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Skipping of exons by premature termination of transcription and alternative splicing within intron-5 of the sheep SCF gene: a novel splice variant.

Saravanaperumal SA, Pediconi D, Renieri C, La Terza A - PLoS ONE (2012)

Bottom Line: In contrast, the shorter (835 and/or 725 bp) cDNA was found to be a 'novel' mRNA splice variant.We also demonstrated that the Northern blot analysis at transcript level is mediated via an intron-5 splicing event.This work provides a basis for understanding the functional role and regulation of SCF in hair follicle melanogenesis in sheep beyond what was known in mice, humans and other mammals.

View Article: PubMed Central - PubMed

Affiliation: School of Environmental Sciences, University of Camerino, via Gentile III da Varano, Camerino, MC, Italy. sivabiotech2002@yahoo.com

ABSTRACT
Stem cell factor (SCF) is a growth factor, essential for haemopoiesis, mast cell development and melanogenesis. In the hematopoietic microenvironment (HM), SCF is produced either as a membrane-bound (-) or soluble (+) forms. Skin expression of SCF stimulates melanocyte migration, proliferation, differentiation, and survival. We report for the first time, a novel mRNA splice variant of SCF from the skin of white merino sheep via cloning and sequencing. Reverse transcriptase (RT)-PCR and molecular prediction revealed two different cDNA products of SCF. Full-length cDNA libraries were enriched by the method of rapid amplification of cDNA ends (RACE-PCR). Nucleotide sequencing and molecular prediction revealed that the primary 1519 base pair (bp) cDNA encodes a precursor protein of 274 amino acids (aa), commonly known as 'soluble' isoform. In contrast, the shorter (835 and/or 725 bp) cDNA was found to be a 'novel' mRNA splice variant. It contains an open reading frame (ORF) corresponding to a truncated protein of 181 aa (vs 245 aa) with an unique C-terminus lacking the primary proteolytic segment (28 aa) right after the D(175)G site which is necessary to produce 'soluble' form of SCF. This alternative splice (AS) variant was explained by the complete nucleotide sequencing of splice junction covering exon 5-intron (5)-exon 6 (948 bp) with a premature termination codon (PTC) whereby exons 6 to 9/10 are skipped (Cassette Exon, CE 6-9/10). We also demonstrated that the Northern blot analysis at transcript level is mediated via an intron-5 splicing event. Our data refine the structure of SCF gene; clarify the presence (+) and/or absence (-) of primary proteolytic-cleavage site specific SCF splice variants. This work provides a basis for understanding the functional role and regulation of SCF in hair follicle melanogenesis in sheep beyond what was known in mice, humans and other mammals.

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Expression of ovine SCF in skin.Northern blot analysis show ovine SCF (+) and (−) mRNA expression. Ovine 18S rRNA was used as an internal control. Northern blot analysis was carried out with a DIG-labeled cDNA probe for SCF and 18S rRNA (see Table S2) as described in Materials and Methods section. Br, Bl, Wh represents individual of Brown, Black and White merino sheep, respectively.
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pone-0038657-g007: Expression of ovine SCF in skin.Northern blot analysis show ovine SCF (+) and (−) mRNA expression. Ovine 18S rRNA was used as an internal control. Northern blot analysis was carried out with a DIG-labeled cDNA probe for SCF and 18S rRNA (see Table S2) as described in Materials and Methods section. Br, Bl, Wh represents individual of Brown, Black and White merino sheep, respectively.

Mentions: Initially, to verify any eventual difference(s) between the expression level of two different splice variants of oSCF (+/−) four sets of primer (summarized in Table S2) were used as described in materials and methods. Three individuals of white, black and brown animals were subjected to a single round RT-PCR amplification. The RT-PCR reactions gave fragments (see Table S2 for details) exhibiting almost the same level of band intensity for both the (+) and (−) form (data not shown). In contrast, Northern blot analysis showed substantial differences in the expression oSCF between (+) and (−) form (Figure 7). At this juncture, we propose that the oSCF gene expression in white, black and brown animals at mRNA transcript level is mediated via an intron-5 AS event (Figure 3(c,d). However, both forms (+/−) are biologically active and reported to have different effects on cells [9]–[11], [20]. The regulation of processing of the proposed secondary proteolytic cleavage site encoded by exon 7, could play a critical role in the function of membrane-associated SCF (−) protein [10].


