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Functional variants of 5S rRNA in the ribosomes of common sea urchin Paracentrotus lividus
Gene 508 (2012) 21–25
Contents lists available at SciVerse ScienceDirect
Gene journal homepage: www.elsevier.com/locate/gene
Functional variants of 5S rRNA in the ribosomes of common sea urchin Paracentrotus lividus Eufrosina Dimarco, Eleonora Cascone, Daniele Bellavia ⁎, Fabio Caradonna Dipartimento di Scienze e Tecnologie Molecolari e Biomolecolari (STEMBIO), Sezione di Biologia Cellulare, Università degli Studi di Palermo, V.le delle Scienze—90128 Palermo, Italy
a r t i c l e
i n f o
Article history: Accepted 30 July 2012 Available online 8 August 2012 Keywords: Sea urchin Paracentrotus lividus 5S gene 5S rRNA variants Single-strand conformation polymorphism (SSCP)
a b s t r a c t We have previously reported a molecular and cytogenetic characterization of three different 5S rDNA clusters in the sea urchin Paracentrotus lividus; this study, performed at DNA level only, lends itself as starting point to verify that these clusters could contain transcribed genes, then, to demonstrate the presence of heterogeneity at functional RNA level, also. In the present work we report in P. lividus ribosomes the existence of several transcribed variants of the 5S rRNA and we associate all transcribed variants to the cluster to which belong. Our finding is the first demonstration of the presence of high heterogeneity in functional 5S rRNA molecules in animal ribosomes, a feature that had been considered a peculiarity of some plants. © 2012 Elsevier B.V. All rights reserved.
1. Introduction Ribosomal RNAs (rRNAs) are highly evolutionarily conserved molecules, and their genes, belonging to tandem repeated multigenic families, have been extensively studied. 5S ribosomal RNA is a component of the large ribosomal subunit in the ribosomes of all the eukaryotes, and the genes coding for this small rRNA have been studied extensively both in animals and in plants. Although distant from its ancestral cluster, 5S ribosomal DNA has maintained the original organization of a head-to-tail arrangement (Appels et al., 1980; Ellis et al., 1988; Long and Dawid, 1980; Lu et al., 1980, 1981) in which a spacer sequence of variable length separates highly conserved RNA coding sequences. 5S rDNA clusters may be localized on one or several chromosomal loci, generally different from the ones occupied by the genes encoding for the “large” rRNAs (28S, 18S and 5.8S). Heterogeneity in the genomic sequence of 5S clusters has been demonstrated in a number of different organisms, such as fungi (Wildeman and Nazar, 1982), arthropods (Komiya et al., 1980), amphibians (Ford and Southern, 1973), fish (Martins and Galetti, 2001; Pasolini et al., 2006; Pinhal et al., 2011) and mammals (Sørensen and Frederiksen, 1991), but up to now, heterogeneity in functional 5S rRNA, has been demonstrated only in Xenopus laevis. In this organism, a model of developmental regulation has been characterized which involves only two 5S rRNA variants codified by two different rDNA clusters: a so-called oocytespecific 5S rRNA, found in oocytes and silenced in early developmental
stages, and a somatic 5S rRNA synthesized in adult cells (Douet and Tormente, 2007; Ghose et al., 2004; Peterson et al., 1980). Until now 5S rRNA functional variants had only been described in the plant kingdom (Cloix et al., 2000, 2002; Negi et al., 2002; Singh et al., 1994), and this “high” (more of two variants) heterogeneity is related to speciation phenomena (Cronn et al., 1996). Although 5S rDNA clusters are among the most studied repeated genes, little data are available on these genes in echinoderms. A recent work from our laboratory indicates the existence of three 5S repeated units of different lengths in the sea urchin Paracentrotus lividus genome (Caradonna et al., 2007). These units are of about 700 base pairs (bp), 900 bp and 950 bp, respectively (EMBL accession numbers: AJ417697, AJ417698, and AJ417699) and contain about 40 units per cluster. The longest repeats (900 bp and 950 bp) show identical transcribed sequences and similar intervening spacer sequences (the 950 bp unit spacer presents additional CT di-nucleotides repeats), whereas the shorter repeat (700 bp) shows both 9 mutation points in its transcribed region and a spacer sequence that totally diverges from the longest repeats. In the present paper we join from genomic to transcription level and describe the finding of functional 5S rRNA molecules variants found in P. lividus ribosomes and then, codified by active 5S genes. These data represent the first example of high heterogeneity of functional 5S RNA in animal kingdom.
