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Cell Biology International (2007) 31, 1331–1335 (Printed in Great Britain)
Hsp56 mRNA in Paracentrotus lividus embryos binds to a mitochondrial protein
Carlo Maria Di Liegro and Anna Maria Rinaldi*
Dipartimento di Biologia Cellulare e dello Sviluppo “A. Monroy”, Università di Palermo, Viale delle Scienze, Parco d'Orleans, 90128 Palermo, Italy


Abstract

We previously demonstrated that Paracentrotus lividus Hsp56 mitochondrial chaperonin is constitutively expressed during development, that it has a specific territorial distribution, both in normal and heat-shocked embryos, and that its amount increases after heat shock [Roccheri MC, Patti M, Agnello M, Gianguzza F, Carra E, Rinaldi AM. Localization of mitochondrial Hsp56 chaperonin during sea urchin development. Biochem Biophys Res Commun 2001;287:1093–98] and cadmium treatment [Roccheri MC, Agnello M, Boneventura R, Matranga V. Cadmium induces the expression of specific stress proteins in sea urchin embryos. Biochem Biophys Res Commun 2004;321:80–7]. In this study, we looked at Hsp56 mRNA during normal development and under stress conditions. The messenger is almost constantly expressed at all stages of development and its amount is steadily increased in stressed embryos. Moreover, we found, using T1 RNase protection assay, that the most proximal region of the 3′-UTR of the Hsp56 mRNA binds a 40kDa protein: this factor is more abundant in the mitochondrial extract and, more specifically, in the outer membrane of the organelle.


Keywords: Cadmium, Chaperonin, Embryo development, Heat shock, Mitochondria.

*Corresponding author. Fax: +39 0916577430.


1 Introduction

Heat-shock proteins (Hsps) have an essential role in most organisms, participating in many basic processes, such as protein folding (Ellis and Hartl, 1999). Hsps expression is activated by different stress conditions and Hsps activity probably counteracts apoptosis pathways (Beere, 2004). Mitochondrial Hsp60 chaperonin was studied in different organisms and shown to be involved in the folding of proteins imported into the mitochondrial matrix, assembly of protein complexes when required, and sorting of proteins to different locations in mitochondria, favoring the transport of imported proteins across membranes (Soltys and Gupta, 1999). Marine invertebrates are directly exposed to ever-changing environmental conditions and the induction of Hsps may constitute an important defense mechanism.

As shown by sequence similarity at protein as well as at mRNA level, Paracentrotus lividus mitochondrial Hsp56 is the homologue of vertebrate Hsp60 chaperonin and, given the evolutionary conservation of the sequence, it is believed to perform similar functions in sea urchin embryos.

In a previous study, we showed that: (i) Hsp56 is constitutively expressed at all stages of sea urchin development, (ii) in heat-shocked embryos, the chaperonin concentration increases, as shown by Western blot analysis and in situ (whole mount) immunoreaction, and (iii) its presence becomes evident also in the ectodermal layer (Roccheri et al., 2001). As previously reported, sublethal cadmium concentrations also have a clear effect on Hsp56 expression (Roccheri et al., 2004). In this paper, we analyzed the Hsp56 mRNA content, both during normal sea urchin development and in embryos that underwent heat-shock or cadmium treatment. Moreover, we identified an RNA-binding protein, more abundant in the outer mitochondrial membrane, that interacts with the Hsp56 mRNA 3′-UTR.

2 Materials and methods

2.1 Embryo cultures

Embryo cultures were carried out as described by Roccheri et al. (2001).

2.2 Protein extracts

Cell fractionation was carried out as described elsewhere (Cannino et al., 2004). Preparation of mitochondrial sub-fractions was carried out essentially as described by Schnaitman and Greenawalt (1968). Protein concentration was calculated by the method of Bradford (1976).

2.3 Western analyses

Western blotting was performed as described by Cannino et al. (2004). Protein markers were from Fermentas, polyclonal anti-rabbit Mn-SOD antibodies were from Stressgen and polyclonal anti-rabbit GAPDH antibodies were from Santa Cruz Biotechnologies. Goat anti-rabbit secondary antibodies were from Stressgen.

