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J. Biol. Chem., Vol. 275, Issue 24, 18153-18159, June 16, 2000

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Mammalian Mitochondrial Ribosomal Proteins (4)

AMINO ACID SEQUENCING, CHARACTERIZATION, AND IDENTIFICATION OF CORRESPONDING GENE SEQUENCES^*

From the ^a Department of Biochemistry and Molecular Biology, University of Florida, Gainesville, Florida 32610-0245, ^d Nemours Children's Clinic, Jacksonville, Florida 32207, ^f Ahmanson Department of Pediatrics, Cedars-Sinai Medical Center and UCLA School of Medicine, Los Angeles, California 90048-1865, ^g Max-Delbrück-Center for Molecular Medicine, Robert-Rössle-Sraße 10, D-13125 Berlin, Germany, and ^h Institute for Biology-Genetics, AG Kress, Free University of Berlin, Arnimallee 7, D-14195 Berlin, Germany

Received for publication, December 9, 1999, and in revised form, April 4, 2000

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

Mitochondrial ribosomal proteins (MRPs) are required for the translation of all 13 mitochondrial encoded genes in humans. It has been speculated that mutations and polymorphisms in the human MRPs may be a primary cause of some oxidative phosphorylation disorders or modulate the severity and tissue specificity of pathogenic mitochondrial DNA mutations. Although the sequences of most of the yeast MRPs are known, only very few mammalian and nearly no human MRPs have been completely characterized. MRPs differ greatly in sequence, and sometimes biochemical properties, between different species, not allowing easy recognition by sequence homology. Therefore, the Mammalian Mitochondrial Ribosomal Consortium is using a direct approach of purifying individual mammalian (bovine) MRPs, determining their N-terminal and/or internal peptide sequences using different protein sequencing techniques, and using the resulting sequence information for screening expressed sequence tags and genomic data bases to determine human, mouse, and rat homologues of the bovine proteins. Two proteins of the large and three proteins of the small ribosomal subunit have been analyzed in this manner. Three of them represent "new," i.e. formerly unknown mammalian mitochondrial ribosomal protein classes. Only one of these three different MRPs shows significant sequence similarities to known ribosomal proteins. In one case, the corresponding human genomic DNA sequences were found in the data bases, and the exon/intron structure was determined.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

Human oxidative phosphorylation disorders have been shown over the last decade to be the cause of a great variety of inherited and acquired diseases, including such different clinical entities as systemic neuromuscular disorders, diabetes, aplastic anemia, deafness, and degenerative disorders by either mitochondrial or nuclear gene mutations (2). It has been speculated that mutations or polymorphisms in proteins involved in mitochondrial RNA processing and translation can be involved in some of these diseases, either as the primary cause or as factors modulating the severity and/or tissue specificity of them (3).

The proper expression of the mitochondrial encoded protein genes depends on the nuclear-encoded components of the mitochondrial translational system (4). In yeast, knockout mutants of nuclear encoded MRPs¹ lose their mitochondrial DNA, changing the mitochondrial genetic status to ^o (5), an effect also observed in mammalian nuclear mutations affecting mitochondria (6). Mammalian mitochondrial ribosomes differ significantly from other known bacterial and eukaryotic cytoplasmic ribosomes when analyzed by biochemical methods (7-10). Numbers and sizes of rRNA molecules are reduced as compared with bacterial ribosomes, and numbers of ribosomal proteins are elevated (11). However, the differences turned out to be even greater than expected when molecular biological and protein biochemical investigations began to reveal the amino acid and gene sequences of MRPs. These efforts are supported by the recent genome projects. Most of the yeast MRPs were identified by the comparison of peptide sequences obtained from purified mature yeast MRPs by N-terminal sequencing, with the protein sequences deduced from the yeast genome project data (reviewed in Ref. 12). However, the assumption that yeast MRPs and mammalian MRPs are similar to each other, as is the case for yeast and rat cytoplasmic ribosomal proteins, and that it may be possible to identify mammalian MRPs by using yeast MRP probes as screening devices is not valid. When the current project of mammalian MRP gene identification was launched in 1997, only very few mammalian MRPs were known on the molecular biological level, rather by chance than by a systematic approach. The mammalian mitochondrial homologue of the bacterial L3 ribosomal protein was identified as an overexpressed protein in Mahlavu hepatocarcinoma cells (13, 14). The mammalian homologues RPMS12 of the EcoS12 protein, a protein strongly conserved through evolution, were cloned by sequence similarities to the EcoS12 ribosomal protein and the S12 homologue of Drosophila melanogaster mitochondria (15, 16). L23MRP was identified by its sequence similarity to the EcoL23 ribosomal protein (17). The mammalian mitochondrial homologue of the strongly conserved EcoL7/L12 gene was cloned as a delayed-early expressed gene (18). An HSMRPS14 cDNA similar to the EcoS14 ribosomal protein was cloned (GenBank^TM accession number Z99297) but not further characterized. However, none of the identified mammalian MRPs was "new" in terms of lacking sequence similarities to known ribosomal proteins.

