Comparative genomic analysis reveals a novel mitochondrial isoform of human rTS protein and unusual phylogenetic distribution of the rTS gene
© Liang et al; licensee BioMed Central Ltd. 2005
Received: 20 April 2005
Accepted: 14 September 2005
Published: 14 September 2005
The rTS gene (ENOSF1), first identified in Homo sapiens as a gene complementary to the thymidylate synthase (TYMS) mRNA, is known to encode two protein isoforms, rTSα and rTSβ. The rTSβ isoform appears to be an enzyme responsible for the synthesis of signaling molecules involved in the down-regulation of thymidylate synthase, but the exact cellular functions of rTS genes are largely unknown.
Through comparative genomic sequence analysis, we predicted the existence of a novel protein isoform, rTS, which has a 27 residue longer N-terminus by virtue of utilizing an alternative start codon located upstream of the start codon in rTSβ. We observed that a similar extended N-terminus could be predicted in all rTS genes for which genomic sequences are available and the extended regions are conserved from bacteria to human. Therefore, we reasoned that the protein with the extended N-terminus might represent an ancestral form of the rTS protein. Sequence analysis strongly predicts a mitochondrial signal sequence in the extended N-terminal of human rTSγ, which is absent in rTSβ. We confirmed the existence of rTS in human mitochondria experimentally by demonstrating the presence of both rTSγ and rTSβ proteins in mitochondria isolated by subcellular fractionation. In addition, our comprehensive analysis of rTS orthologous sequences reveals an unusual phylogenetic distribution of this gene, which suggests the occurrence of one or more horizontal gene transfer events.
The presence of two rTS isoforms in mitochondria suggests that the rTS signaling pathway may be active within mitochondria. Our report also presents an example of identifying novel protein isoforms and for improving gene annotation through comparative genomic analysis.
The rTS (ENOSF1) gene, a member of the enolase family, was initially identified in Homo sapiens by the discovery of an RNA with extensive complementarity to the mRNA for the DNA biosynthetic enzyme thymidylate synthase[1, 2]. The rTS gene was later shown to code for two proteins (rTSα and rTSβ) through alternative RNA splicing [2, 3]. The mRNA for rTSα is complementary to thymidylate synthase mRNA, while the mRNA for rTSβ is not [2, 3]. The rTSβ protein is the major protein product of the rTS gene and its expression is associated with the down-regulation of thymidylate synthase protein as cultured cells enter growth arrest . Expression of rTSβ correlates with the production of small molecules that appear to mediate the down-regulation of thymidylate synthase protein by a novel intercellular signaling mechanism . Overproduction of rTSβ occurs in some cells resistant to inhibitors of thymidylate synthase or dihydrofolate reductase, indicating a role for the rTS gene in folate and nucleotide metabolism, as well as anticancer drug resistance [2–6].
While the specific function(s) of the rTS gene products are currently under investigation, we now report a new rTS protein isoform and its association with mitochondria. The existence of this new isoform, rTSγ, was first predicted using a computational comparative genomic sequence analysis approach and was then verified experimentally. This unexpected observation suggests that rTS may have functions in addition to intercellular signaling.