2.4 Northern analyses

Total RNA was prepared from P. lividus eggs or embryos at different stages of development, by the method of Chomczynski and Sacchi (1987). Northern analyses were performed according to Castiglia et al. (1994). DNA fragments used to make probes for hybridizations were radiolabeled using the “random primed DNA labeling kit” purchased from Roche, following the manufacturer's instructions. Autoradiographs were scanned and planimetries of scans were used to calculate the relative abundance of mRNAs using the ImageJ program.

2.5 Preparation of in vitro transcribed RNAs for T1 RNase assay

Different regions of the P. lividus Hsp56 cDNA (Gianguzza et al., 2000; Genbank accession no. AJ249625) were amplified by PCR and cloned between KpnI and EcoR1 or HindIII sites of Bluescript (+) SK plasmid. The oligonucleotides used for PCR amplification were: 5′-GCGCGGTACCTGAGGTGCTTATGAACCCCA-3′ forward and 5′-GCGCGGTTCGAATTCCCCTGACTTTTCCCC-3′ reverse (fragment 1906–2413); 5′-GCGCGGTACCGGCACGAGCTCAGCCGTA-3′ forward and 5′-GCGCGGGAATTCTCTCCTGCCTCCTCGTTG-3′ reverse (fragment 54–510); 5′-GCGCGGTACCGTTTTGATAAGGAGCAGAAC-3′ forward and 5′-GCGCGGAAGCTTTGAATGAAGTGCTGATAGAT-3′ reverse (fragment 2932–3422). The cloned fragments were in vitro transcribed using Riboprobe Sistem-T7 (Promega). The PEP-19 unlabeled RNA was prepared by using as a template a PEP-19 cDNA cloned by Sala et al. (2007) and identical to the one reported by Sangameswaran et al. (1989; EMBL accession number: M24852).

2.6 T1 RNase protection assay

T1 RNase protection assay was carried out according to the method described by Zaidi and Malter (1994), as modified by Izquierdo and Cuezva (1997). Briefly, cell extracts (15μg of total proteins) were incubated for 10min, at 30°C, with 1×106cpm (specific activity: 1–2×107cpm/pmol of RNA) of in vitro transcribed, radiolabeled RNA. Samples were then incubated for 30min at 37°C, with 100units of T1 RNase (Roche) to degrade the whole of the RNA except the regions protected by bound proteins; the extracts were finally exposed to a spectroline UV (254nm) lamp (Aldrich Chemical Co., Inc.), for 20min at 4°C, to cross-link RNA to proteins. The covalent radioactive complexes were analyzed by denaturing electrophoresis on sodiumdodecylsulfate (SDS)-polyacrylamide slab gels (PAGE), according to Laemmli (1970) and the gels were directly exposed to X-ray film for autoradiography, with intensifying screens, for 12–18h, at −70°C. At the end of exposure, the gels were stained with Coomassie brilliant Blue R-250 (Sigma) to confirm the loading of equal amounts of proteins per lane.

3 Results

First, we analyzed by Northern analysis total RNA from sea urchin P. lividus eggs, and embryos at different stages of development, using as a probe the 32P-labeled cDNA encoding the P. lividus Hsp56, previously isolated (Gianguzza et al., 2000; Genbank accession no. AJ249625).

As shown in Fig. 1, upper panel, the probe identifies a unique band (about 4000nt) in all the samples: Hsp56 messenger appears to be almost constantly expressed during embryonic development, from fertilization up to pluteus stage. In order to be sure that identical amounts of RNA had been loaded per lane, the membranes were stripped and re-hybridized with a 32P-labeled fragment of the gene encoding the 18S P. lividus rRNA (a gift from Dr. R. Barbieri, Fig. 1, lower panel).


