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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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Sequencing chromatogram of cDNA in comparison with the gDNA amplification of oSCF gene.(a) Complementary DNA (cDNA) chromatogram shows the CDS, GT repeats and p(A)18 tail adapter primer as ‘black dotted oval mark’ and a premature termination codon (PTC) as ‘red dotted oval mark’ on the 3′ RACE product (336 bp, see Figure 1B(b5)); (b) Genomic DNA (gDNA) chromatogram shows the counter part of the above cDNA illustration (a) on exon 5 to exon 6 intervened by intron-5 sequences of the oSCF gene; (c) Amplification scheme of 948 bp splice junction covering exon(5)-intron-5-exon(6) of the oSCF gene with reference to human and mouse. The two exons 5, 6 are differentiated by ‘open and shaded box’ respectively. Arrows over the boxes indicate the fwd and rev primer. Different symbols on the intron-5 indicate the part of retained intronic sequences (161 bp) by a PTC along with the stretch of p(A)11 signal (see key to symbols below the diagram); (d) Gel picture shows the PCR amplification of 948 bp fragment corresponding to the above schema (c) of oSCF gene from blood gDNA; In the picture, arrow mark indicates the exact size of amplicon; M1 indicates DNA size marker of λ-DNA EcoRI/HindIII digest; and (−)ve represents PCR negative control.
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pone-0038657-g002: Sequencing chromatogram of cDNA in comparison with the gDNA amplification of oSCF gene.(a) Complementary DNA (cDNA) chromatogram shows the CDS, GT repeats and p(A)18 tail adapter primer as ‘black dotted oval mark’ and a premature termination codon (PTC) as ‘red dotted oval mark’ on the 3′ RACE product (336 bp, see Figure 1B(b5)); (b) Genomic DNA (gDNA) chromatogram shows the counter part of the above cDNA illustration (a) on exon 5 to exon 6 intervened by intron-5 sequences of the oSCF gene; (c) Amplification scheme of 948 bp splice junction covering exon(5)-intron-5-exon(6) of the oSCF gene with reference to human and mouse. The two exons 5, 6 are differentiated by ‘open and shaded box’ respectively. Arrows over the boxes indicate the fwd and rev primer. Different symbols on the intron-5 indicate the part of retained intronic sequences (161 bp) by a PTC along with the stretch of p(A)11 signal (see key to symbols below the diagram); (d) Gel picture shows the PCR amplification of 948 bp fragment corresponding to the above schema (c) of oSCF gene from blood gDNA; In the picture, arrow mark indicates the exact size of amplicon; M1 indicates DNA size marker of λ-DNA EcoRI/HindIII digest; and (−)ve represents PCR negative control.

Mentions: In all the above cases, we obtained always the (−) form, hence we designed a splice variant specific Nested forward primer (Table S2) with higher Tm for the (+) form. The primer was designed in between two exonic junctions (see Figure 1A and 2(c)) spanning into the proteolytic site viz. exon 5 into exon 6 in reference to the human, mouse, dog, horse SCF (source: Ensembl). The second round 3′ RACE amplification (Nested 1; see Table S2) was performed with 1 µl of the primary reaction product using (+) form specific forward primer (Table S2) and oligo(dT)18 modified reverse primer into a final PCR volume of 50 µl. The RT-PCR yielded an amplicon size of 855 bp (Figure 1A(b1)). Further third round amplification (Nested 2; see Table S2) yielded the expected 793 bp amplicon with some non-specific amplicons. The purified fragment of 793 bp (Figure 1A(b2)) was then cloned and sequenced. Sequence analyses by BLASTN and BLASTP confirmed the oSCF and named as ‘SCF isoform-1’, hereafter referred as (+) form, which is the counterpart of previously reported ‘soluble’ SCF (s-SCF) sequences in other vertebrate species [99]–[103] (source: GenBank, NCBI). Overlapping and editing of the 793 bp 3′ UTR fragment with the 621 bp CDS fragment, we obtained a total length of 1330 bp (without the 5′ UTR). The ORF of 825 bp corresponding to the deduced amino acid sequence of 274 aa revealed it as the s-SCF (+) form, indicating that this cDNA encodes the ‘soluble’ form of oSCF. This ovine s-SCF (+) form included the stretch of 28 aa recognized as a putative primary proteolytic site (Figure S1B) right after the D175 at its C-terminus as observed in the previously reported sequences [99]–[103]. The remaining long 505 bp (after removing the adapter sequences from the oligo(dT)18 modified primer) including the polyA nucleotides belong to the 3′ UTR of ovine s-SCF (+) form. The other two amplicons (data not shown) were found to be non-specific and omitted from further characterization.