A conserved extended protein N-terminus can be deduced from all available rTS genes
The extended N-terminus contributes a mitochondrial signal in human rTSγ protein
Predicted mitochondrial signal for rTSβ and γ proteins
rTSγ protein exists and is associated with mitochondria in human cells
Functional implications for the rTS gene based on its mitochondrial location
It has recently been determined that rTSβ is an enzyme responsible for the synthesis of small signaling molecules involved with the down-regulation of thymidylate synthase as cells enter growth arrest in vitro . The signaling associated with rTSβ was also shown to act intercellularly . Sequence analysis of the rTS gene suggested the presence of a mitochondrial leader sequence that would be expected to increase the length of rTSβ from 416 to 443 amino acids in rTSγ with an expected change in molecular mass from 46,892 to 49,742 Da. Based upon the prediction of a mitochondrial leader sequence, we evaluated whether there is a mitochondrial form of the rTS protein. The results shown in Fig. 3 indicate that this is the case, with two species being present in the mitochondria. These two protein species have apparent molecular masses of 52.7 ± 1.8 and 47.6 ± 0.7 kDa. The smaller species corresponds well to the predicted size expected for rTSβ with the mitochondrial leader sequence cleaved off, while the larger species differs by 3 kDa from the predicted molecular mass for the isoform with the leader sequence. The amounts of rTSγ and rTSβ in the combined cytoplasmic and mitochondrial fractions differ from that expected to result from combining equal amounts of each fraction. The increased abundance of rTSβ, relative to rTSγ in the combined fraction may be the result of in situ cleavage of the rTSγ mitochondrial leader sequence by cytosolic proteases, which has been observed to occur in yeast cytosol , although transfer of protein may also contribute to the change in signal strength as the MnSOD signal is weaker in the lane with the combined fractions than in the lane loaded with just the organelle protein. Although the migration of proteins in SDS-PAGE gives only approximate estimation of molecular masses, there is a possibility that other post-translational modifications may contribute to this discrepancy. The co-localization of rTSβ and rTSγ with GDH and MnSOD after subcellular fractionation strongly suggests that the extended N-terminal sequence serves as the leader sequence for mitochondrial location of the protein and that it is likely this is partially cleaved to generate the β isoform, once rTSγ is transported into the mitochondria. The presence of the rTSβ protein in mitochondria raises the question of what role this enzyme and the rTS signaling pathway may play there. Mitochondria are a major site for folate metabolism in mammalian cells . Thymidylate synthase is also found within mitochondria , despite the absence of a canonical mitochondrial leader sequence, and the relationship of rTS signaling to thymidylate synthase and folate metabolism may ultimately provide the explanation for this phenomenon. Recent evidence indicates that treatment of cells with an rTSβ signaling mimic can affect the cytoskeleton and cause down-regulation of TYMS [10, 13]. The co-localization of rTS and thymidylate synthase in the mitochondria may indicate that rTSβ, in addition to its role in intercellular signaling, also provides intracellular signals that regulate thymidylate synthase levels in the mitochondria.
Since the extended N-termini of rTS are conserved from bacteria to human, we believe that the rTSγ form of the rTS gene represents the ancestral form of this gene, while rTSβ, which seems to be the predominantly expressed form in the cytosol, represents an isoform that appeared later during evolution and came to co-exist with the ancestral isoform, at least in Homo sapiens. Based on a recent study which shows that suboptimal AUG codons can support translation via leaky scanning and reinitiation , the co-existence of protein products translated using different AUG codons in the same reading frame may not be a rare phenomena. In fact, the starting AUG codons for rTSγ and rTSβ are both qualified as optimal start codons according to recent studies of translation initiation in mammalian and plant genes [14–16], providing an explanation for the co-existence of rTSγ and rTSβ. A recent study suggests that many alternative splicing forms cause differential subcellular localization, especially in targeting either peroxisomes or mitochondria , and our data serves as evidence supporting such a notion. An interesting subject for future studies will be to determine when the shorter rTSβ isoform appeared during evolution.
Phylogenetic distribution and origin of rTS gene
In addition to the discovery of this novel mitochondrial isoform of rTS, our report also represents a good illustration of the use of comparative genomic analysis for identifying novel protein isoforms of genes and for improving existing gene predictions. Through this study, we have made a comprehensive collection of rTS orthologous sequences and have made a list of suggested modifications to the existing gene annotation (see detailed modifications in the supplemental materials). In addition, by searching all annotated human genes, we identified a few other genes in which evolutionarily conserved alternative upstream AUG codons exist (similar situation as rTSβ). These genes include SELL [NM_000655], SPCS1 [NM_014041.1], NT5C3 [NM_016489], and SDN1 [NM_014390] (data not shown).
In summary, through comparative genomic analyses we revealed an unusual phylogenetic distribution of the rTS gene and identified a novel mitochondria isoform of this gene and verified it experimentally. A mitochondrial location of rTS protein and phylogenetic distribution of this gene provide us with new information that will assist in elucidating its function.