Fig. 1

Northern blot analysis of P. lividus Hsp56 mRNA during development. Total RNA from eggs, and embryos at different stages of development, hybridized to Hsp56 probe (upper panel), or 18S rRNA probe (lower panel): (1) unfertilized egg; (2) four blastomeres; (3) 16 blastomeres; (4) blastula; (5) gastrula; and (6) pluteus.


We previously showed by Western blot (Roccheri et al., 2001; Roccheri et al., 2004) that Hsp56 protein is more abundant in P. lividus gastrulae grown in stress conditions. To analyze the changes in Hsp56 mRNA expression under stress conditions, normal, heat-shocked and cadmium-treated embryos were grown until gastrula or pluteus stage, and total RNAs were prepared and analyzed by Northern blot.

The results of one representative experiment are shown in Fig. 2A (upper panels): the mRNA concentration sharply increases in both heat-shocked gastrulae (lane 2) and in cadmium-treated gastrulae (lane 3), when compared with control gastrulae (lane 1). An increase was also evident when plutei under stress conditions (lanes 5 and 6) were compared with control plutei (lane 4).


Fig. 2

Hsp56 mRNA expression in normal and stressed P. lividus embryos. (A) Northern blot analyses of total RNA, hybridized to Hsp56 probe (upper panels) and 26S (lower panels). RNAs were prepared from (1) control gastrulae; (2) heat-shocked gastrulae; (3) cadmium-treated gastrulae; (4) control plutei; (5) heat-shocked plutei; and (6) cadmium-treated plutei. (B) Graphic representation of Northern analyses data (mean of three experiments for gastrulae and five experiments for plutei). Bars indicate mean values for each sample. Standard deviations are indicated. *p<0.025 when compared to sample 1; **p<0.05 when compared to sample 4.


In order to be sure that identical amounts of RNA had been loaded per lane, the membranes were stripped and re-hybridized with a 32P-labeled fragment of the gene encoding the P. lividus 26S rRNA (a gift from Dr. R. Barbieri, Fig. 2A, lower panels). A graphic representation of the results of three (gastrulae) and five (plutei) independent experiments is shown in Fig. 2B. In heat-shocked and cadmium-treated gastrulae the amount of Hsp56 mRNA is, respectively, 2 and 1.8 times greater (p<0.025; n=3) than in control gastrulae, while in heat-shocked plutei and plutei grown in the presence of cadmium, the increase is, respectively, 1.8- (p<0.05; n=5) and 2-fold (p<0.05; n=5).

Like Hsp56 protein (Roccheri et al., 2001), Hsp56 mRNA shows a peculiar distribution in the embryo (manuscript in preparation), which suggests that its localization could be regulated, possibly through interaction with RNA-binding proteins. To test such a hypothesis, different regions of the Hsp56 cDNA were in vitro transcribed, and the corresponding radioactive transcripts were incubated with cytoplasmic extracts from P. lividus embryos and analyzed by T1 RNase protection assay. As shown in Fig. 3A, the proximal region of the 3′-UTR (nt 1906–2413 of the cDNA) binds a protein, present in the post-nuclear extracts from 16-cells embryos, blastulae and gastrulae (lanes 2, 3 and 4, respectively), the concentration of which apparently increases during development. The RNA-protein complex has a mass of about 40kDa and is specific: the signal indeed tends to disappear in the presence of increasing amounts of the unlabeled specific competitor (Fig. 3B, lanes 2 and 3), while other non-specific probes do not affect complex formation (lanes 4 and 5).


Fig. 3

Identification of an Hsp56 RNA-binding protein in P. lividus embryos by T1 RNase protection assay. (A) An Hsp56 cDNA region (nt 1906–2413) was in vitro transcribed, incubated with post-nuclear extracts, prepared from 16 blastomeres (lane 2), blastulae (lane 3) and gastrulae (lane 4), and treated with RNase T1. 1×106cpm of radiolabeled riboprobe, treated with T1 RNase in the absence of proteins, was loaded on lane 1, as control. (B) Fifteen microgram of proteins from post-nuclear extract prepared from gastrulae were incubated with 1×106cpm of Hsp56 riboprobe and treated as in (A), in the absence (lane 1) or in the presence of a 10:1 (lane 2) or 50:1 (lane 3) excess of specific unlabeled competitor. As a control, the mix was also incubated with a 50:1 excess of unspecific unlabeled probes: (4) mix incubated with an unlabeled riboprobe corresponding to nt 54–510 of Hsp56 cDNA; (5) mix incubated with an unlabeled riboprobe corresponding to nt 2932–3422 of Hsp56 cDNA.