2. Materials and methods Abbreviations: rRNA, ribosomal ribonucleic acids; rDNA, ribosomal deoxyribonucleic acids; SSCP, single-strand conformation polymorphism; bp, base pairs; RT, reverse transcription; PCR, polymerase chain reaction. ⁎ Corresponding author. Tel.: +39 09123897443; fax: +39 0916577210. E-mail address: [email protected] (D. Bellavia). 0378-1119/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.gene.2012.07.067
2.1. RNA extraction Oocytes, eggs and different P. lividus developmental stages were prepared as described elsewhere (Cantone et al., 1993). Ribosomes are
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E. Dimarco et al. / Gene 508 (2012) 21–25
then isolated as previously described (Bellavia and Barbieri, 2010). RNAs from isolated ribosomes were obtained by acid guanidinium thiocyanate–phenol–chloroform extraction (Chomczynski and Sacchi, 1987). RNAs were quantized on the spectrophotometer “Life Science UV/Vis Spectrophotometer DU 730” (Beckman Coulter), and the RNA quality was assessed by the electrophoretic analysis. 2.2. Reverse transcription and PCR reactions Reverse transcription reactions were carried out as described elsewhere (Sambrook et al., 1989) using the SuperScript™ II Reverse Transcriptase, according to manufacturer's (Invitrogen™, USA) protocol, in the presence of the Cod5Srev oligonucleotide (5′-AGCCTACAA CACCCGGTA), complementary to the 3′ ends of P. lividus 5S rRNA. Cluster-specific genomic clones (900 rDNA genomic clone, 950 rDNA genomic clone, 700 rDNA genomic clone), 5S rRNA variants clones (900/950 bp 5S rRNA, 700 bp 5S rRNA, 5S rRNA MT1, 5S rRNA MT2 and 5S rRNA MT3) and cDNAs obtained from specific reverse transcription of 5S rRNA were used as a templates in polymerase chain reaction (PCR) using Cod5Sdir (5′-GCCTACGACCATACCATG) and Cod5Srev oligonucleotides, complementary to the 5′ and 3′ ends of P. lividus 5S rRNA respectively. The PCR conditions for 5S transcribed region amplification were carried out as reported below. The reaction was initially denatured at 95 °C for 2 minutes, and followed by 30 cycles of: denaturation at 94 °C for 30″, annealing at 55 °C for 30″, and extension at 72 °C for 30″. The final extension step at 72 °C was prolonged to 30 minutes, to complete the extremities and create overhangs of dATP required for cloning in Topo-TA vector (Invitrogen™). Negative control PCRs are effected at the same conditions using RNA without RT reaction. 2.3. Elution, cloning and sequencing of amplicons Elution of double strand amplified 5S cDNA was performed as described by Barbieri et al. (1996). Eluted PCR-amplified cDNAs were cloned in TOPO-TA plasmid, using the TOPO-TA Cloning Kit, according to manufacturer's (Invitrogen™) specifications. Sequencing of recombinant plasmids was performed using Sanger's procedure (Sanger et al., 1977), in the presence of T7 DNA polymerase (Invitrogen™). 2.4. Computational analysis Hypothetical 5S rRNA secondary structures were obtained using a RNADRAW software. 2.5. Single strand conformation polymorphism (SSCP) analysis Samples for SSCP analysis were prepared in 1 × loading buffer (10 × loading buffer contains 0.25% bromophenol blue, 0.25% xylene cyanol, and 50% glycerol), and heated at 95 °C for 5 minutes, then rapidly chilled on ice for 2 minutes. The SSCP analysis was then completed by electrophoretic fractionation performed in horizontal polyacrylamide gels (Acrylamide/Bis 37.5:1, 20% w/v solution, 1× TBE, pH 8.3, 2.5% glycerol) as described by Izzo et al. (2006), at 7 V/cm in 1× TBE buffer (89 mM Tris, 89 mM boric acid, and 2 mM EDTA, pH 8.0) for 7 h. 3. Results Previous observations on the genomic organization of P. lividus 5S rDNA (Caradonna et al., 2007) indicate the existence of three clusters of different lengths (of about 950 bp, 900 bp and 700 bp, respectively), which map to different positions on the sea urchin P. lividus chromosomes. The transcribed sequences of two of these three clusters
(900 bp and 950 bp) are identical, while the 700 bp cluster shows 9 point mutations in the transcribed region with respect to the longer clusters. To verify which of the two 5S rRNA variants is recruited in P. lividus ribosomes, we carried out RT-PCR experiments on RNA extracted from ribosomes of oocytes, eggs and different developmental stages (post-hatching blastula, gastrula and pluteus) using primers complementary to the 5′- and 3′-ends of 5S rRNA. The lack of amplified DNA in negative control PCR, using RNA without reverse transcription reaction as template, demonstrated the total absence of contaminant genomic DNA. Aliquots of cDNAs, purified as described in Materials and methods, were then cloned in a TOPO-TA vector, used to transform E. coli XL1-Blue strain. TOPO-TA inserted sequences from 217 different recombinant clones were sequenced. Sequence analysis revealed the presence in P. lividus ribosomes in all stages analyzed of the two expected