Identification of an extended N-terminal region for all available rTS genes
To search for all available rTS orthologous sequences, we first queried all protein sequences deposited in the NCBI non-redundant protein database (nr) by BLAST search . Among all identified rTSβ orthologous protein sequences, the ones with an N-terminus equivalent to human rTSβ were identified and their corresponding genomic sequences, including sufficient 5'-end upstream sequences for analysis, were retrieved. Extended N-termini were deduced by extending the start codon to the next available "ATG" upstream of the start codon used by the existing annotation. In addition to the rTS orthologous genomic sequences, we also searched the NCBI EST database by performing a TBLASTN search with the extended human rTSβ protein sequence to collect rTS cDNA sequences from additional species. To predict any potential new function or cellular location contributed by the extended N-terminus, we analyzed the human rTSγ and rTSβ protein sequences with Mitoprot , TargetP , PSORTII , and Mitopredict [25, 26].
Subcellular fractionation of CCRF-CEM cells
The human T-lymphoblastic cell line CCRF-CEM was cultured as described . Subcellular fractions of CCRF-CEM cells were isolated essentially as described , except that protease (0.5 mM Pefabloc) and phosphatase inhibitors (1 mM sodium metavanadate and 1 mM NaF) were included in all buffers. All steps were performed at 0 – 4°C. Briefly, 1-liter of CCRF-CEM cells (~3.5 × 105 cells/ml) was centrifuged at 1000 × g for 5 min and the pellet was washed with iced 0.9% NaCl, and then suspended in 5 pellet volumes of ice-cold hypotonic buffer  and allowed to swell for 5 min on ice. The suspension was homogenized in an ice-cold 7-ml glass Dounce homogenizer with 15 strokes of the tight pestle to obtain > 95% cell disruption. The homogenate was immediately made up to 250 mM sucrose and centrifuged at 1000 × g for 5 min. The pellet was washed once with 2.5 original pellet volumes of cold isotonic buffer (1 mM Na2-EDTA, 250 mM sucrose; pH 6.9). The two supernatants were combined to generate the post-nuclear supernatant (PNS). The PNS was centrifuged at 17,000 × g, 15 min to generate a cytosolic fraction (supernatant) and an organellar pellet containing both mitochondria and lysosomes. The organellar pellet was suspended in 1 ml of HES (20 mM HEPES-NaOH, 1 mM Na2-EDTA, 250 mM sucrose; pH 7.4). Subcellular fractions were assayed in duplicate for activity of the cytosolic enzyme lactate dehydrogenase (LDH)  and mitochondrial matrix enzyme glutamate dehydrogenase (GDH)  to ensure that fractionation was successful. To separate mitochondria and lysosomes, this organellar suspension was made up to 30% (w/v) iodixanol [8, 32] in a final volume of 2.2 ml and placed on a 5–20% linear iodixanol gradient (9 ml), centrifuged at 70,000 × g for 1.5 hr, and collected as 0.5-ml fractions, which were then diluted with 0.5 ml ice-cold HES and centrifuged at 30,000 × g for 15 min. The resulting organelle pellets were suspended in 200 μl of isotonic buffer (as above, except that NaF was 50 mM). Protein concentration was determined using the BioRad (Hercules, CA) protein assay kit.
Proteins were resolved by denaturing gel electrophoresis using 10% polyacrylamide and transferred to PVDF membranes essentially as described . The primary antibodies used were: D3 (mouse monoclonal to rTSβ), LAMP-1 (mouse monoclonal to lysosome-associated membrane protein-1; Santa Cruz Biotechnology), and MnSOD (rabbit polyclonal to manganese superoxide dismutase; Stressgen). Secondary antibodies consisted of horseradish peroxidase conjugated F(ab')2 fragments and were obtained from Jackson ImmunoResearch Laboratories. Probed blots were imaged using West Pico Dura chemiluminescent reagent (Pierce) and X-OMAT AR X-ray film (Kodak). Pre-stained protein molecular weight markers (BioRad) were included during electrophoresis to allow determination of apparent molecular weights of detected antigens. All experiments were repeated at least twice with similar results.
The authors would like to thank Dr. John M. Graham, JG Research Consultancy, UK, for his suggestions and advice for the preparation of iodixanol gradients. This work is partially supported by a developmental fund from Roswell Park Cancer Institute (PL) and NIH grants CA101515 (PL), EB002116 (BJD), CA43500 (JJM), and CA16056 (RPCI).
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