To characterize mammalian MRPs systematically, the Mammalian Mitochondrial Ribosomal Consortium was formed, and the initial primary experimental approach was to be based on the N-terminal sequencing of purified mature bovine MRPs. By using the obtained peptide sequence information, EST and genomic DNA data bases are screened, and cDNA sequences are assembled in silico. This approach takes advantage of the existing bovine model for MRPs (7, 19) and the rapidly growing sequence data bases of human and other organisms. The obtained sequences were characterized by comparison to known ribosomal protein sequences, and corresponding genomic DNA sequences were identified. Nineteen different groups of homologous mammalian MRPs have been determined so far (20-22).² Only 10 of them show significant sequence similarities to yeast MRPs and/or bacterial ribosomal proteins. This paper describes the identification of five mammalian MRPs.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

Analysis of Bovine MRPs-- Isolation and purification of bovine MRPs have been described (19). The numbering of the proteins follows (7). Large subunit proteins were stained with Coomassie Blue after two-dimensional PAGE. Individual spots were excised from the gel and subjected to in-gel digestion by trypsin according to the method of Otto et al. (23). Resulting peptides were isolated and concentrated, purified by reverse phase high pressure liquid chromatography, and subjected to Edman sequencing (23) or individual peptides were subjected to tandem mass spectroscopic analysis with a Q-TOF Micromass (Manchester, UK) equipped with a nanoflow Z-spray ion source. It should be noted that the latter method is not able to differentiate between leucine and isoleucine because of the identical molecular mass of these two amino acids. Small subunit proteins were electroblotted from a two-dimensional PAGE gel onto polyvinylidene difluoride membrane, visualized by Coomassie Blue staining, and cut from the membrane. Individual spots obtained were subjected to N-terminal amino acid sequencing in an Applied Biosystems model 494 Protein Sequencer.

Computing-- Virtual screening of public EST data bases was performed using the blast program of Altschul et al. (24) and the NCBI server. For screening of short peptides the advanced BLAST program was performed using the modified options "expected," 1000, and "other options," e2. The analysis of the obtained sequences, the sequence comparison, and the assembly of virtual consensus cDNAs was performed as described (20) using several analytic programs of the GCG DNA analysis computer software package (25). Alignments were performed using the "bestfit" program, and multiple alignments were assembled manually. The algorithm used in the "pileup" program classifies weak or strong "similarities" of amino acid residues by the frequencies of exchanges in a given collection of sequences and not by chemical similarities. Therefore, classifications of amino acid exchanges termed "weak" or "strong" in the presented alignment figures were made manually regarding chemical similarities. For example, Ser versus Thr are strongly conserved amino acid residues, and glutamic acid versus glutamine represent weakly conserved amino acid residues. BLAST searches for homologous Caenorhabditis elegans protein sequences were performed on the server at the Sanger Center. Analysis of N-terminal peptide sequences for signal peptide properties was done using the Signal V1.1 Center for Biological Sequence Analysis server (26).

