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Identification of three prominin homologs and characterization of their messenger RNA expression in Xenopus laevis tissues.

Han Z, Papermaster DS - Mol. Vis. (2011)

Bottom Line: Two of these homologs are likely to be the X. laevis orthologs of mammalian prominin-1 and 2, respectively, while the third homolog is likely to be the X. laevis ortholog of prominin-3, which was only found in nonmammalian vertebrates and the platypus.Similar to mammalian prominin-1, we found that exons 26b, 27, and 28a of the X. laevis prominin-1 gene are alternatively spliced, and that the splice isoforms of mRNA show tissue-specific expression profiles.Our results suggest that the mRNAs of prominin homologs are expressed in many tissues of X. laevis, but differ in their expression levels and mRNA splicing.

View Article: PubMed Central - PubMed

Affiliation: Department of Neuroscience, University of Connecticut Health Center, Farmington, CT 06030, USA.

ABSTRACT

Purpose: Prominin is a family of pentaspan transmembrane glycoproteins. They are expressed in various types of cells, including many stem/progenitor cells. Prominin-1 plays an important role in generating and maintaining the structure of the photoreceptors. In this study, we identified three prominin homologs in Xenopus laevis, a model animal widely used in vision research, and characterized their messenger RNA (mRNA) expression in selected tissues of this frog.

Methods: Reverse-transcription PCR (RT-PCR) and rapid amplification of cDNA ends (RACE) were used to isolate cDNAs of prominin homologs. Semiquantitative RT-PCR was used to measure the relative expression levels of mRNAs of the three prominin homologs in four X. laevis tissues, specifically those of the retina, brain, testis, and kidney. Sequences of prominin homologs were analyzed with bioinformatic software.

Results: We isolated cDNAs of three prominin homologs from X. laevis tissues and compared their sequences with previously described prominin-1, 2, and 3 sequences from other species using phylogenetic analysis. Two of these homologs are likely to be the X. laevis orthologs of mammalian prominin-1 and 2, respectively, while the third homolog is likely to be the X. laevis ortholog of prominin-3, which was only found in nonmammalian vertebrates and the platypus. We identified alternatively spliced exons in mRNAs of all three prominin homologs. Similar to mammalian prominin-1, we found that exons 26b, 27, and 28a of the X. laevis prominin-1 gene are alternatively spliced, and that the splice isoforms of mRNA show tissue-specific expression profiles. We found that prominin-1 was the most abundant homolog expressed in the retina, brain, and testis, while prominin-3 was the most abundant homolog in the kidney. The expression level of prominin-2 was the lowest of the three prominin homologs in all four examined tissues of this frog.

Conclusions: Our results suggest that the mRNAs of prominin homologs are expressed in many tissues of X. laevis, but differ in their expression levels and mRNA splicing. Prominin-1 is the most abundant of the three prominin homologs expressed in the frog retina.

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Alignment of the predicted protein sequences of three X. laevis prominin homologs, showing characteristic features of prominins, including the pentaspan transmembrane topology, a conserved cysteine rich domain, conserved leucine residues and N-glycosylation sites. We designated the three X. laevis prominin homologs as xlProminin-1, 2, and 3, respectively. Identical and conserved residues are indicated by differentially shaded boxes. Predicted transmembrane domains are marked with M#. The predicted signal peptides of xlProminin-1 and 2 are boxed. No signal peptide was predicted for xlProminin-3. Positions of alternatively spliced exons are indicated by arrows and the sequences of the alternatively spliced exons are given. Note that the N- and C-termini of the three X. laevis prominin homologs are less similar than other regions. Cysteine residues at the junction of M1 and the first intracellular domain (I1) are marked with yellow. Conserved leucine residues of the three xlProminin homologs are marked with red. Conserved glycosylation sites are marked with green.
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f1: Alignment of the predicted protein sequences of three X. laevis prominin homologs, showing characteristic features of prominins, including the pentaspan transmembrane topology, a conserved cysteine rich domain, conserved leucine residues and N-glycosylation sites. We designated the three X. laevis prominin homologs as xlProminin-1, 2, and 3, respectively. Identical and conserved residues are indicated by differentially shaded boxes. Predicted transmembrane domains are marked with M#. The predicted signal peptides of xlProminin-1 and 2 are boxed. No signal peptide was predicted for xlProminin-3. Positions of alternatively spliced exons are indicated by arrows and the sequences of the alternatively spliced exons are given. Note that the N- and C-termini of the three X. laevis prominin homologs are less similar than other regions. Cysteine residues at the junction of M1 and the first intracellular domain (I1) are marked with yellow. Conserved leucine residues of the three xlProminin homologs are marked with red. Conserved glycosylation sites are marked with green.

