Conservation of CD44 exon v3 functional elements in mammals
© Vela et al; licensee BioMed Central Ltd. 2008
Received: 07 March 2008
Accepted: 29 July 2008
Published: 29 July 2008
The human CD44 gene contains 10 variable exons (v1 to v10) that can be alternatively spliced to generate hundreds of different CD44 protein isoforms. Human CD44 variable exon v3 inclusion in the final mRNA depends on a multisite bipartite splicing enhancer located within the exon itself, which we have recently described, and provides the protein domain responsible for growth factor binding to CD44.
We have analyzed the sequence of CD44v3 in 95 mammalian species to report high conservation levels for both its splicing regulatory elements (the 3' splice site and the exonic splicing enhancer), and the functional glycosaminglycan binding site coded by v3. We also report the functional expression of CD44v3 isoforms in peripheral blood cells of different mammalian taxa with both consensus and variant v3 sequences.
CD44v3 mammalian sequences maintain all functional splicing regulatory elements as well as the GAG binding site with the same relative positions and sequence identity previously described during alternative splicing of human CD44. The sequence within the GAG attachment site, which in turn contains the Y motif of the exonic splicing enhancer, is more conserved relative to the rest of exon. Amplification of CD44v3 sequence from mammalian species but not from birds, fish or reptiles, may lead to classify CD44v3 as an exclusive mammalian gene trait.
Human variable exon v3 can follow a specific alternative splicing route different from that affecting other variable exons so it can be included in the mRNA together with other variable exons or independently from them [6, 7]. This inclusion is regulated by a multisite bipartite exonic splicing enhancer (ESE) consisting in a tandem nonamer (XX motif) and a heptamer (Y motif) that act cooperatively for the efficient recognition of the splice sites . The XX motif is located centrally in the exon while the Y motif is located within the sequence coding for the glycosaminglycan (GAG) binding site immediately downstream from the SGSG motif in v3 (figure 1B).
In order to address the existence and functional nature of the XXY ESE in non-human species we have evaluated the overall level of conservation of CD44 exon v3, including its splicing regulatory elements-the 3' splice site (3'ss), the XXY splicing enhancer- and the GAG binding site, in 95 mammalian species. We also provide data of CD44v3 inclusion into mRNA from peripheral blood samples, by means of RT-PCR, in some representative mammalian taxa with differing levels of conservation of the sequence elements analyzed.
Exon v3-intron 7
CD44 RT-PCR was performed from total RNA extracted from frozen blood samples with a modification of the QIAamp RNA Blood Mini kit protocol (QIAGEN). Briefly, 150 μl of frozen blood were lysed at 70°C for 10 min with RLT/β-mercaptoethanol buffer containing 4 mg/ml Proteinase K and centrifuged at 10,000 × g for 3 min. 450 μl of the lysate supernatant were mixed with 225 μl of absolute ethanol and loaded in a QIAamp spin column following manufacturer's instructions. Eluted RNA was treated with RQ1 RNase-free DNase (Promega) at 37°C for 30 min and purified following the QIAamp RNA Mini protocol for RNA cleanup (QIAGEN). The first-strand reaction was performed with random primers (Promega) and SuperScript II Reverse Transcriptase (Invitrogen). As control of RNA quality, total CD44 isoforms were amplified with degenerate E20F-VI and E20R-QEM primer set (table 1) using GC-Rich PCR System (Roche).
In order to amplify CD44v3 containing isoforms, PCR primers were designed based on a multiple sequence alignment containing the sequences corresponding to the 95 mammalian species. Exon v3 positions that showed full conservation were identified and selected to locate the 3' ends of the primers ensuring perfect matches. According to this, v3 amplification was perfomed with primers 13v3F and 100v3R (table 1) and PCR Master Mix (Promega). As control of complete genomic DNA digestion, non reverse-transcribed RNAs were tested amplification negative with primers 13v3F and 100v3R.
There is little sequence data available for CD44 variable exons from most animal species. The orthologue prediction for human CD44 in Ensembl release 48 provides v3 exon sequence for 16 species of mammals. In order to increase the data available we studied CD44 exon v3 in most of the animal samples stored in our tissue bank.
