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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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Graphical representation of evolutionary conservation of sheep SCF isoforms.(a) Percent of conservation was calculated for sheep, goat, cow, pig, cat, dog, panda, horse, human, chimpanzee, marmoset, mouse, rat, rabbit, chicken, zebra finch and fishes, such as zebra fish, gold fish using the multiple sequence alignment (MSA) tool, ClustalW2 with four different datasets (provided on request). The Circos graphical table view represents the sheep soluble, s-SCF (+) and membrane-bound SCF, m-SCF (−) nucleotide (nt) and protein (aa) as the query sequences (in black dotted left bracket) against 17 other vertebrate species. Four different colour small bars on the query sequences represnts the four different data sets of sheep s-SCF (+) and m-SCF (−) nt/aa sequences. The 15 different colour ribbons passing through each other represent respective vertebrate species and the percent identity is indicated outside as the boundary. The four different colour small bars over the 15 vertebrate species as against 15 different colour small bars above the sheep query sequences represnts the percent identity among each other. The scale over each species (above small bar) represents the total score obtained from the sequence coverage; (b) Graphical logo representing the conservation of oSCF splice junction (intron-5) which was generated by MUSCLE alignment (manually predicted for other species), depicting the GT repeats (black oval dotted lines) proximal to the poly(A)11 stretch (black dotted right brace symbol). The constitutive splice donor (GT) and acceptor (AG) sites are circled by black dotted lines along with one of the proposed usage of alternative/cryptic splice donor site (GT) (see Figure 3f). Numbers below the logo indicate the nucleotide/amino acid position of the MUSCLE aligned sequences; (c) Logo representing the 23 nt conservation of the m-SCF (−) form (novel sequence reported in this study) and its deduced 7 aa new C-terminus is shown; (d) Graphical logo representing the 84 nt conservation of the s-SCF (+) form and its deduced 28 aa proteolytic site is shown. Numbers below the graphical representation of (c), (d) indicate the actual nucleotide/amino acid position. The height of the letters on each logo represents the relative frequency of each nucleotide/amino acid in a given position.
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pone-0038657-g006: Graphical representation of evolutionary conservation of sheep SCF isoforms.(a) Percent of conservation was calculated for sheep, goat, cow, pig, cat, dog, panda, horse, human, chimpanzee, marmoset, mouse, rat, rabbit, chicken, zebra finch and fishes, such as zebra fish, gold fish using the multiple sequence alignment (MSA) tool, ClustalW2 with four different datasets (provided on request). The Circos graphical table view represents the sheep soluble, s-SCF (+) and membrane-bound SCF, m-SCF (−) nucleotide (nt) and protein (aa) as the query sequences (in black dotted left bracket) against 17 other vertebrate species. Four different colour small bars on the query sequences represnts the four different data sets of sheep s-SCF (+) and m-SCF (−) nt/aa sequences. The 15 different colour ribbons passing through each other represent respective vertebrate species and the percent identity is indicated outside as the boundary. The four different colour small bars over the 15 vertebrate species as against 15 different colour small bars above the sheep query sequences represnts the percent identity among each other. The scale over each species (above small bar) represents the total score obtained from the sequence coverage; (b) Graphical logo representing the conservation of oSCF splice junction (intron-5) which was generated by MUSCLE alignment (manually predicted for other species), depicting the GT repeats (black oval dotted lines) proximal to the poly(A)11 stretch (black dotted right brace symbol). The constitutive splice donor (GT) and acceptor (AG) sites are circled by black dotted lines along with one of the proposed usage of alternative/cryptic splice donor site (GT) (see Figure 3f). Numbers below the logo indicate the nucleotide/amino acid position of the MUSCLE aligned sequences; (c) Logo representing the 23 nt conservation of the m-SCF (−) form (novel sequence reported in this study) and its deduced 7 aa new C-terminus is shown; (d) Graphical logo representing the 84 nt conservation of the s-SCF (+) form and its deduced 28 aa proteolytic site is shown. Numbers below the graphical representation of (c), (d) indicate the actual nucleotide/amino acid position. The height of the letters on each logo represents the relative frequency of each nucleotide/amino acid in a given position.