coding sequence variants (EMBL accession numbers: FM242579.1 and FM242580.1): in particular, we normally observed the expected 700 bp 5S rRNA and 900/950 bp 5S rRNA; surprisingly, we found also 3 additional, less frequent, 5S rRNA sequence variants, called 5S ribosomal RNA Minor Transcript 1 (MT1), 5S ribosomal RNA Minor Transcript 2 (MT2) and ribosomal RNA Minor Transcript 3 (MT3) (EMBL accession numbers: FM242581.1, FM242582.1 and FM242583.1). In Table 1 is shown the amount of clones of all 5S rRNA sequences found in every stages. Reading the table horizontally, it is evident that each 5S rRNA sequence has approximately the same percentage in every stage. In particular, the two major 5S RNA (700 bp 5S rRNA and 900/950 bp 5S rRNA) represent about 70% of total 5S RNA pool, while each minor transcript (MT1, MT2 and MT3) has approximately only a frequency of 10% in all stage analyzed. The nucleotide sequence of all these variants is reported in Fig. 1A. As 5S rRNA gene is an evolutionarily conserved gene both in the primary sequence and in the secondary structure, we investigated the theoretical conformation of the 5S molecules we found in P. lividus ribosomes using DNA-DRAW program. As shown in Fig. 1B, both the 700 bp and 900/950 bp transcribed regions give rise to very similar secondary structures which are also compatible with the canonical 5S rRNA structure present in literature (Sørensen and Frederiksen, 1991). A secondary structure, obtained from sequence of 5S minor transcripts, also shows a similar conformation. Since this approach is only theoretical we analyzed the native secondary structures from these variants by single strand conformation polymorphism (SSCP) analysis. SSCP detects point mutations through the electrophoretic behavior of short (200 nucleotides or less) mutated single strand molecules. To associate every single variant to its own relative cluster, we carried out a SSCP analysis comparing the migration of every single cloned variant, with the migration of PCR-product obtained from cluster-specific genomic clones as template (Fig. 2). The result in Fig. 2 demonstrates that 900/950 bp 5S rRNA sequence is the unique variant found in the largest cluster, that 700 bp 5S rRNA and all minor variants are exclusively associated to the cluster of 700 bp. Finally, we carried out a further SSCP analysis comparing all single clones with RT-PCR fragments obtained from ribosomes-extracted RNA by P. lividus oocytes (O), eggs (E) and Plutei (P). This SSCP analysis (Fig. 3) show that all variants are present in all analyzed stages. 4. Discussion As indicated above, we reported in a precedent paper the existence of two different 5S transcribed regions belonging to three different 5S rDNA clusters, which map in different positions on the sea urchin P. lividus chromosomes (Caradonna et al., 2007). The presence of 5S rRNA pseudo-genes is reported in a variety of eukaryotes (Del Pino et al., 1992; Doran et al., 1987; Leah et al., 1990; Vahidi et al., 1991), but a high number of functional 5S variants have, up to now, been considered a peculiarity of the plants (Cloix et al., 2000,
E. Dimarco et al. / Gene 508 (2012) 21–25
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Table 1 Percentage of clones of all 5S rRNA sequences found in every stages. In brackets are indicated the number of found clones. 5S rRNA sequences
Found clones per stage (%)
Total clones per sequences
(Total clones n = 217)
900/950 bp 5S rRNA 700 bp 5S rRNA 5S rRNA MT1 5S rRNA MT2 5S rRNA MT3
Oocytes (n = 51)
Eggs (n = 44)
Post-hatching blastula (n = 38)
Gastrula (n = 34)
Pluteus (n = 50)
33.33 35.27 11.76 9.80 9.80
34.09 34.09 11.36 11.36 9.09
34.02 36.08 10.52 7.89 10.52
38.00 32.35 8.82 8.82 11.76
36.00 38.00 10.00 8.00 8.00
(n = 17) (n = 18) (n = 6) (n = 5) (n = 5)
(n = 15) (n = 15) (n = 5) (n = 5) (n = 4)
(n = 13) (n = 14) (n = 4) (n = 3) (n = 4)
2002; Negi et al., 2002; Singh et al., 1994), and has never been described in animals. An intriguing feature of the nucleotide variants reported in Fig. 1A is the occurrence of point mutations in just those 9 positions of the
(n = 13) (n = 11) (n = 3) (n = 3) (n = 4)
(n = 18) (n = 19) (n = 5) (n = 4) (n = 4)
35.02 35.48 10.59 9.21 9.67
(n = 76) (n = 77) (n = 23) (n = 20) (n = 21)
sequence mentioned. These positions are indicated in the 5S canonical secondary structure shown in Fig. 1B. RNA-DRAW construction of theoretical secondary structures formed by these variants show very similar drawings with slight differences in regions II and III, and in the loop B
Fig. 1. A—alignment of P. lividus 5S rRNA sequence from clones obtained by RT-PCR on RNA templates derived from ribosome as described in the text. B—hypothetical secondary structure of the canonical 5S molecule (Szymanski et al., 2002) and of the five P. lividus variants we found. The arrows indicate the position of mutation points in the 5S canonical secondary structure.
