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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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Analysis of the xlProminin-1 alternatively spliced isoforms in four tissues: the retina, brain, testis, and kidney. We found that profiles of the xlProminin-1 alternative splicing are different in these tissues. A: Products of reverse-transcription PCR (RT–PCR) from a region of extensive alternative splicing on the xlProminin-1 gene (exon 20 to 28) were separated on a 1% agarose gel and visualized with ethidium bromide staining. Five discrete PCR products were excised from the gel, cloned, and sequenced. Their exon compositions were determined and each product was designated with a Greek letter (α, β, γ, δ, and ε) for identification when used in the quantitative analysis shown in panel B. The predominant isoforms of xlProminin-1 expressed in the retina lack the alternatively spliced exons 26b, 27, and 28a, whereas the predominant isoform of xlProminin-1 expressed in the kidney retains these exons. The predominant isoforms of xlProminin-1 expressed in the brain and kidney retain exon 28a. Exon 27 was retained only when exon 26b was retained as well. B: Quantification of the xlProminin-1 alternatively spliced isoforms in four tissues: the retina, brain, testis, and kidney. The resolved RT–PCR products were linearly scanned and the optical densities were plotted. Green arrows indicate peaks that represent isoforms resulting from alternative splicing. The major isoform of xlProminin-1 expressed in the retina, ε, lacks the alternatively spliced exons 26b, 27, and 28a, whereas the major form of xlProminin-1 expressed in the kidney, α, retains all possible exons. The majority of xlProminin-1 expressed in the brain and kidney retains exon 28a.
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f5: Analysis of the xlProminin-1 alternatively spliced isoforms in four tissues: the retina, brain, testis, and kidney. We found that profiles of the xlProminin-1 alternative splicing are different in these tissues. A: Products of reverse-transcription PCR (RT–PCR) from a region of extensive alternative splicing on the xlProminin-1 gene (exon 20 to 28) were separated on a 1% agarose gel and visualized with ethidium bromide staining. Five discrete PCR products were excised from the gel, cloned, and sequenced. Their exon compositions were determined and each product was designated with a Greek letter (α, β, γ, δ, and ε) for identification when used in the quantitative analysis shown in panel B. The predominant isoforms of xlProminin-1 expressed in the retina lack the alternatively spliced exons 26b, 27, and 28a, whereas the predominant isoform of xlProminin-1 expressed in the kidney retains these exons. The predominant isoforms of xlProminin-1 expressed in the brain and kidney retain exon 28a. Exon 27 was retained only when exon 26b was retained as well. B: Quantification of the xlProminin-1 alternatively spliced isoforms in four tissues: the retina, brain, testis, and kidney. The resolved RT–PCR products were linearly scanned and the optical densities were plotted. Green arrows indicate peaks that represent isoforms resulting from alternative splicing. The major isoform of xlProminin-1 expressed in the retina, ε, lacks the alternatively spliced exons 26b, 27, and 28a, whereas the major form of xlProminin-1 expressed in the kidney, α, retains all possible exons. The majority of xlProminin-1 expressed in the brain and kidney retains exon 28a.

Mentions: Fargeas et al. previously reported that splicing of the prominin-1 gene in mice results in great variations in the sequences of alternative C-termini [32]. We therefore examined the splicing profile of xlProminin-1 within a region from exon 20 to 28 by RT–PCR (Figure 5). We found that the two most abundant isoforms of xlProminin-1 in the X. laevis retina did not contain exon 27; their exon compositions were: (20–24)+26b-27-28a-28+ (ε in Figure 5) or (20–24)+26b+27-28a-28+ (δ in Figure 5). Isoforms containing exon 28a were also detected in the retina; their exon compositions were: (20–24)+26b-27-28a+28+ (γ in Figure 5), (20–24)+26b+27-28a+28+ (β in Figure 5) and (20–24)+26b+27+28a+28+ (α in Figure 5). However, isoforms containing exon 28a only accounted for a very small proportion of all xlProminin-1 in the retina (Figure 5). This result was unlikely to be biased by preferential amplification of smaller PCR products, since profiling of xlProminin-1 alternative splicing in this region gave very different results in the kidney, testis, and brain: Those tissues preferentially express the isoforms with exon 28a (Figure 5).


