms, but not for the presumed Cd/CuMT isoform.

Fig. 7. Comparison of the MT gene transcription levels in unexposed embryonic snails and hepatopancreatic tissue of adult snails from the two helicid species Helix pomatia and Cantareus aspersus. CdMT mRNA concentrations (factor = 1) were compared to the CuMT and the Cd/CuMT mRNA transcription levels and the corresponding factors were calculated (A) for the hepatopancreatic of unexposed adult individuals as well as for (B) 7 and 20 dpf old unexposed embryonic snails. In addition, a table shows if the respective MT protein was detected (tick) or not (cross) in the hepatopancreas of adult snails for both species (see Fig. 4; Dallinger et al., 2004; Hispard et al., 2008; Palacios et al., 2011). MT gene expression data of Cantareus aspersus embryos was modiﬁed from Baurand et al. (2016a). For the hepatopancreatic tissue of adult individuals from both species data from this study and from Palacios et al. (2011) were combined. Abbreviations: C.a. = Cantareus aspersus;H.p.=Helix pomatia.

Fig. 8. Schematic comparison of the 3′UTR sequences of the Cantareus aspersus and Helix pomatia Cd/CuMT mRNAs. In the aligned sequences, regulatory elements and theoretical attaching sites for RNA binding proteins are highlighted in different colours as follows: = KH domain-containing, RNA-binding, signal transduction-associated protein 3 (KHDRBS3); = ELAV-like protein 1; = Pumilio homolog 2 (Pum2); = Y box binding protein 2 (ybx2-α); = K-box; XXX = Musashi binding element (MBE); = stem loop structure; = polyadenylate-binding protein 1 (PABPC1); = pseudoknot like structure. degree of amino acid sequence identity indicates that the metal binding Also in adult snails of Cantareus aspersus, the expression level of the features of the Cd/CuMT homologs may not vary significantly between Cd/CuMT isoform is rather low (Hispard et al., 2008)(Fig. 7A), and even Helix pomatia and Cantareus aspersus. Cd exposure has no significant impact on the transcription of the Cd/ CuMT gene (Höckner et al., 2011; Palacios et al., 2011). It was concluded that the contribution of this isoform to the metal balance and detoxifica- 4.3. The Cd/CuMT isoform shows similar mRNA transcript concentrations tion in adult Cantareus aspersus snails may be negligible (Höckner et al., but different protein expression levels in the hepatopancreas of adult helicid 2011). However, it cannot be excluded that the Cd/CuMT gene of adult snails helicid snails may be induced by exposure to non-metallic stressors or by stressful physiological conditions. Further studies of the Cd/CuMT In the present study, the transcription levels of the Cd/CuMT mRNA isoform at both, the transcriptional and translational levels and in di- of untreated and metal-exposed adult Helix pomatia snails were quanti- verse tissues of adult helicid snails, will be required to test these fied for the first time. Cd/CuMT mRNA concentrations in the hepatopan- hypotheses. creas of adult Roman snails were consistently lower than the expression levels of the CdMT gene, irrespective of the kind of exposure (control or 4.4. Species-specific differences in Cd/CuMT mRNA structure: a possible rea- Cd-exposed snails) (Fig. 5). As already shown in previous studies, low son for varying mRNA translation efficiencies between Helix pomatia and transcription levels are normally also detected for the CuMT gene of Cantareus aspersus? adult Roman snails (Palacios et al., 2011)(Fig. 7). Moreover, the visual pattern shows an upregulation of the Cd/CuMT gene due to Cd stress An important difference exists between adult individuals of Helix and a significant one tailed P-value (Fig. 5A). In contrast to adult pomatia and Cantareus aspersus in their capacity to express the Cd/ Cantareus aspersus snails (Hispard et al., 2008), however, no expression CuMT protein. As already shown above, no Cd/CuMT protein could be of the Cd/CuMT protein could be detected in the hepatopancreas of detected in adult Roman snails (Fig. 6) in spite of the proven transcrip- adult Roman snails after HPLC fractionation, in spite of the isoform-spe- tion of its gene, at least at a low mRNA level. In contrast, a distinct Cd/ cific sensitivity of the method applied (Fig. 6). Instead, the presence of CuMT peak containing both, Cd2+ and Cu+ ions, was observed previ- distinct expression peaks of the CdMT and CuMT isoforms was observed ously in adult Cd-exposed individuals of Cantareus aspersus (Hispard (Fig. 6). In earlier studies too, the absence of a Cd/CuMT protein was et al., 2008). A possible, although speculative, explanation for this differ- confirmed in several organs of adult Helix pomatia, under both control ence may reside in the differential structure of the Cd/CuMT mRNAs be- andCdexposureconditions(Dallinger et al., 2004; Palacios et al., 2011). tween the two species. While the 5’UTR and the coding region of the Cd/ CuMT mRNA are almost identical between the two species (87.21% identity), there are distinct species-specific sequence differences in Table 2 the 3’UTR. In particular, the Cd/CuMT mRNA 3’UTR of Helix pomatia is Comparison of the abundance of regulatory mRNA features within helicid MT isoforms. significantly longer compared to the respective mRNA region of The abundance of regulatory elements, theoretical sites for RNABPs and miRNAs of the 3′ untranslated region (3′UTR) are listed. Abb.: H.p. Helix pomatia (KY420171); C.a. Cantareus aspersus containing a higher number of regulatory elements Cantareus aspersus (EF206312). as well as potential binding sites for RNA binding proteins (Fig. 8). In fact, hypothetical binding sites for proteins with a potential to inhibit Name 3′UTR [bp] RegEl RNABPs miRNA the translational process are present only in the Cd/CuMT mRNA C.a. Cd/CuMT 110 7 9 2 3’UTR of Helix pomatia but not in that of Cantareus aspersus. These fac- H.p. Cd/CuMT 227 9 16 15 tors include, for example, suggested binding sites for the mRNA binding