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E. Dimarco et al. / Gene 508 (2012) 21–25
The presence of several 5S rRNA functional variants in P. lividus ribosomes is remarkable novelty, especially as all these 5S rRNA variants have been found in all analyzed stages, excluding a switching on/off mechanism of cell/stage-specific 5S rDNA clusters during sea urchin development, as seen in Xenopus (Douet and Tormente, 2007; Ghose et al., 2004; Peterson et al., 1980). Acknowledgments This work was supported by funds of Italian MIUR (Fondo di Ateneo, 2007 R.B.). All the authors remember professor Rainer Barbieri, died prematurely, true inspirer and promoter for this work. References
Fig. 2. SSCP analysis of PCR obtained from cluster-specific genomic clones as template (900 bp rDNA genomic clone, 950 bp rDNA genomic clone and 700 bp rDNA genomic clones). 700, 900/950, MT1, MT2, and MT3 correspond to every single cloned variants.
and the helix 4 of the canonical 5S secondary structure (Fig. 1B). The fact that these variants were found in P. lividus ribosomes, and are then fully functional, indicates that these different conformations are consistent with the intermolecular relationships of 5S rRNAs with other structural components of the ribosome. SSCP analyses of these variants confirm the theoretical observations, showing that the analyzed molecules adopt different conformations depending on each specific nucleotide sequence. A first SSCP analysis, performed in cluster-specific genomic clones, demonstrated the association between minor transcript (MT1, MT2, MT3) exclusively to 700 bp rDNA cluster. This linkage would suggest that MT variants have originated from 700 bp 5S rRNA sequence. This indicates that these mutations have not been selected against, probably because they do not lead to critical disturbance of the secondary structure that would make their interactions with the other structural elements of the ribosome difficult or impossible; as such, these variants are both conserved and propagated. Moreover, the further SSCP analysis, carried-out on RT-PCR obtained using ribosomes-extracted RNA, established that all the variants are expressed in oocytes, eggs and pluteus (Table 1 and Fig. 3).
Fig. 3. SSCP analysis of cDNA obtained by RT-PCR of ribosome-derived RNA from oocytes (O), eggs (E) and pluteus (P); in these tracks, all P. lividus 5S variants are visible. 700, 900/950, MT1, MT2, and MT3 corresponds to every single cloned variants.
Appels, R., Gerlach, W.L., Dennis, E.S., Swift, H., Peacock, W.J., 1980. Molecular and chromosomal organization of DNA sequences coding for the ribosomal RNAs in cereals. Chromosoma 78, 293–311. Barbieri, R., Duro, G., Morgante, A., Izzo, V., 1996. Rapid DNA elution procedure from agarose gels. Anal. Biochem. 235, 106–107. Bellavia, D., Barbieri, R., 2010. Evidence for a novel cytoplasmic processing event in ribosome maturation in the sea urchin Paracentrotus lividus. Cell. Mol. Life Sci. 67, 1871–1879. Cantone, M., Barbieri, R., Duro, G., Izzo, V., Giudice, G., 1993. Sequence analysis of the rDNA spacer of Paracentrotus lividus and observations about pre-rRNA processing. Mol. Biol. Rep. 18, 177–182. Caradonna, F., Bellavia, D., Clemente, A.M., Sisino, G., Barbieri, R., 2007. Chromosomal localization and molecular characterization of three different 5S ribosomal DNA clusters in the sea urchin Paracentrotus lividus. Genome 50, 867–870. Chomczynski, P., Sacchi, N., 1987. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal. Biochem. 162, 156–159. Cloix, C., et al., 2000. Analysis of 5S rDNA Arrays in Arabidopsis thaliana—mapping e chromosome specific polymorph. Genome Res. 10, 679–690. Cloix, C., et al., 2002. Analysis of the 5S RNA pool in Arabidopsis thaliana: RNAs are heterogeneous and only two of the genomic 5S loci produce mature 5S RNA. Genome Res. 12, 132–144. Cronn, R.C., Zhao, X., Paterson, A.H., Wendel, J.F., 1996. Polymorphism and concerted evolution in a tandemly repeated gene family: 5S ribosomal DNA in diploid and allopolyploid cottons. J. Mol. Evol. 42, 685–705. Del Pino, E.M., Murphy, C., Masson, P.H., Gall, J.G., 1992. 5S rRNA-encoding genes of the marsupial frog Gastrotheca riobambae. Gene 111, 235–238. Doran, J.L., Bingle, W.H., Roy, K.L., 1987. The nucleotide sequences of two human 5S rRNA pseudogenes. Nucleic Acids Res. 15, 6297. Douet, J., Tormente, S., 2007. Transcription of the 5S rRNA heterochromatic genes is epigenetically controlled in Arabidopsis thaliana and Xenopus laevis. Heredity 99, 5–13. Ellis, T.N., et al., 1988. 5S rRNA genes in pisum: sequence, long range and chromosomal organization. Mol. Gen. Genet. 