To study the localization of the RNA-binding protein inside the cells, we performed an RNase T1 assay using the same riboprobe, and extracts prepared from mitochondrial and post-mitochondrial fractions of P. lividus gastrulae. As shown in Fig. 4A, the factor is almost exclusively present in the mitochondrial fraction (lane Mit). To confirm the binding specificity, we carried out competition experiments with increasing amounts of unlabeled RNA (corresponding to nt 1906–2413 of the cDNA); representative results are shown in Fig. 4C. The radioactive signal decreases dramatically in the presence of a 20-fold excess of the unlabeled RNA, and disappears when the concentration of the specific competitor is raised up to 100-fold. To test the purity of the mitochondrial extract, we performed western analysis with an anti-GAPDH (glyceraldehyde 3-phosphate dehydrogenase) antibody. As it can be seen in Fig. 4B, this antibody immunostains a protein only in the post-mitochondrial extract, as expected.


Fig. 4

Localization of the Hsp56 RNA-binding protein. (A) Fifteen microgram of proteins from mitochondrial (Mit) or post-mitochondrial (P-Mit) extracts, prepared from gastrulae, were incubated with the Hsp56 riboprobe and treated with RNase T1, as described in the legend to Fig. 3. (B) Western analysis of mitochondrial (Mit) and post-mitochondrial (P-mit) extracts (15μg) incubated with anti-GAPDH antibody. (C) Fifteen microgram of proteins from the mitochondrial extract were incubated and treated as in (A) in the absence (No) or in the presence of a 20:1, 50:1 or 100:1 excess of specific competitor.


Next, we analyzed the distribution of the Hsp56 mRNA-binding protein in the organelle. Extracts prepared from the matrix, inner membrane, outer membrane or intermembrane space, respectively, were incubated with the aforementioned Hsp56 3′-UTR riboprobe, and the RNase T1 assay was performed. Fig. 5A shows the results of this kind of experiment: the factor interacting with Hsp56 RNA is more abundant in the mitochondrial outer membrane (OM), but is also detectable in other fractions, especially in the matrix (MX). After exposure, the gel was stained with Coomassie brilliant blue: the loaded amounts of proteins were similar for all the samples, as shown in Fig. 5C. The Coomassie staining also shows that the protein pattern is different for each fraction. Further confirmation of the purity of the mitochondrial sub-fractions came from western analyses with anti-Mn-SOD (manganese superoxide dismutase) antibodies, which immunostain a protein only in the mitochondrial matrix, as expected (Fig. 5B).


Fig. 5

Localization of the Hsp56 RNA-binding protein in different mitochondrial sub-fractions. (A) Fifteen microgram of proteins from different mitochondrial sub-fractions were incubated with the Hsp56 riboprobe and treated with RNase T1, as described in the legend to Fig. 3. (B) Western analysis of the same mitochondrial sub-fractions as in (A) (15μg) incubated with anti-Mn-SOD antibody. (C) Coomassie blue staining of the whole gel partially showed in (A). Molecular weight markers migration is indicated. Mx=matrix; O.M.=outer membrane; I.M.=inner membrane; I.S.=intermembrane space.


Finally, to confirm the specificity of the binding, we performed a T1 RNase protection assay with the mitochondrial outer membrane extract, in the presence of unlabeled competitors (Fig. 6A and B). As shown in the figure, the radioactive signal progressively decreases in the presence of increasing amounts of unlabeled Hsp56 RNA fragment, while a non-specific RNA, PEP-19 (Sangameswaran et al., 1989), does not compete.