	RESULTS AND DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

Protein Sequencing of Individual MRPs-- Proteins of the bovine small and large mitochondrial ribosomal subunits were purified from bovine liver and sequenced (see "Experimental Procedures"). Seven different peptide sequences were obtained from five individual proteins (Table I). The MRP-L43_bov protein turned out to be the corresponding bovine MRP to the previously characterized MRP-L32_rat (20). The bovine MRP-L43 peptide 1 makes a perfect match in the C-terminal part of the mammalian MRP-L32s and the bovine peptide 2 matches close to the mature N termini of the MRP-L32_mammalians (see Ref. 20 for comparison). The MRP-S18_bov protein corresponds to the previously published MRP-S13_mammalian (20). However, comparison of the mature N termini of MRP-S18_bov and MRP-S13_rat showed that the true N terminus of mammalian MRP-S13s may be positioned one amino acid residue further upstream since the MRP-S18_bov sequencing seems to be more reliable (20). Thus, we can combine the different model systems used for mammalian MRPs by the presented method (7, 20).

Characterization of Gene Sequences Corresponding to Individual Mammalian MRPs-- Peptide sequence information obtained was used to screen public EST data bases. By multiple screenings and comparison of ESTs from human, mouse, and rat sources consensus cDNAs were assembled electronically (Table II). Due to the identification of false positives for MRP-S12_human by the initial EST data base screening using the MRP-S12_bov peptide sequence as virtual screening probe, the MRP-S12_mouse amino acid sequence (below) was used to screen for corresponding human ESTs. For all three new MRPs (MRP-L18, MRP-S12, and MRP-S28) complete ORFs were deduced of mouse and human origin. Only partial sequences were found for the corresponding rat MRPs (Table II and Fig. 2). Additionally, two corresponding genomic clones were found in the data bases for MRP-S12_human. The gene is located in chromosomal position 16q13-21 very close to the DR-nm23 nucleoside diphosphate kinase homologous gene (27). Genomic and EST sequences were assembled (Table II) and compared, permitting the exon/intron structure of the MRP-S12_human gene to be determined (Fig. 1). The gene spans approximately 1500 bp. However, the 5' exon A containing 53 bp is not covered by the published genomic sequence. Exon B extends from bp 54 to 302, exon C from bp 303 to 342, exon D from bp 343 to 662, and exon E from bp 663 to 804. Other human MRP genes whose genomic structure and chromosomal location have been determined similarly are the MRP-L22_human (20), located in position 22q11, and the MRP-L5_human gene in position 21q22.1 (22). The chromosomal positions are being examined for putative linkages to known inherited diseases caused by mitochondrial disorders.

View larger version (12K): [in this window] [in a new window]   Fig. 1.   Exon/intron structure of the human MRP-S12 gene. The genomic DNA is shown as a horizontal line. Boxes mark location of cDNA sequences; black boxes represent translated cDNA sequence, open boxes show untranslated cDNA sequences. Exons are termed consecutively B, C, D, and E. Exon A is missing in this figure. Numbers give genomic DNA positions in the corresponding clone (GenBankTM accession number Z34802).

For human MRP-S28 a full-length cDNA clone HSPC007 (GenBank^TM accession number AF070663) was identified. This clone was sequenced, and an ORF of 188 amino acid residues was determined by the authors but it wasn't assigned to a specific function. Similarly, other mammalian MRP genes that had been sequenced prior to this project of systematic MRP gene characterization were identified the same way. The human RLX1 gene was sequenced (28), but the function of the deduced protein remained unknown until the annotation of this gene to be the human MRP-L15 homologue (21). The KIAA0264 cDNA (29) was shown to encode the human MRP-S4,² and the cDNA CGI-22 (GenBank^TM accession number AF132956) corresponds to the MRP-L14_human1 cDNA (22), although a sequencing error in the CGI-22 cDNA made the annotation more difficult. Except for the MRP-L14_human1/CGI-22 protein, which is homologous to the EcoL2 ribosomal protein, all other genes mentioned are "new" in respect that they have no counterparts in known ribosomal systems. These results (i) demonstrate the power of the methodology applied to determine the identity of some ESTs as coding for MRPs, (ii) identify unknown new mammalian MRP genes, and (iii) shed some light on the numbers of new genes to be expected among the mammalian MRPs in total.