214, 333–342. Ford, P.J., Southern, E.M., 1973. Different sequences for 5S RNA in kidney cells and ovaries of Xenopus laevis. Nat. New Biol. 241, 7–12. Ghose, R., Malik, M., Huber, P.W., 2004. Restricted specificity of Xenopus TFIIIA for Transcription of Somatic 5S rRNA Genes. Mol. Cell. Biol. 24, 2467–2477. Izzo, V., et al., 2006. Electrophoresis of proteins and DNA on horizontal sodium dodecyl sulfate polyacrylamide gels. Immun. Ageing http://dx.doi.org/10.1186/1742-49333-7. Komiya, H., Shimizu, N., Kawakami, M., Takemura, S., 1980. Nucleotide sequence of 5S ribosomal RNA from Lingula anatina. A study on the molecular evolution of 5S ribosomal RNA from a living fossil. J. Biochem. 88, 1449–1456. Leah, R., Frederiksen, S., Engberg, J., Sørensen, P.D., 1990. Nucleotide sequence of a mouse 5S rRNA variant gene. Nucleic Acids Res. 18, 7441. Long, E.O., Dawid, I.B., 1980. Repeated genes in eukaryotes. Annu. Rev. Biochem. 49, 727–764. Lu, A.L., Steege, D.A., Stafford, D.W., 1980. Nucleotide sequence of a 5S ribosomal RNA gene in the sea urchin Lytechinus variegatus. Nucleic Acids Res. 8, 1839–1853. Lu, A.L., Blin, N., Stafford, D.W., 1981. Cloning and organization of genes for 5S ribosomal RNA in the sea urchin. Lytechinus variegatus. Gene 14, 51–62. Martins, C., Galetti Jr., P.M., 2001. Organization of 5S rDNA in species of the fish Leporinus: two different genomic locations are characterized by distinct nontranscribed spacers. Genome 44, 903–910. Negi, M.S., Rajagopal, J., Chauhan, N., Cronn, R., Lakshmikumaran, M., 2002. Length and sequence heterogeneity in 5S rDNA of Populus deltoides. Genome 45, 1181–1188. Pasolini, P., Costagliola, D., Rocco, L., Tinti, F., 2006. Molecular organization of 5S rDNAs in Rajidae (Chondrichthyes): structural features and evolution of piscine 5S rRNA genes and nontranscribed intergenic spacers. J. Mol. Evol. 62, 564–574. Peterson, R.C., Doering, J.L., Brown, D.D., 1980. Characterization of two Xenopus somatic 5S DNAs and one minor oocyte-specific 5S DNA. Cell 20, 131–141. Pinhal, D., Yoshimura, T.S., Araki, C.S., Martins, C., 2011. The 5S rDNA family evolves through concerted and birth-and-death evolution in fish genomes: an example from freshwater stingrays. BMC Evol. Biol. http://dx.doi.org/10.1186/1471-214811-151.
E. Dimarco et al. / Gene 508 (2012) 21–25 Sambrook, J., Fritsch, E.F., Maniatis, T., 1989. Molecular Cloning: A Laboratory Manual, 2nd edn. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. Sanger, F., Nicklen, S., Coulson, A.R., 1977. DNA sequencing with chain terminating inhibitors. Proc. Natl. Acad. Sci. 77, 5463–5467. Singh, K., Bhatia, S., Lakshmikumaran, M., 1994. Novel variants of the 5S rRNA genes in Eruca sativa. Genome 37, 121–128. Sørensen, P.D., Frederiksen, S., 1991. Characterization of human 5S rRNA genes. Nucleic Acids Res. 19, 4147–4151.
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Szymanski, M., Barciszewska, M.Z., Erdmann, V.A., Barciszewski, J., 2002. 5S Ribosomal RNA database. Nucleic Acids Res. 30, 176–178. Vahidi, H., Purac, A., LeBlanc, J.M., Honda, B.M., 1991. Characterization of potentially functional 5S rRNA-encoding genes within ribosomal DNA repeats of the nematode Meloidogyne arenaria. Gene 108, 281–284. Wildeman, A.G., Nazar, R.N., 1982. Structural studies of 5S ribosomal RNAs from a thermophilic fungus, Thermomyces lanuginosus. A comparison of generalized models for eukaryotic 5S RNAs. J. Biol. Chem. 257, 11395–11404.
Contents lists available at SciVerse ScienceDirect
Gene journal homepage: www.elsevier.com/locate/gene
Functional variants of 5S rRNA in the ribosomes of common sea urchin Paracentrotus lividus Eufrosina Dimarco, Eleonora Cascone, Daniele Bellavia ⁎, Fabio Caradonna Dipartimento di Scienze e Tecnologie Molecolari e Biomolecolari (STEMBIO), Sezione di Biologia Cellulare, Università degli Studi di Palermo, V.le delle Scienze—90128 Palermo, Italy
a r t i c l e
i n f o
Article history: Accepted 30 July 2012 Available online 8 August 2012 Keywords: Sea urchin Paracentrotus lividus 5S gene 5S rRNA variants Single-strand conformation polymorphism (SSCP)
a b s t r a c t We have previously reported a molecular and cytogenetic characterization of three different 5S rDNA clusters in the sea urchin Paracentrotus lividus; this study, performed at DNA level only, lends itself as starting point to verify that these clusters could contain transcribed genes, then, to demonstrate the presence of heterogeneity at functional RNA level, also. In the present work we report in P. lividus ribosomes the existence of several transcribed variants of the 5S rRNA and we associate all transcribed variants to the cluster to which belong. Our finding is the first demonstration of the presence of high heterogeneity in functional 5S rRNA molecules in animal ribosomes, a feature that had been considered a peculiarity of some plants. © 2012 Elsevier B.V. All rights reserved.