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

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

Analysis of the xlProminin-1 alternatively spliced isoforms in four tissues: the retina, brain, testis, and kidney. We found that profiles of the xlProminin-1 alternative splicing are different in these tissues. A: Products of reverse-transcription PCR (RT–PCR) from a region of extensive alternative splicing on the xlProminin-1 gene (exon 20 to 28) were separated on a 1% agarose gel and visualized with ethidium bromide staining. Five discrete PCR products were excised from the gel, cloned, and sequenced. Their exon compositions were determined and each product was designated with a Greek letter (α, β, γ, δ, and ε) for identification when used in the quantitative analysis shown in panel B. The predominant isoforms of xlProminin-1 expressed in the retina lack the alternatively spliced exons 26b, 27, and 28a, whereas the predominant isoform of xlProminin-1 expressed in the kidney retains these exons. The predominant isoforms of xlProminin-1 expressed in the brain and kidney retain exon 28a. Exon 27 was retained only when exon 26b was retained as well. B: Quantification of the xlProminin-1 alternatively spliced isoforms in four tissues: the retina, brain, testis, and kidney. The resolved RT–PCR products were linearly scanned and the optical densities were plotted. Green arrows indicate peaks that represent isoforms resulting from alternative splicing. The major isoform of xlProminin-1 expressed in the retina, ε, lacks the alternatively spliced exons 26b, 27, and 28a, whereas the major form of xlProminin-1 expressed in the kidney, α, retains all possible exons. The majority of xlProminin-1 expressed in the brain and kidney retains exon 28a.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3108902&req=5

f5: Analysis of the xlProminin-1 alternatively spliced isoforms in four tissues: the retina, brain, testis, and kidney. We found that profiles of the xlProminin-1 alternative splicing are different in these tissues. A: Products of reverse-transcription PCR (RT–PCR) from a region of extensive alternative splicing on the xlProminin-1 gene (exon 20 to 28) were separated on a 1% agarose gel and visualized with ethidium bromide staining. Five discrete PCR products were excised from the gel, cloned, and sequenced. Their exon compositions were determined and each product was designated with a Greek letter (α, β, γ, δ, and ε) for identification when used in the quantitative analysis shown in panel B. The predominant isoforms of xlProminin-1 expressed in the retina lack the alternatively spliced exons 26b, 27, and 28a, whereas the predominant isoform of xlProminin-1 expressed in the kidney retains these exons. The predominant isoforms of xlProminin-1 expressed in the brain and kidney retain exon 28a. Exon 27 was retained only when exon 26b was retained as well. B: Quantification of the xlProminin-1 alternatively spliced isoforms in four tissues: the retina, brain, testis, and kidney. The resolved RT–PCR products were linearly scanned and the optical densities were plotted. Green arrows indicate peaks that represent isoforms resulting from alternative splicing. The major isoform of xlProminin-1 expressed in the retina, ε, lacks the alternatively spliced exons 26b, 27, and 28a, whereas the major form of xlProminin-1 expressed in the kidney, α, retains all possible exons. The majority of xlProminin-1 expressed in the brain and kidney retains exon 28a.
Mentions: Fargeas et al. previously reported that splicing of the prominin-1 gene in mice results in great variations in the sequences of alternative C-termini [32]. We therefore examined the splicing profile of xlProminin-1 within a region from exon 20 to 28 by RT–PCR (Figure 5). We found that the two most abundant isoforms of xlProminin-1 in the X. laevis retina did not contain exon 27; their exon compositions were: (20–24)+26b-27-28a-28+ (ε in Figure 5) or (20–24)+26b+27-28a-28+ (δ in Figure 5). Isoforms containing exon 28a were also detected in the retina; their exon compositions were: (20–24)+26b-27-28a+28+ (γ in Figure 5), (20–24)+26b+27-28a+28+ (β in Figure 5) and (20–24)+26b+27+28a+28+ (α in Figure 5). However, isoforms containing exon 28a only accounted for a very small proportion of all xlProminin-1 in the retina (Figure 5). This result was unlikely to be biased by preferential amplification of smaller PCR products, since profiling of xlProminin-1 alternative splicing in this region gave very different results in the kidney, testis, and brain: Those tissues preferentially express the isoforms with exon 28a (Figure 5).

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