A region that enabled amplification of CD44v3, was located by a Blast search against multiple species using a human genomic fragment spanning intron6-v3-intron7. Alignment of sequences corresponding to the 10 nt at the 3' end of the INT6SF primer matched perfectly in Macaca mulatta, Canis familiaris, Oryctolagus cuniculus and Pan troglodytes and presented a single nucleotide missmatch in Mus musculus, Rattus norvegicus, Loxodonta africana and Bos taurus. Likewise, primer I7wtR matched perfectly with the exception of Mus musculus where there was a single nucleotide missmatch. We tested primers INT6SF and I7wtR in Loxodonta africana, Canis familiaris, Bos taurus, Oryctolagus cuniculus and in non-mammalian species such as Spheniscus humboldti, Psittacus erithacus and Varanus niloticus. Sequence confirmation of PCR products showed that only mammalian species amplified CD44v3. We failed to amplify v3 (exonic and/or flanking intronic sequences) from Spheniscus humboldti, Psittacus erithacus, Cygnus atratus, Threskiornis aethiopicus, Dacelo novaguineae, Ciconia ciconia, Amazona aestiva, Guaruba guarouba, Varanus niloticus, Sparus aurata, Merluccious merlucius and Plesionika edwarsii. In the absence of v3 specific PCR amplification we cannot evaluate the presence/absence of v3 nor the lack of conservation of primer regions. Lack of v3 amplification from bird, fish or reptile DNA is in agreement with the absence of exon v3 from available genomes of Gallus gallus [GenBank: NC_006092], Xenopus tropicalis [Ensembl: ENSXETG00000007556] and Gasterosteus aculeatus [Ensembl: ENSGACG00000011430].
In view of this, our sample set has been restricted to 95 mammalian species distributed in 29 families. The region amplified comprises, relative to the known human CD44v3 sequence, a 5' partial fragment of intron 6 and a 3' partial fragment of exon v3 (117 nucleotides out of 126) (see figure 1B). The resulting sequences are shown in Additional file 1.
CD44v3 splicing regulatory elements conservation
The 3'ss is fully conserved in 84 out of 95 species. The rest of species (11 out of 95) have single-nucleotide substitutions at positions -5 (n = 1), -6 (n = 2), -7 (n = 6) or -8 (n = 2) (see figure 2B). The functional significance of these varying positions in the splice site is addressed below by means of v3 expression analysis in peripheral blood.
Sequences representing the XXY ESE in CD44v3
CD44v3 GAG binding site conservation
Human exon v3 contains acidic residues both upstream and downstream of the SGSG motif. The eight amino acids located downstream of the SGSG site consist of acidic residues flanked by hydrophobic residues that are necessary for the specific addition of HS at this site . The species analyzed maintain a conserved GAG binding site both at the nucleotide (see figure 2A) and the amino acid level (figure 3B) implying that the secondary and/or tertiary structure around the SGSG motif is critical to initiate HS attachment, and this may have further contributed to the conservation of the Y ESE motif contained therein in all species tested.
CD44v3 expression in mammalian species
CD44v3 has been reported to be constitutively expressed in human peripheral blood cells, irrespective of their activation status [13–15]. We have used peripheral blood accordingly as a model to evaluate the expression of CD44v3 isoforms in some taxon-representative mammalian species. The RT-PCR results (see Additional files 1 and 2) show that there is no correlation between sequence variation within the 3'ss or the ESE and lack of v3 expression in peripheral blood. All species tested have revealed v3 expression implying that the conservation observed is sufficient to maintain v3 inclusion. The human CD44 protein contains an unique HS binding site coded by CD44 exon v3 , therefore enabling only CD44v3 containing isoforms to carry HS side chains and to bind and present heparin-binding growth factors and cytokines. On this basis, presence of a conserved GAG attachment site in all mammals studied may reflect a similar function [17, 18] for CD44v3 in these species.
In addition to mammals, CD44 constitutive exons are also found in birds, amphibians and fish as described in public databases although their expression in certain tissues in such taxa has not been addressed. In conclusion, we have obtained CD44v3 sequence from 95 mammalian species but have failed to amplify the homologous fragment from bird, reptile or fish species, in agreement with the lack of CD44 variable exons from available genome sequences of model organisms in these taxa. This implies that CD44v3 appears to be an exclusive mammalian gene trait. The sequence conservation observed in our dataset would support a common origin and function for this exon in all mammals. Furthermore, CD44v3 sequence conservation in mammalian species enables maintenance of functional splicing regulatory elements and the GAG binding site. The level of conservation of the sequence encoding the GAG binding site, which in turn contains the Y motif of the ESE analysed, is higher than the overall level found for the rest of the exon. Whether this phenomenon is due to purifying selection pressure contributed by the GAG attachment domain alone or in conjunction with the Y motif of the ESE remains undetermined. Functional inclusion of CD44v3 has also been demonstrated in peripheral blood from mammalian species representative of the different sequence variations observed, implying in vivo use of exon v3 in these species. Further work is required to search for the exact evolutionary origin of CD44 exon v3 in mammals.
exonic splicing enhancer
3' splice site.
This work was supported in part by PROFIT-01000-2004-212 from the Spanish Ministry of Industry, Tourism and Trade. EV is registered at the Department of Cellular Biology, Physiology and Immunology at the Autonomous University of Barcelona, Spain, for PhD programme administration purposes.
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