1. Introduction Ribosomal RNAs (rRNAs) are highly evolutionarily conserved molecules, and their genes, belonging to tandem repeated multigenic families, have been extensively studied. 5S ribosomal RNA is a component of the large ribosomal subunit in the ribosomes of all the eukaryotes, and the genes coding for this small rRNA have been studied extensively both in animals and in plants. Although distant from its ancestral cluster, 5S ribosomal DNA has maintained the original organization of a head-to-tail arrangement (Appels et al., 1980; Ellis et al., 1988; Long and Dawid, 1980; Lu et al., 1980, 1981) in which a spacer sequence of variable length separates highly conserved RNA coding sequences. 5S rDNA clusters may be localized on one or several chromosomal loci, generally different from the ones occupied by the genes encoding for the “large” rRNAs (28S, 18S and 5.8S). Heterogeneity in the genomic sequence of 5S clusters has been demonstrated in a number of different organisms, such as fungi (Wildeman and Nazar, 1982), arthropods (Komiya et al., 1980), amphibians (Ford and Southern, 1973), fish (Martins and Galetti, 2001; Pasolini et al., 2006; Pinhal et al., 2011) and mammals (Sørensen and Frederiksen, 1991), but up to now, heterogeneity in functional 5S rRNA, has been demonstrated only in Xenopus laevis. In this organism, a model of developmental regulation has been characterized which involves only two 5S rRNA variants codified by two different rDNA clusters: a so-called oocytespecific 5S rRNA, found in oocytes and silenced in early developmental
stages, and a somatic 5S rRNA synthesized in adult cells (Douet and Tormente, 2007; Ghose et al., 2004; Peterson et al., 1980). Until now 5S rRNA functional variants had only been described in the plant kingdom (Cloix et al., 2000, 2002; Negi et al., 2002; Singh et al., 1994), and this “high” (more of two variants) heterogeneity is related to speciation phenomena (Cronn et al., 1996). Although 5S rDNA clusters are among the most studied repeated genes, little data are available on these genes in echinoderms. A recent work from our laboratory indicates the existence of three 5S repeated units of different lengths in the sea urchin Paracentrotus lividus genome (Caradonna et al., 2007). These units are of about 700 base pairs (bp), 900 bp and 950 bp, respectively (EMBL accession numbers: AJ417697, AJ417698, and AJ417699) and contain about 40 units per cluster. The longest repeats (900 bp and 950 bp) show identical transcribed sequences and similar intervening spacer sequences (the 950 bp unit spacer presents additional CT di-nucleotides repeats), whereas the shorter repeat (700 bp) shows both 9 mutation points in its transcribed region and a spacer sequence that totally diverges from the longest repeats. In the present paper we join from genomic to transcription level and describe the finding of functional 5S rRNA molecules variants found in P. lividus ribosomes and then, codified by active 5S genes. These data represent the first example of high heterogeneity of functional 5S RNA in animal kingdom.
2. Materials and methods Abbreviations: rRNA, ribosomal ribonucleic acids; rDNA, ribosomal deoxyribonucleic acids; SSCP, single-strand conformation polymorphism; bp, base pairs; RT, reverse transcription; PCR, polymerase chain reaction. ⁎ Corresponding author. Tel.: +39 09123897443; fax: +39 0916577210. E-mail address: [email protected] (D. Bellavia). 0378-1119/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.gene.2012.07.067
2.1. RNA extraction Oocytes, eggs and different P. lividus developmental stages were prepared as described elsewhere (Cantone et al., 1993). Ribosomes are
22
E. Dimarco et al. / Gene 508 (2012) 21–25
then isolated as previously described (Bellavia and Barbieri, 2010). RNAs from isolated ribosomes were obtained by acid guanidinium thiocyanate–phenol–chloroform extraction (Chomczynski and Sacchi, 1987). RNAs were quantized on the spectrophotometer “Life Science UV/Vis Spectrophotometer DU 730” (Beckman Coulter), and the RNA quality was assessed by the electrophoretic analysis. 2.2. Reverse transcription and PCR reactions Reverse transcription reactions were carried out as described elsewhere (Sambrook et al., 1989) using the SuperScript™ II Reverse Transcriptase, according to manufacturer's (Invitrogen™, USA) protocol, in the presence of the Cod5Srev oligonucleotide (5′-AGCCTACAA CACCCGGTA), complementary to the 3′ ends of P. lividus 5S rRNA. Cluster-specific genomic clones (900 rDNA genomic clone, 950 rDNA genomic clone, 700 rDNA genomic clone), 5S rRNA variants clones (900/950 bp 5S rRNA, 700 bp 5S rRNA, 5S rRNA MT1, 5S rRNA MT2 and 5S rRNA MT3) and cDNAs obtained from specific reverse transcription of 5S rRNA were used as a templates in polymerase chain reaction (PCR) using Cod5Sdir (5′-GCCTACGACCATACCATG) and Cod5Srev oligonucleotides, complementary to the 5′ and 3′ ends of P. lividus 5S rRNA respectively. The PCR conditions for 5S transcribed region amplification were carried out as reported below. The reaction was initially denatured at 95 °C for 2 minutes, and followed by 30 cycles of: denaturation at 94 °C for 30″, annealing at 55 °C for 30″, and extension at 72 °C for 30″. The final extension step at 72 °C was prolonged to 30 minutes, to complete the extremities and create overhangs of dATP required for cloning in Topo-TA vector (Invitrogen™). Negative control PCRs are effected at the same conditions using RNA without RT reaction. 