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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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Exon organization of xlProminin-1, 2, and 3. Homologous exons of xlProminin-1, 2, and 3 are aligned. Genes of all three xlProminins are alternatively spliced and their exon organization is evolutionarily conserved. Constitutive exons are marked in black. Alternatively spliced exons are marked orange. Spliced forms identified in cDNA clones are indicated by joining lines. Translation start sites (ATG) are marked with green lines and the positions of translational stop codons are marked with white lines. Note the complex splicing of exons 26b, 27, and 28a, which generates several distinct isoforms of xlProminin-1. Positions of primers used in reverse-transcription (RT)-PCR for semiquantitative analysis of mRNA expression and for alternative splicing are indicated on the diagram.
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f4: Exon organization of xlProminin-1, 2, and 3. Homologous exons of xlProminin-1, 2, and 3 are aligned. Genes of all three xlProminins are alternatively spliced and their exon organization is evolutionarily conserved. Constitutive exons are marked in black. Alternatively spliced exons are marked orange. Spliced forms identified in cDNA clones are indicated by joining lines. Translation start sites (ATG) are marked with green lines and the positions of translational stop codons are marked with white lines. Note the complex splicing of exons 26b, 27, and 28a, which generates several distinct isoforms of xlProminin-1. Positions of primers used in reverse-transcription (RT)-PCR for semiquantitative analysis of mRNA expression and for alternative splicing are indicated on the diagram.

Mentions: A total of seven full-length splice variants (s1–s7) of xlProminin-1 were cloned and sequenced, and the presence or absence of certain alternatively spliced exons is summarized in Table 4. These splice variants were not named following the nomenclature for splice variants of human and mouse prominin-1 [31], because some alternative exons in xlProminin-1 do not have corresponding exons in human and mouse, and vice versa. Comparison of exon organization of X. laevis, mouse, and human prominin-1 is described in detail below. Alignment of these cloned cDNAs reveals that there are at least seven alternatively spliced exons in the xlProminin-1 gene (Figure 4). We numbered exons following the established nomenclature [31]. Exons 3a (“a” for “alternative”), 8a, 11a, and 28a are newly discovered alternative exons found in X. laevis. They were numbered based on their positions relative to the constitutive exons. Exons 19, 26b, and 27 are alternative exons in the human and mouse that were alternatively spliced in X. laevis. Alternatively spliced exons 26b to 28a of xlProminin-1 reside in a region encoding the C-terminus of the protein. Exons 3 and 28 of xlProminin-1 were always retained, despite the fact that they appear to be alternative in the mouse and human [2,31]. We noticed that the inclusion or deletion of these alternatively spliced exons does not change the downstream reading frame, except for exon 28a, within which an in-frame stop codon resides. Inclusion of exon 28a results in a longer but different protein sequence for the C-terminus of xlProminin-1 relative to other isoforms.


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

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

Exon organization of xlProminin-1, 2, and 3. Homologous exons of xlProminin-1, 2, and 3 are aligned. Genes of all three xlProminins are alternatively spliced and their exon organization is evolutionarily conserved. Constitutive exons are marked in black. Alternatively spliced exons are marked orange. Spliced forms identified in cDNA clones are indicated by joining lines. Translation start sites (ATG) are marked with green lines and the positions of translational stop codons are marked with white lines. Note the complex splicing of exons 26b, 27, and 28a, which generates several distinct isoforms of xlProminin-1. Positions of primers used in reverse-transcription (RT)-PCR for semiquantitative analysis of mRNA expression and for alternative splicing are indicated on the diagram.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f4: Exon organization of xlProminin-1, 2, and 3. Homologous exons of xlProminin-1, 2, and 3 are aligned. Genes of all three xlProminins are alternatively spliced and their exon organization is evolutionarily conserved. Constitutive exons are marked in black. Alternatively spliced exons are marked orange. Spliced forms identified in cDNA clones are indicated by joining lines. Translation start sites (ATG) are marked with green lines and the positions of translational stop codons are marked with white lines. Note the complex splicing of exons 26b, 27, and 28a, which generates several distinct isoforms of xlProminin-1. Positions of primers used in reverse-transcription (RT)-PCR for semiquantitative analysis of mRNA expression and for alternative splicing are indicated on the diagram.
Mentions: A total of seven full-length splice variants (s1–s7) of xlProminin-1 were cloned and sequenced, and the presence or absence of certain alternatively spliced exons is summarized in Table 4. These splice variants were not named following the nomenclature for splice variants of human and mouse prominin-1 [31], because some alternative exons in xlProminin-1 do not have corresponding exons in human and mouse, and vice versa. Comparison of exon organization of X. laevis, mouse, and human prominin-1 is described in detail below. Alignment of these cloned cDNAs reveals that there are at least seven alternatively spliced exons in the xlProminin-1 gene (Figure 4). We numbered exons following the established nomenclature [31]. Exons 3a (“a” for “alternative”), 8a, 11a, and 28a are newly discovered alternative exons found in X. laevis. They were numbered based on their positions relative to the constitutive exons. Exons 19, 26b, and 27 are alternative exons in the human and mouse that were alternatively spliced in X. laevis. Alternatively spliced exons 26b to 28a of xlProminin-1 reside in a region encoding the C-terminus of the protein. Exons 3 and 28 of xlProminin-1 were always retained, despite the fact that they appear to be alternative in the mouse and human [2,31]. We noticed that the inclusion or deletion of these alternatively spliced exons does not change the downstream reading frame, except for exon 28a, within which an in-frame stop codon resides. Inclusion of exon 28a results in a longer but different protein sequence for the C-terminus of xlProminin-1 relative to other isoforms.

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