Mentions: By performing a BLAST search of the X. laevis EST database, we retrieved a sequence (GenBank accession: BJ061435) with 62% similarity to human prominin-1. BLAST search of the NCBI nucleotide database and the Ensembl X. tropicalis genomic database using the available sequences of prominin homologs from different species as query sequences revealed three different prominin homologs in X. tropicalis. These sequences have the assigned GenBank accession numbers of BC127277, XM_002932561, and XM_002940570. Sequence BC127277 showed a high degree of protein sequence identity (95%) to the above mentioned X. laevis sequence BJ061435. However, X. laevis homologs of sequences XM_002932561 and XM_002940570 could not be retrieved from any public cDNA or genomic database by BLAST search, perhaps due to incomplete coverage of the X. laevis sequences by those databases. They were instead cloned by RT–PCR using specific primer pairs based on X. tropicalis prominin homolog sequences, employing cDNA synthesized from total RNA isolated from X. laevis tissues as templates. Multiple clones were obtained and sequenced. Full-length cDNA sequences of the three X. laevis prominin homologs were obtained by performing 5′ and 3′ RACE. Predicted protein sequences are aligned in Figure 1.


Identification of three prominin homologs and characterization of their messenger RNA expression in Xenopus laevis tissues.

Han Z, Papermaster DS - Mol. Vis. (2011)

Alignment of the predicted protein sequences of three X. laevis prominin homologs, showing characteristic features of prominins, including the pentaspan transmembrane topology, a conserved cysteine rich domain, conserved leucine residues and N-glycosylation sites. We designated the three X. laevis prominin homologs as xlProminin-1, 2, and 3, respectively. Identical and conserved residues are indicated by differentially shaded boxes. Predicted transmembrane domains are marked with M#. The predicted signal peptides of xlProminin-1 and 2 are boxed. No signal peptide was predicted for xlProminin-3. Positions of alternatively spliced exons are indicated by arrows and the sequences of the alternatively spliced exons are given. Note that the N- and C-termini of the three X. laevis prominin homologs are less similar than other regions. Cysteine residues at the junction of M1 and the first intracellular domain (I1) are marked with yellow. Conserved leucine residues of the three xlProminin homologs are marked with red. Conserved glycosylation sites are marked with green.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3108902&req=5