2.3. Elution, cloning and sequencing of amplicons Elution of double strand amplified 5S cDNA was performed as described by Barbieri et al. (1996). Eluted PCR-amplified cDNAs were cloned in TOPO-TA plasmid, using the TOPO-TA Cloning Kit, according to manufacturer's (Invitrogen™) specifications. Sequencing of recombinant plasmids was performed using Sanger's procedure (Sanger et al., 1977), in the presence of T7 DNA polymerase (Invitrogen™). 2.4. Computational analysis Hypothetical 5S rRNA secondary structures were obtained using a RNADRAW software. 2.5. Single strand conformation polymorphism (SSCP) analysis Samples for SSCP analysis were prepared in 1 × loading buffer (10 × loading buffer contains 0.25% bromophenol blue, 0.25% xylene cyanol, and 50% glycerol), and heated at 95 °C for 5 minutes, then rapidly chilled on ice for 2 minutes. The SSCP analysis was then completed by electrophoretic fractionation performed in horizontal polyacrylamide gels (Acrylamide/Bis 37.5:1, 20% w/v solution, 1× TBE, pH 8.3, 2.5% glycerol) as described by Izzo et al. (2006), at 7 V/cm in 1× TBE buffer (89 mM Tris, 89 mM boric acid, and 2 mM EDTA, pH 8.0) for 7 h. 3. Results Previous observations on the genomic organization of P. lividus 5S rDNA (Caradonna et al., 2007) indicate the existence of three clusters of different lengths (of about 950 bp, 900 bp and 700 bp, respectively), which map to different positions on the sea urchin P. lividus chromosomes. The transcribed sequences of two of these three clusters
(900 bp and 950 bp) are identical, while the 700 bp cluster shows 9 point mutations in the transcribed region with respect to the longer clusters. To verify which of the two 5S rRNA variants is recruited in P. lividus ribosomes, we carried out RT-PCR experiments on RNA extracted from ribosomes of oocytes, eggs and different developmental stages (post-hatching blastula, gastrula and pluteus) using primers complementary to the 5′- and 3′-ends of 5S rRNA. The lack of amplified DNA in negative control PCR, using RNA without reverse transcription reaction as template, demonstrated the total absence of contaminant genomic DNA. Aliquots of cDNAs, purified as described in Materials and methods, were then cloned in a TOPO-TA vector, used to transform E. coli XL1-Blue strain. TOPO-TA inserted sequences from 217 different recombinant clones were sequenced. Sequence analysis revealed the presence in P. lividus ribosomes in all stages analyzed of the two expected coding sequence variants (EMBL accession numbers: FM242579.1 and FM242580.1): in particular, we normally observed the expected 700 bp 5S rRNA and 900/950 bp 5S rRNA; surprisingly, we found also 3 additional, less frequent, 5S rRNA sequence variants, called 5S ribosomal RNA Minor Transcript 1 (MT1), 5S ribosomal RNA Minor Transcript 2 (MT2) and ribosomal RNA Minor Transcript 3 (MT3) (EMBL accession numbers: FM242581.1, FM242582.1 and FM242583.1). In Table 1 is shown the amount of clones of all 5S rRNA sequences found in every stages. Reading the table horizontally, it is evident that each 5S rRNA sequence has approximately the same percentage in every stage. In particular, the two major 5S RNA (700 bp 5S rRNA and 900/950 bp 5S rRNA) represent about 70% of total 5S RNA pool, while each minor transcript (MT1, MT2 and MT3) has approximately only a frequency of 10% in all stage analyzed. The nucleotide sequence of all these variants is reported in Fig. 1A. As 5S rRNA gene is an evolutionarily conserved gene both in the primary sequence and in the secondary structure, we investigated the theoretical conformation of the 5S molecules we found in P. lividus ribosomes using DNA-DRAW program. As shown in Fig. 1B, both the 700 bp and 900/950 bp transcribed regions give rise to very similar secondary structures which are also compatible with the canonical 5S rRNA structure present in literature (Sørensen and Frederiksen, 1991). A secondary structure, obtained from sequence of 5S minor transcripts, also shows a similar conformation. Since this approach is only theoretical we analyzed the native secondary structures from these variants by single strand conformation polymorphism (SSCP) analysis. SSCP detects point mutations through the electrophoretic behavior of short (200 nucleotides or less) mutated single strand molecules. To associate every single variant to its own relative cluster, we carried out a SSCP analysis comparing the migration of every single cloned variant, with the migration of PCR-product obtained from cluster-specific genomic clones as template (Fig. 2). The result in Fig. 2 demonstrates that 900/950 bp 5S rRNA sequence is the unique variant found in the largest cluster, that 700 bp 5S rRNA and all minor variants are exclusively associated to the cluster of 700 bp. Finally, we carried out a further SSCP analysis comparing all single clones with RT-PCR fragments obtained from ribosomes-extracted RNA by P. lividus oocytes (O), eggs (E) and Plutei (P). This SSCP analysis (Fig. 3) show that all variants are present in all analyzed stages. 