Mentions: Using the default settings of NCBI, BLASTN and BLASTP search was conducted with ovine s-SCF (+) form of 825 bp CDS and its deduced 274 aa as query sequences, respectively. Multiple sequence alignments (MSA) [64] (Figure S3(a,b,d)) of the nucleotide and the deduced amino acid sequences belonging to different mammalian representatives indicated that the sheep SCF was highly conserved and found to have between 57% and 99% nucleotide similarity and 19% to 99% protein identity (Figure 6(a,d)). The highest identity was with the goat SCF where as the lowest was with gold fish and zebra fish SCF for nucleotide and protein respectively viz. goat (99/99%), cow (97/98%), pig (95/94%), cat (94/90%), panda (93/90%), horse (93/89%), dog (92/88%), human and chimpanzee (91/86%), rabbit (90/84%), marmoset (90/83%), rat (88/82%), mouse (87/80%), zebra finch (74/55%), chicken (73/53%), zebra fish(59/19%) and gold fish (57/26%). The graphical logo representing the conservation of oSCF splice junction (intron-5) with GT repeats, poly(A)11 stretch and the constitutive splice donor (GT) and acceptor (AG) sites are shown in Figure 6(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)

Graphical representation of evolutionary conservation of sheep SCF isoforms.(a) Percent of conservation was calculated for sheep, goat, cow, pig, cat, dog, panda, horse, human, chimpanzee, marmoset, mouse, rat, rabbit, chicken, zebra finch and fishes, such as zebra fish, gold fish using the multiple sequence alignment (MSA) tool, ClustalW2 with four different datasets (provided on request). The Circos graphical table view represents the sheep soluble, s-SCF (+) and membrane-bound SCF, m-SCF (−) nucleotide (nt) and protein (aa) as the query sequences (in black dotted left bracket) against 17 other vertebrate species. Four different colour small bars on the query sequences represnts the four different data sets of sheep s-SCF (+) and m-SCF (−) nt/aa sequences. The 15 different colour ribbons passing through each other represent respective vertebrate species and the percent identity is indicated outside as the boundary. The four different colour small bars over the 15 vertebrate species as against 15 different colour small bars above the sheep query sequences represnts the percent identity among each other. The scale over each species (above small bar) represents the total score obtained from the sequence coverage; (b) Graphical logo representing the conservation of oSCF splice junction (intron-5) which was generated by MUSCLE alignment (manually predicted for other species), depicting the GT repeats (black oval dotted lines) proximal to the poly(A)11 stretch (black dotted right brace symbol). The constitutive splice donor (GT) and acceptor (AG) sites are circled by black dotted lines along with one of the proposed usage of alternative/cryptic splice donor site (GT) (see Figure 3f). Numbers below the logo indicate the nucleotide/amino acid position of the MUSCLE aligned sequences; (c) Logo representing the 23 nt conservation of the m-SCF (−) form (novel sequence reported in this study) and its deduced 7 aa new C-terminus is shown; (d) Graphical logo representing the 84 nt conservation of the s-SCF (+) form and its deduced 28 aa proteolytic site is shown. Numbers below the graphical representation of (c), (d) indicate the actual nucleotide/amino acid position. The height of the letters on each logo represents the relative frequency of each nucleotide/amino acid in a given position.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0038657-g006: Graphical representation of evolutionary conservation of sheep SCF isoforms.(a) Percent of conservation was calculated for sheep, goat, cow, pig, cat, dog, panda, horse, human, chimpanzee, marmoset, mouse, rat, rabbit, chicken, zebra finch and fishes, such as zebra fish, gold fish using the multiple sequence alignment (MSA) tool, ClustalW2 with four different datasets (provided on request). The Circos graphical table view represents the sheep soluble, s-SCF (+) and membrane-bound SCF, m-SCF (−) nucleotide (nt) and protein (aa) as the query sequences (in black dotted left bracket) against 17 other vertebrate species. Four different colour small bars on the query sequences represnts the four different data sets of sheep s-SCF (+) and m-SCF (−) nt/aa sequences. The 15 different colour ribbons passing through each other represent respective vertebrate species and the percent identity is indicated outside as the boundary. The four different colour small bars over the 15 vertebrate species as against 15 different colour small bars above the sheep query sequences represnts the percent identity among each other. The scale over each species (above small bar) represents the total score obtained from the sequence coverage; (b) Graphical logo representing the conservation of oSCF splice junction (intron-5) which was generated by MUSCLE alignment (manually predicted for other species), depicting the GT repeats (black oval dotted lines) proximal to the poly(A)11 stretch (black dotted right brace symbol). The constitutive splice donor (GT) and acceptor (AG) sites are circled by black dotted lines along with one of the proposed usage of alternative/cryptic splice donor site (GT) (see Figure 3f). Numbers below the logo indicate the nucleotide/amino acid position of the MUSCLE aligned sequences; (c) Logo representing the 23 nt conservation of the m-SCF (−) form (novel sequence reported in this study) and its deduced 7 aa new C-terminus is shown; (d) Graphical logo representing the 84 nt conservation of the s-SCF (+) form and its deduced 28 aa proteolytic site is shown. Numbers below the graphical representation of (c), (d) indicate the actual nucleotide/amino acid position. The height of the letters on each logo represents the relative frequency of each nucleotide/amino acid in a given position.
Mentions: Using the default settings of NCBI, BLASTN and BLASTP search was conducted with ovine s-SCF (+) form of 825 bp CDS and its deduced 274 aa as query sequences, respectively. Multiple sequence alignments (MSA) [64] (Figure S3(a,b,d)) of the nucleotide and the deduced amino acid sequences belonging to different mammalian representatives indicated that the sheep SCF was highly conserved and found to have between 57% and 99% nucleotide similarity and 19% to 99% protein identity (Figure 6(a,d)). The highest identity was with the goat SCF where as the lowest was with gold fish and zebra fish SCF for nucleotide and protein respectively viz. goat (99/99%), cow (97/98%), pig (95/94%), cat (94/90%), panda (93/90%), horse (93/89%), dog (92/88%), human and chimpanzee (91/86%), rabbit (90/84%), marmoset (90/83%), rat (88/82%), mouse (87/80%), zebra finch (74/55%), chicken (73/53%), zebra fish(59/19%) and gold fish (57/26%). The graphical logo representing the conservation of oSCF splice junction (intron-5) with GT repeats, poly(A)11 stretch and the constitutive splice donor (GT) and acceptor (AG) sites are shown in Figure 6(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