Characterization of Consensus cDNAs-- Assembly of consensus cDNAs from EST sequences is limited by the poor quality of EST sequences, especially in the further 5'-coding regions. Therefore, we suspect most of the assembled consensus cDNAs are incomplete at their respective 5' ends. This phenomenon is observed generally when using ESTs for cDNA assembly (20-22). From experience, the decreasing numbers and diminishing qualities of ESTs derived from the 5' regions of long mRNAs pose problems for the successful assembly of cDNAs longer than about 1500 bp from ESTs of public (!) data bases. At their respective 3' ends, the consensus cDNAs frequently show canonical polyadenylation signals, AATAAA, and parts of the corresponding poly(A) tracks. The ORFs presented (Table II) are complete, and in the cases of MRP-L18 and MRP-S12 5' preceding in-frame stop codons were found 5' to the initiation codons.

It turned out that MRP-S12_human exists in two different splice variants (Fig. 2c). For MRP-S12_human1 a consensus cDNA of 847 bp was assembled. Since the N terminus of the MRP-S12_human1 sequence was only found in the genomic DNAs, it explains why the gene was not identified by the initial EST data base search. For MRP-S12_human2 a consensus cDNA of 662 bp was assembled (Table II). Although its ORF is open at its 5' end, it shows strong sequence similarity to both MRP-S12_human1 and MRP-S12_mouse except for a deletion of 26 amino acid residues in length. Putatively, this is a result of alternative splicing due to the fact that the respective DNA sequences show the common features of intron sequences including canonical donor and acceptor splice sites and a polypyrimidine tract close to the 3' end of the putative intron (data not shown). However, among more than 20 independent EST sequences discovered for MRP-S12_mouse, no sequence corresponding to the splice variant of MRP-S12_human2 was determined. Similar to mouse, MRP-S12_rat shows no splicing variant corresponding to MRP-S12_human2. Therefore, we can only speculate whether MRP-S12_human2 is a naturally occurring splice variant different from human and mouse if it belongs to an incomplete, non-functional variant of MRP-S12_human or if it is a cloning artifact.

View larger version (54K): [in this window] [in a new window]   Fig. 2.   Alignment of deduced mammalian MRP amino acid sequences and with ribosomal protein sequences from other sources. Obtained deduced mammalian MRP amino acid sequences are aligned with bovine peptide sequences (Table I) and corresponding ribosomal protein sequences of E. coli. Numbers give the respective amino acid positions. Vertical lines mark identical amino acid positions; colons mark strongly conserved amino acid residues; and periods mark weakly conserved amino acid residues. Dashes show N- or C-terminal ends of incomplete amino acid sequences, and asterisks mark stop codons. Amino acid residues in lowercase letters within the bovine peptide sequences are uncertain in their determination by amino acid sequencing (see Table I). x marks unidentified amino acids at particular positions in the sequence. a, alignment of mammalian MRP-L18 sequences with the E. coli EcoL24 sequence (Swiss-Prot accession number Sw:rl24_ecoli). b, alignment of the mammalian MRP-S12 protein sequences. c, alignment of the mammalian MRP-S28 protein sequences.