f1: Alignment of the predicted protein sequences of three X. laevis prominin homologs, showing characteristic features of prominins, including the pentaspan transmembrane topology, a conserved cysteine rich domain, conserved leucine residues and N-glycosylation sites. We designated the three X. laevis prominin homologs as xlProminin-1, 2, and 3, respectively. Identical and conserved residues are indicated by differentially shaded boxes. Predicted transmembrane domains are marked with M#. The predicted signal peptides of xlProminin-1 and 2 are boxed. No signal peptide was predicted for xlProminin-3. Positions of alternatively spliced exons are indicated by arrows and the sequences of the alternatively spliced exons are given. Note that the N- and C-termini of the three X. laevis prominin homologs are less similar than other regions. Cysteine residues at the junction of M1 and the first intracellular domain (I1) are marked with yellow. Conserved leucine residues of the three xlProminin homologs are marked with red. Conserved glycosylation sites are marked with green.
Mentions: By performing a BLAST search of the X. laevis EST database, we retrieved a sequence (GenBank accession: BJ061435) with 62% similarity to human prominin-1. BLAST search of the NCBI nucleotide database and the Ensembl X. tropicalis genomic database using the available sequences of prominin homologs from different species as query sequences revealed three different prominin homologs in X. tropicalis. These sequences have the assigned GenBank accession numbers of BC127277, XM_002932561, and XM_002940570. Sequence BC127277 showed a high degree of protein sequence identity (95%) to the above mentioned X. laevis sequence BJ061435. However, X. laevis homologs of sequences XM_002932561 and XM_002940570 could not be retrieved from any public cDNA or genomic database by BLAST search, perhaps due to incomplete coverage of the X. laevis sequences by those databases. They were instead cloned by RT–PCR using specific primer pairs based on X. tropicalis prominin homolog sequences, employing cDNA synthesized from total RNA isolated from X. laevis tissues as templates. Multiple clones were obtained and sequenced. Full-length cDNA sequences of the three X. laevis prominin homologs were obtained by performing 5′ and 3′ RACE. Predicted protein sequences are aligned in Figure 1.

Bottom Line: Two of these homologs are likely to be the X. laevis orthologs of mammalian prominin-1 and 2, respectively, while the third homolog is likely to be the X. laevis ortholog of prominin-3, which was only found in nonmammalian vertebrates and the platypus.Similar to mammalian prominin-1, we found that exons 26b, 27, and 28a of the X. laevis prominin-1 gene are alternatively spliced, and that the splice isoforms of mRNA show tissue-specific expression profiles.Our results suggest that the mRNAs of prominin homologs are expressed in many tissues of X. laevis, but differ in their expression levels and mRNA splicing.

View Article: PubMed Central - PubMed

Affiliation: Department of Neuroscience, University of Connecticut Health Center, Farmington, CT 06030, USA.

ABSTRACT

Purpose: Prominin is a family of pentaspan transmembrane glycoproteins. They are expressed in various types of cells, including many stem/progenitor cells. Prominin-1 plays an important role in generating and maintaining the structure of the photoreceptors. In this study, we identified three prominin homologs in Xenopus laevis, a model animal widely used in vision research, and characterized their messenger RNA (mRNA) expression in selected tissues of this frog.

Methods: Reverse-transcription PCR (RT-PCR) and rapid amplification of cDNA ends (RACE) were used to isolate cDNAs of prominin homologs. Semiquantitative RT-PCR was used to measure the relative expression levels of mRNAs of the three prominin homologs in four X. laevis tissues, specifically those of the retina, brain, testis, and kidney. Sequences of prominin homologs were analyzed with bioinformatic software.

Results: We isolated cDNAs of three prominin homologs from X. laevis tissues and compared their sequences with previously described prominin-1, 2, and 3 sequences from other species using phylogenetic analysis. Two of these homologs are likely to be the X. laevis orthologs of mammalian prominin-1 and 2, respectively, while the third homolog is likely to be the X. laevis ortholog of prominin-3, which was only found in nonmammalian vertebrates and the platypus. We identified alternatively spliced exons in mRNAs of all three prominin homologs. Similar to mammalian prominin-1, we found that exons 26b, 27, and 28a of the X. laevis prominin-1 gene are alternatively spliced, and that the splice isoforms of mRNA show tissue-specific expression profiles. We found that prominin-1 was the most abundant homolog expressed in the retina, brain, and testis, while prominin-3 was the most abundant homolog in the kidney. The expression level of prominin-2 was the lowest of the three prominin homologs in all four examined tissues of this frog.

Conclusions: Our results suggest that the mRNAs of prominin homologs are expressed in many tissues of X. laevis, but differ in their expression levels and mRNA splicing. Prominin-1 is the most abundant of the three prominin homologs expressed in the frog retina.

Show MeSH
Related in: MedlinePlus