4. Discussion As indicated above, we reported in a precedent paper the existence of two different 5S transcribed regions belonging to three different 5S rDNA clusters, which map in different positions on the sea urchin P. lividus chromosomes (Caradonna et al., 2007). The presence of 5S rRNA pseudo-genes is reported in a variety of eukaryotes (Del Pino et al., 1992; Doran et al., 1987; Leah et al., 1990; Vahidi et al., 1991), but a high number of functional 5S variants have, up to now, been considered a peculiarity of the plants (Cloix et al., 2000,
E. Dimarco et al. / Gene 508 (2012) 21–25
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Table 1 Percentage of clones of all 5S rRNA sequences found in every stages. In brackets are indicated the number of found clones. 5S rRNA sequences
Found clones per stage (%)
Total clones per sequences
(Total clones n = 217)
900/950 bp 5S rRNA 700 bp 5S rRNA 5S rRNA MT1 5S rRNA MT2 5S rRNA MT3
Oocytes (n = 51)
Eggs (n = 44)
Post-hatching blastula (n = 38)
Gastrula (n = 34)
Pluteus (n = 50)
33.33 35.27 11.76 9.80 9.80
34.09 34.09 11.36 11.36 9.09
34.02 36.08 10.52 7.89 10.52
38.00 32.35 8.82 8.82 11.76
36.00 38.00 10.00 8.00 8.00
(n = 17) (n = 18) (n = 6) (n = 5) (n = 5)
(n = 15) (n = 15) (n = 5) (n = 5) (n = 4)
(n = 13) (n = 14) (n = 4) (n = 3) (n = 4)
2002; Negi et al., 2002; Singh et al., 1994), and has never been described in animals. An intriguing feature of the nucleotide variants reported in Fig. 1A is the occurrence of point mutations in just those 9 positions of the
(n = 13) (n = 11) (n = 3) (n = 3) (n = 4)
(n = 18) (n = 19) (n = 5) (n = 4) (n = 4)
35.02 35.48 10.59 9.21 9.67
(n = 76) (n = 77) (n = 23) (n = 20) (n = 21)
sequence mentioned. These positions are indicated in the 5S canonical secondary structure shown in Fig. 1B. RNA-DRAW construction of theoretical secondary structures formed by these variants show very similar drawings with slight differences in regions II and III, and in the loop B
Fig. 1. A—alignment of P. lividus 5S rRNA sequence from clones obtained by RT-PCR on RNA templates derived from ribosome as described in the text. B—hypothetical secondary structure of the canonical 5S molecule (Szymanski et al., 2002) and of the five P. lividus variants we found. The arrows indicate the position of mutation points in the 5S canonical secondary structure.
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E. Dimarco et al. / Gene 508 (2012) 21–25
The presence of several 5S rRNA functional variants in P. lividus ribosomes is remarkable novelty, especially as all these 5S rRNA variants have been found in all analyzed stages, excluding a switching on/off mechanism of cell/stage-specific 5S rDNA clusters during sea urchin development, as seen in Xenopus (Douet and Tormente, 2007; Ghose et al., 2004; Peterson et al., 1980). Acknowledgments This work was supported by funds of Italian MIUR (Fondo di Ateneo, 2007 R.B.). All the authors remember professor Rainer Barbieri, died prematurely, true inspirer and promoter for this work. References
Fig. 2. SSCP analysis of PCR obtained from cluster-specific genomic clones as template (900 bp rDNA genomic clone, 950 bp rDNA genomic clone and 700 bp rDNA genomic clones). 700, 900/950, MT1, MT2, and MT3 correspond to every single cloned variants.
and the helix 4 of the canonical 5S secondary structure (Fig. 1B). The fact that these variants were found in P. lividus ribosomes, and are then fully functional, indicates that these different conformations are consistent with the intermolecular relationships of 5S rRNAs with other structural components of the ribosome. SSCP analyses of these variants confirm the theoretical observations, showing that the analyzed molecules adopt different conformations depending on each specific nucleotide sequence. A first SSCP analysis, performed in cluster-specific genomic clones, demonstrated the association between minor transcript (MT1, MT2, MT3) exclusively to 700 bp rDNA cluster. This linkage would suggest that MT variants have originated from 700 bp 5S rRNA sequence. This indicates that these mutations have not been selected against, probably because they do not lead to critical disturbance of the secondary structure that would make their interactions with the other structural elements of the ribosome difficult or impossible; as such, these variants are both conserved and propagated. Moreover, the further SSCP analysis, carried-out on RT-PCR obtained using ribosomes-extracted RNA, established that all the variants are expressed in oocytes, eggs and pluteus (Table 1 and Fig. 3).
Fig. 3. SSCP analysis of cDNA obtained by RT-PCR of ribosome-derived RNA from oocytes (O), eggs (E) and pluteus (P); in these tracks, all P. lividus 5S variants are visible. 700, 900/950, MT1, MT2, and MT3 corresponds to every single cloned variants.
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