Please cite this article as: Pedrini-Martha, V., et al., The physiological role and toxicological significance of the non-metal-selective cadmium/ copper-metallothionein isoform differ bet..., Comp. Biochem. Physiol., C (2017), http://dx.doi.org/10.1016/j.cbpc.2017.02.009 V. Pedrini-Martha et al. / Comparative Biochemistry and Physiology, Part C xxx (2017) xxx–xxx 9 proteins Pum2 and ybx2-α,aswellasPABPC1(Fig. 8). It was shown that methodical background. Environ. Toxicol. Chem. 23:890–901. http://dx.doi.org/10. 1897/03-100. these proteins are able to repress mRNA translation, with subsequent Dallinger, R., Chabicovsky, M., Hödl, E., Prem, C., Hunziker, P., Manzl, C., 2005. Copper in accumulation of the respective transcript (Matsumoto and Wolffe, Helix pomatia (Gastropoda) is regulated by one single cell type: differently responsive 1998; Miles et al., 2012; Van Etten et al., 2012). Moreover, a K-box metal pools in rhogocytes. Am. J. Phys. Regul. Integr. Comp. Phys. 289:R1185–R1195. fi ′ http://dx.doi.org/10.1152/ajpregu.00052.2005. was identi ed in the 3 UTR of the Cd/CuMT mRNA from Helix pomatia. Druart, C., Scheifler, R., de Vaufleury, A., 2010. Towards the development of an This conserved hexamer nucleotide sequence can be part of miRNA seed embryotoxicity bioassay with terrestrial snails: screening approach for cadmium sequences (Lewis et al., 2005) and is involved in negative transcription- and pesticides. J. Hazard. Mater. 184:26–33. http://dx.doi.org/10.1016/j.jhazmat. al regulation, too (Lai et al., 2005). The most prominent finding, howev- 2010.07.099. Druart, C., Scheifler, R., Millet, M., De Vaufleury, A., 2012. Landsnail eggs bioassays: a new er, was the abundance of miRNA binding sites to a much greater extent tool to assess embryotoxicity of contaminants in the solid, liquid or gaseous phase of in the 3’UTR of the Helix pomatia Cd/CuMT mRNA compared to its near soil. Appl. Soil Ecol. 53:56–64. http://dx.doi.org/10.1016/j.apsoil.2011.11.006. relative (Supplementary file 1). Together with the above-mentioned Egg, M., Höckner, M., Brandstätter, A., Schuler, D., Dallinger, R., 2009. Structural and bioinformatic analysis of the Roman snail Cd-metallothionein gene uncovers putative inhibitory binding sites, this could indicate that Cd/CuMT molecular adaptation towards plasticity in coping with multifarious environ- translation in the Roman snail is repressed giving rise to the detected ac- mental stress. Mol. Ecol. 18:2426–2443. http://dx.doi.org/10.1111/j.1365-294X. cumulation of mRNA (Table 2; Supplementary file 1). As a consequence, 2009.04191.x. ’ Egli, D., Yepiskoposyan, H., Selvaraj, A., Balamurugan, K., Rajaram, R., Simons, A., the respective 3 UTR of the accumulated Cd/CuMT transcript may as- Multhaup, G., Mettler, S., Vardanyan, A., Georgiev, O., et al., 2006. A family knockout sume regulatory functions (Hughes and Jones, 2008; Karapetyan et al., of all four Drosophila metallothioneins reveals a central role in copper homeostasis 2013) by acting, for example, as a natural miRNA sponge (Fang et al., and detoxification. Mol. Cell. Biol. 26:2286. http://dx.doi.org/10.1128/MCB.26.6.2286. Espart, A., Marín, M., Gil-moreno, S., Palacios, Ò., Amaro, F., Gutiérrez, J.C., Capdevila, M., 2013). Translational inhibition of mRNAs affecting protein abundance Atrian, S., 2015. Hints for Metal-Preference Protein Sequence Determinants: Different was also described in previous studies for Caenorhabditis elegans Metal Binding Features of the Five Tetrahymena thermophila Metallothioneins. :p. 11 (Seggerson et al., 2002), Drosophila (Miles et al., 2012) or human cells http://dx.doi.org/10.7150/ijbs.11060. Fang, L., Du, W.W., Yang, X., Chen, K., Ghanekar, A., Levy, G., Yang, W., Yee, A.J., Lu, W.Y., (Van Etten et al., 2012). 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