Properties of the Deduced ORFs-- Comparison of the N-terminal peptides determined by amino acid sequencing with the deduced ORFs revealed the existence and extensions of the putative MISPs (Fig. 2). As was proposed,² mammalian MRP import may be mediated through different import signal recognition mechanisms and N-terminal processing during or after import. Also, the MISPs presented in this paper belong to different classes according to the classification of Branda and Isaya (30). MRP-L18s show MISPs of the R-none class; MRP-S28_mouse has an R-3 MISP, and MRP-S28_human possesses an R-none MISP. Mammalian mitochondrial protein import seems to be at least as heterogeneous as in yeast (12). For human and mouse MRP-L18, we postulate MISPs of 9 amino acid residues (Fig. 2a). Although such short MISPs are rather unusual for mammalian imported mitochondrial proteins, the stop codons preceding the respective initiator methionines and the N-terminal sequence of the MRP-L18_bovine give no other choice. The MISPs do not fit the general features of an import signal peptide, and the cleavage site obtained experimentally was not identified by the SignalP computer program (26). In contrast, mammalian MRP-S12s have no substantial cleavable MISPs; only the N-terminal methionine residues are cleaved off in the mature forms (Fig. 2b). Although this feature is not unusual with yeast MRPs (12), it is the first example for a mammalian MRP that is only processed on the N-terminal methionine (Fig. 2b). For mouse and human MRP-S28 and HSPC007, respectively, we postulate cleavable MISPs of 72 amino acid residues. Interestingly, the MISPs of these two proteins are much less conserved as compared with the conservation of the mature proteins, i.e. after cleavage of the MISPs (Fig. 2c). Both MISPs show the common features of mitochondrial import signal sequences such as large numbers of hydroxylated, hydrophobic, and positively charged amino acid residues. However, the SignalP program identified most probable cleavage sites between amino acid residues numbers 31 (Thr) and 32 (Glu) of mouse and human MRP-S28, respectively (26). These results do not fit the biochemical data. Both MISPs contain the N-terminal MAA motif that has been shown to be common among N termini of mammalian MISPs for MRPs.² However, only one of the presented three new mammalian MRP classes shows the "MAA" motif in their respective N termini (MRP-S28, Fig. 2c).

After N-terminal processing, the presented MRPs show more (pI 11.08, MRP-S12_mouse) or less (pI 7.44, MRP-S28_mouse) basic characters, as is common for ribosomal proteins. The calculated molecular masses of the mature human and mouse proteins correspond very well to the molecular masses of the bovine MRPs (Table I) determined by SDS-PAGE, showing that MRPs of mammals are closely related in their molecular properties.

Evolution of MRPs-- Human and mouse MRPs deduced pairwise from the presented cDNAs are identical in length (Fig. 2). They share between 72.5 (MRP-S28) and 87.5% (MRP-L18) identical amino acid residues, comparable to the MRP sequence conservation previously observed among mammalian MRPs (20-22, 31). These results are in strong contrast to the sequence conservation observed between cytoplasmic ribosomal proteins of the same species. Cytoplasmic ribosomal proteins of rat and man are close to 100% identical (32). The rate of evolutionary divergence among mammalian MRPs was estimated to be 13 times higher than that of mammalian cytoplasmic ribosomal proteins (33). Consequently, MRPs themselves are very heterogeneous among species (34). This is reflected in the low sequence identities determined between yeast and mammalian MRPs and by the experimental problem to identify mammalian MRP genes using their homologous MRP probes from yeast for screening (Refs. 20-22, 31, and this work). Curiously, mitochondrial ribosomes of distantly related species like yeast and mammals have similar numbers of different MRPs, but only a subset of their respective MRPs is really homologous. Instead, mitochondrial ribosomes of yeast and mammals have many MRPs that are unique to one or the other (group of) organism(s). Two of the 3 new MRPs presented in this paper have no counterparts in other ribosomal protein systems at all (MRP-S12 and MRP-S28). Only the mammalian MRP-L18 proteins showed significant sequence similarities to a known ribosomal protein from Escherichia coli (EcoL24). However, the sequence conservation is poor. 30.5% of a stretch of 87 amino acid residues of EcoL24 are identical to the MRP-L18_human sequence. The mammalian counterparts are elongated into both N- and C-terminal directions resulting in a protein more than twice the size of the EcoL24 protein (Fig. 2b). These values are comparable to those that have been determined for other homologous MRPs of mammals and yeast (20-22, 31). However, only half of the mammalian MRPs known on the molecular level to date have counterparts in the yeast mitochondrial and/or E. coli ribosomal system at all (Table III). Interestingly, mammalian MRPs that are similar to new yeast MRPs that have no bacterial counterparts are very rare. MRP-L27 (mammals)/YmL27 (yeast; Ref. 20) is the only example. The yeast MRPs seem to be much more distantly related to the mammalian MRPs than the cytoplasmic ribosomal proteins of the same species, rat and yeast cytoplasmic ribosomal proteins share 40-80% identical amino acid residues (32). For several of the mammalian MRPs that have been investigated so far, corresponding sequences have been identified in the C. elegans data bases (Table III). However, percentages of sequence identities between the worm and mammalian MRPs do not exceed 45% and are confined to relatively short positions and match only stretches of the complete mammalian MRP sequences. Mitochondrial ribosomes must be much more divergent among species than the cytoplasmic ribosomes from the same species. Finally, these results raise questions why and how two different multimolecular complexes that perform the same function, synthesis of proteins, but are separated only by two (mitochondrial) membranes evolved two different rates of evolution. The explanation of poor DNA repair mechanisms of the mitochondria is not satisfactory since all mitochondrial and cytoplasmic ribosomal proteins are encoded by nuclear genes (in mammals). How did mitochondrial ribosomes of yeast and mammals adopt so many additional and different proteins? Did they acquire additional proteins or did they simply not lose proteins during evolution which have been omitted by e.g. the bacteria to keep a streamlined, high-throughput but poorly regulated ribosome? We hope that the discovery of the complete set of mammalian MRPs in the future will provide some insight to these questions.