Skipping of exons by premature termination of transcription and alternative splicing within intron-5 of the sheep SCF gene: a novel splice variant.

Saravanaperumal SA, Pediconi D, Renieri C, La Terza A - PLoS ONE (2012)

Expression of ovine SCF in skin.Northern blot analysis show ovine SCF (+) and (−) mRNA expression. Ovine 18S rRNA was used as an internal control. Northern blot analysis was carried out with a DIG-labeled cDNA probe for SCF and 18S rRNA (see Table S2) as described in Materials and Methods section. Br, Bl, Wh represents individual of Brown, Black and White merino sheep, respectively.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3376141&req=5

pone-0038657-g007: Expression of ovine SCF in skin.Northern blot analysis show ovine SCF (+) and (−) mRNA expression. Ovine 18S rRNA was used as an internal control. Northern blot analysis was carried out with a DIG-labeled cDNA probe for SCF and 18S rRNA (see Table S2) as described in Materials and Methods section. Br, Bl, Wh represents individual of Brown, Black and White merino sheep, respectively.
Mentions: Initially, to verify any eventual difference(s) between the expression level of two different splice variants of oSCF (+/−) four sets of primer (summarized in Table S2) were used as described in materials and methods. Three individuals of white, black and brown animals were subjected to a single round RT-PCR amplification. The RT-PCR reactions gave fragments (see Table S2 for details) exhibiting almost the same level of band intensity for both the (+) and (−) form (data not shown). In contrast, Northern blot analysis showed substantial differences in the expression oSCF between (+) and (−) form (Figure 7). At this juncture, we propose that the oSCF gene expression in white, black and brown animals at mRNA transcript level is mediated via an intron-5 AS event (Figure 3(c,d). However, both forms (+/−) are biologically active and reported to have different effects on cells [9]–[11], [20]. The regulation of processing of the proposed secondary proteolytic cleavage site encoded by exon 7, could play a critical role in the function of membrane-associated SCF (−) protein [10].

Bottom Line: In contrast, the shorter (835 and/or 725 bp) cDNA was found to be a 'novel' mRNA splice variant.We also demonstrated that the Northern blot analysis at transcript level is mediated via an intron-5 splicing event.This work provides a basis for understanding the functional role and regulation of SCF in hair follicle melanogenesis in sheep beyond what was known in mice, humans and other mammals.

View Article: PubMed Central - PubMed

Affiliation: School of Environmental Sciences, University of Camerino, via Gentile III da Varano, Camerino, MC, Italy. sivabiotech2002@yahoo.com

ABSTRACT
Stem cell factor (SCF) is a growth factor, essential for haemopoiesis, mast cell development and melanogenesis. In the hematopoietic microenvironment (HM), SCF is produced either as a membrane-bound (-) or soluble (+) forms. Skin expression of SCF stimulates melanocyte migration, proliferation, differentiation, and survival. We report for the first time, a novel mRNA splice variant of SCF from the skin of white merino sheep via cloning and sequencing. Reverse transcriptase (RT)-PCR and molecular prediction revealed two different cDNA products of SCF. Full-length cDNA libraries were enriched by the method of rapid amplification of cDNA ends (RACE-PCR). Nucleotide sequencing and molecular prediction revealed that the primary 1519 base pair (bp) cDNA encodes a precursor protein of 274 amino acids (aa), commonly known as 'soluble' isoform. In contrast, the shorter (835 and/or 725 bp) cDNA was found to be a 'novel' mRNA splice variant. It contains an open reading frame (ORF) corresponding to a truncated protein of 181 aa (vs 245 aa) with an unique C-terminus lacking the primary proteolytic segment (28 aa) right after the D(175)G site which is necessary to produce 'soluble' form of SCF. This alternative splice (AS) variant was explained by the complete nucleotide sequencing of splice junction covering exon 5-intron (5)-exon 6 (948 bp) with a premature termination codon (PTC) whereby exons 6 to 9/10 are skipped (Cassette Exon, CE 6-9/10). We also demonstrated that the Northern blot analysis at transcript level is mediated via an intron-5 splicing event. Our data refine the structure of SCF gene; clarify the presence (+) and/or absence (-) of primary proteolytic-cleavage site specific SCF splice variants. This work provides a basis for understanding the functional role and regulation of SCF in hair follicle melanogenesis in sheep beyond what was known in mice, humans and other mammals.

Show MeSH
Related in: MedlinePlus