Fig. 6

T1 RNase protection assay of mitochondrial outer membrane extract. (A) Fifteen microgram of proteins from outer membrane extract were incubated with the Hsp56 riboprobe and treated with RNase T1, as described in the legend to Fig. 3, in the absence (No), or in the presence of a 20:1, 50:1 or 100:1 excess of the specific unlabeled competitor, or 100:1 excess of non-specific (PEP) unlabeled competitor. (B) Fifteen microgram of proteins from outer membrane extract were incubated with the Hsp56 riboprobe and treated with RNase T1, as described in the legend to Fig. 3, in the absence (No), or in the presence of a 5:1, 10:1 or 15:1 excess of the specific unlabeled competitor.


4 Discussion

We analyzed the expression of Hsp56 mRNA during P. lividus normal development and in stressed embryos, i.e. either heat-shocked or cadmium-treated gastrulae and plutei. The finding that the level of the mRNA is constant at all stages of development is an expected result, as it was previously reported that Hsp56 protein is constitutively expressed and maternally inherited, and that its concentration does not show any appreciable variation throughout P. lividus embryo development (Roccheri et al., 2001). On the other hand, we found that stress conditions induce a sharp increase in mRNA expression.

These results suggest that the stress-induced regulation of hsp56 gene expression is mainly played at the transcriptional level. Experiments are now in progress aimed at studying regulation of the gene promoter. We indeed recently isolated and characterized the hsp56 gene (Genbank accession no. DQ464426) from a P. lividus genomic library.

Interestingly, other experiments (manuscript in preparation) demonstrated asymmetric distribution of Hsp56 mRNA in the embryo, suggesting that its localization could be regulated, possibly through interaction with RNA-binding proteins. It is indeed well known that messenger metabolism and localization can be controlled by different classes of proteins interacting with specific regions of mRNA, mostly localized in the 3′-UTR (for review, see: Derrigo et al., 2000; Colegrove-Otero et al., 2005; St Johnston, 2005).

In order to find out any factor able to bind Hsp56 mRNA, we used T1 RNase protection assays to investigate the ability of the in vitro transcribed RNA to form complexes with embryonic proteins. We found evidence that at least one complex of 40kDa can be formed by a region of the 3′-UTR of the messenger that does not show any particular similarity with known sequences. The complex is specific, as demonstrated by competition experiments.

The concentration of the protein increases from 16 blastomere embryos to gastrulae, suggesting that it could be involved in the regulation of Hsp56 expression during development. Studies are in progress to clarify this point. Interestingly, the binding protein is almost exclusively present in the mitochondrial extracts and, more specifically, is more abundant in the outer membrane of the organelle. This finding might implicate a direct involvement of a mitochondrial factor in the localization in the embryo, and/or in the regulation of translation or stability of a messenger of nuclear origin encoding a mitochondrial protein. In agreement with this view, Ginsberg et al. (2003) previously found that the KH (K homology RNA-binding motif) domain of the outer mitochondrial membrane AKAP121 (kinase A anchoring protein) binds Mn-SOD mRNA and regulates its activity. In addition, it was recently reported that Puf3p, a Pumilio family RNA-binding protein, localizes to mitochondria and regulates mitochondrial biogenesis in yeast (Garcia-Rodriguez et al., 2007).

Acknowledgements

We thank R. Barbieri for the kind gift of 18S and 26S probes and F. Gianguzza for Hsp56 cDNA. We thank I. Di Liegro for the kind gift of PEP-19 cDNA and for critical reading of the manuscript. We also thank G. Morici for technical assistance. This work was supported by MIUR (ex 60% and COFIN 2004, grant 2004057282-003).

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Received 13 February 2007/29 March 2007; accepted 12 May 2007

doi:10.1016/j.cellbi.2007.05.006


ISSN Print: 1065-6995
ISSN Electronic: 1095-8355
Published by Portland Press Limited on behalf of the International Federation for Cell Biology (IFCB)