Functions of Mammalian MRP-L18s-- So far, the functions of mammalian MRPs have been elucidated only indirectly. Some analogous functions can be supposed if the MRP is homologous to a known ribosomal protein of bacterial sources for example. MRP-L18_mammalian is homologous to EcoL24. EcoL24 has been characterized as an early assembly protein of the large ribosomal subunit, binding directly to the 13 S rRNA (35). This coincides with the characterization of the MRP-L18_bov as a core protein of the ribosomal subunit that is found bound to the subunit core after washes with 4 M LiCl (1). However, MRP-L18_mammalian is more than twice the size of EcoL24, raising the possibility that additional functions for this protein may exist.

Conclusion-- Five individual bovine MRPs have been purified and partially sequenced. Two of them, MRP-L43_bov and MRP-S18_bov, turned out to be homologous to the recently published mammalian MRP-L32s and MRP-S13s, respectively (20). Based on the obtained peptide information, corresponding EST and genome DNA sequences were identified in silico, and the virtual cDNAs for human and mouse (and partially rat) were assembled. Deduced MRP sequences were characterized corresponding to the biochemical data obtained from bovine MRPs. MISPs postulated by comparison of bovine N-terminal peptide data and the deduced mammalian MRPs suggested different mechanisms for mammalian MRP import as it has been proposed for yeast MRPs (12). Only one (MRP-L18) of three newly characterized mammalian MRPs showed significant sequence similarities to a known ribosomal protein class (EcoL24). Our data demonstrate the poor conservation of MRPs among different distantly related species such as yeast and mammals (Table III), thus raising questions concerning the mechanisms of mitochondrial ribosomal evolution compared with those of cytoplasmic ribosomes.

	FOOTNOTES

^* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The peptide sequences reported in this paper have been submitted to the MIPS Data Bank with accession numbers S78761 (MRP-L18_bov, fragments), S78772 (MRP-L43_bov, fragments), S78762 (MRP-S12_bov, fragment), S78763 (MRP-S18_bov, fragment), and S78766 (MRP-S28_bov, fragment).

^b Supported by NIDCD Grant RO1-DC04092 from the National Institutes of Health.

^c Member of the Mammalian Mitochondrial Ribosomal Consortium.

^e Supported by the Nemours Research Program.

ⁱ To whom correspondence should be addressed: AG Kress, Institute for Biology-Genetics, Free University of Berlin, Arnimallee 7, D-14195 Berlin, Germany. Tel.: +49-30-838-2629; Fax: +49-30-838-3649; E-mail: graack@zedat.fu-berlin.de.

Published, JBC Papers in Press, April 5, 2000, DOI 10.1074/jbc.M909762199

² J. Anders, H.-R. Graack, J. Liu, and T. W. O'Brien, manuscript in preparation.

	ABBREVIATIONS

The abbreviations used are: MRP, mitochondrial ribosomal protein(s); PAGE, polyacrylamide gel electrophoresis; bp, base pair(s); MISP, mitochondrial import signal peptide; ORF, open reading frame; EST, expressed sequence tag.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

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