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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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Location of potential miRNA target sites on the 3′ UTR sequences of oSCF (+/−). Vertical black bars on the schematic diagram represent miRNA target sites on the 3′ UTR region. Open and dotted boxes represent potential miRNA target sites and sequence conservation, through evolution in sheep, goat, cow, dog, horse and pig. (a) The predicted potential binding site of miR-27a,b on the 3′ UTR of ovine s-SCF (+) and stemloop structure (mfold) of the miR-27a,b is shown. The seed sequences (nt. 2 to nt. 8) of the miR-27a,b is shown in red, bold letters. The target nucleotides involved in pairing are shown in blue, bold on the ovine s-SCF (+). The nucleotides involved in pairing outside the seed sequence are underlined in black; (b) The predicted potential binding site of miR-669f-3p on the 3′ UTR of ovine m-SCF (−) and stemloop structure (mfold) of the mature miR-669f-3p is shown. The miR-669f-3p target sequence is located on the non-coding intron-5 closest to exon 5. The mature miR-669f-3p is shown in red, bold letters. The target nucleotides involved in pairing are shown in blue, bold on the ovine m-SCF (−).
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pone-0038657-g009: Location of potential miRNA target sites on the 3′ UTR sequences of oSCF (+/−). Vertical black bars on the schematic diagram represent miRNA target sites on the 3′ UTR region. Open and dotted boxes represent potential miRNA target sites and sequence conservation, through evolution in sheep, goat, cow, dog, horse and pig. (a) The predicted potential binding site of miR-27a,b on the 3′ UTR of ovine s-SCF (+) and stemloop structure (mfold) of the miR-27a,b is shown. The seed sequences (nt. 2 to nt. 8) of the miR-27a,b is shown in red, bold letters. The target nucleotides involved in pairing are shown in blue, bold on the ovine s-SCF (+). The nucleotides involved in pairing outside the seed sequence are underlined in black; (b) The predicted potential binding site of miR-669f-3p on the 3′ UTR of ovine m-SCF (−) and stemloop structure (mfold) of the mature miR-669f-3p is shown. The miR-669f-3p target sequence is located on the non-coding intron-5 closest to exon 5. The mature miR-669f-3p is shown in red, bold letters. The target nucleotides involved in pairing are shown in blue, bold on the ovine m-SCF (−).

Mentions: A number of potential miRNA target sites are found within the longer ∼4.4 kb 3′ UTR sequence of human SCF (data not shown). However, in sheep, the analyzed miRNA sites that are located in the 505 bp 3′ UTR of the ovine s-SCF (+) form belongs to the miRNA families of miR-27a/b, miR-194, miR-128, miR-370, and two sites for miR-132/212, miR-320/320abcd (Figure 9(a)) where as miR-669f/a/o-3p, miR-466b and miR828b are detected on the shorter 3′ UTR segment (144 bp) of ovine m-SCF (−) form (Figure 9(b)). Interestingly, the 8-mer miRNA (miR-669f) has a high context score (87 percentile) which binds to the 21 nt off 23 nt of the 3′ UTR target of the oSCF (−) form (Figure 9(b)).


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)

Location of potential miRNA target sites on the 3′ UTR sequences of oSCF (+/−). Vertical black bars on the schematic diagram represent miRNA target sites on the 3′ UTR region. Open and dotted boxes represent potential miRNA target sites and sequence conservation, through evolution in sheep, goat, cow, dog, horse and pig. (a) The predicted potential binding site of miR-27a,b on the 3′ UTR of ovine s-SCF (+) and stemloop structure (mfold) of the miR-27a,b is shown. The seed sequences (nt. 2 to nt. 8) of the miR-27a,b is shown in red, bold letters. The target nucleotides involved in pairing are shown in blue, bold on the ovine s-SCF (+). The nucleotides involved in pairing outside the seed sequence are underlined in black; (b) The predicted potential binding site of miR-669f-3p on the 3′ UTR of ovine m-SCF (−) and stemloop structure (mfold) of the mature miR-669f-3p is shown. The miR-669f-3p target sequence is located on the non-coding intron-5 closest to exon 5. The mature miR-669f-3p is shown in red, bold letters. The target nucleotides involved in pairing are shown in blue, bold on the ovine m-SCF (−).
© Copyright Policy
Related In: Results  -  Collection

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

pone-0038657-g009: Location of potential miRNA target sites on the 3′ UTR sequences of oSCF (+/−). Vertical black bars on the schematic diagram represent miRNA target sites on the 3′ UTR region. Open and dotted boxes represent potential miRNA target sites and sequence conservation, through evolution in sheep, goat, cow, dog, horse and pig. (a) The predicted potential binding site of miR-27a,b on the 3′ UTR of ovine s-SCF (+) and stemloop structure (mfold) of the miR-27a,b is shown. The seed sequences (nt. 2 to nt. 8) of the miR-27a,b is shown in red, bold letters. The target nucleotides involved in pairing are shown in blue, bold on the ovine s-SCF (+). The nucleotides involved in pairing outside the seed sequence are underlined in black; (b) The predicted potential binding site of miR-669f-3p on the 3′ UTR of ovine m-SCF (−) and stemloop structure (mfold) of the mature miR-669f-3p is shown. The miR-669f-3p target sequence is located on the non-coding intron-5 closest to exon 5. The mature miR-669f-3p is shown in red, bold letters. The target nucleotides involved in pairing are shown in blue, bold on the ovine m-SCF (−).
Mentions: A number of potential miRNA target sites are found within the longer ∼4.4 kb 3′ UTR sequence of human SCF (data not shown). However, in sheep, the analyzed miRNA sites that are located in the 505 bp 3′ UTR of the ovine s-SCF (+) form belongs to the miRNA families of miR-27a/b, miR-194, miR-128, miR-370, and two sites for miR-132/212, miR-320/320abcd (Figure 9(a)) where as miR-669f/a/o-3p, miR-466b and miR828b are detected on the shorter 3′ UTR segment (144 bp) of ovine m-SCF (−) form (Figure 9(b)). Interestingly, the 8-mer miRNA (miR-669f) has a high context score (87 percentile) which binds to the 21 nt off 23 nt of the 3′ UTR target of the oSCF (−) form (Figure 9(b)).

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