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)

Sequencing chromatogram of cDNA in comparison with the gDNA amplification of oSCF gene.(a) Complementary DNA (cDNA) chromatogram shows the CDS, GT repeats and p(A)18 tail adapter primer as ‘black dotted oval mark’ and a premature termination codon (PTC) as ‘red dotted oval mark’ on the 3′ RACE product (336 bp, see Figure 1B(b5)); (b) Genomic DNA (gDNA) chromatogram shows the counter part of the above cDNA illustration (a) on exon 5 to exon 6 intervened by intron-5 sequences of the oSCF gene; (c) Amplification scheme of 948 bp splice junction covering exon(5)-intron-5-exon(6) of the oSCF gene with reference to human and mouse. The two exons 5, 6 are differentiated by ‘open and shaded box’ respectively. Arrows over the boxes indicate the fwd and rev primer. Different symbols on the intron-5 indicate the part of retained intronic sequences (161 bp) by a PTC along with the stretch of p(A)11 signal (see key to symbols below the diagram); (d) Gel picture shows the PCR amplification of 948 bp fragment corresponding to the above schema (c) of oSCF gene from blood gDNA; In the picture, arrow mark indicates the exact size of amplicon; M1 indicates DNA size marker of λ-DNA EcoRI/HindIII digest; and (−)ve represents PCR negative control.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3376141&req=5

pone-0038657-g002: Sequencing chromatogram of cDNA in comparison with the gDNA amplification of oSCF gene.(a) Complementary DNA (cDNA) chromatogram shows the CDS, GT repeats and p(A)18 tail adapter primer as ‘black dotted oval mark’ and a premature termination codon (PTC) as ‘red dotted oval mark’ on the 3′ RACE product (336 bp, see Figure 1B(b5)); (b) Genomic DNA (gDNA) chromatogram shows the counter part of the above cDNA illustration (a) on exon 5 to exon 6 intervened by intron-5 sequences of the oSCF gene; (c) Amplification scheme of 948 bp splice junction covering exon(5)-intron-5-exon(6) of the oSCF gene with reference to human and mouse. The two exons 5, 6 are differentiated by ‘open and shaded box’ respectively. Arrows over the boxes indicate the fwd and rev primer. Different symbols on the intron-5 indicate the part of retained intronic sequences (161 bp) by a PTC along with the stretch of p(A)11 signal (see key to symbols below the diagram); (d) Gel picture shows the PCR amplification of 948 bp fragment corresponding to the above schema (c) of oSCF gene from blood gDNA; In the picture, arrow mark indicates the exact size of amplicon; M1 indicates DNA size marker of λ-DNA EcoRI/HindIII digest; and (−)ve represents PCR negative control.
Mentions: In all the above cases, we obtained always the (−) form, hence we designed a splice variant specific Nested forward primer (Table S2) with higher Tm for the (+) form. The primer was designed in between two exonic junctions (see Figure 1A and 2(c)) spanning into the proteolytic site viz. exon 5 into exon 6 in reference to the human, mouse, dog, horse SCF (source: Ensembl). The second round 3′ RACE amplification (Nested 1; see Table S2) was performed with 1 µl of the primary reaction product using (+) form specific forward primer (Table S2) and oligo(dT)18 modified reverse primer into a final PCR volume of 50 µl. The RT-PCR yielded an amplicon size of 855 bp (Figure 1A(b1)). Further third round amplification (Nested 2; see Table S2) yielded the expected 793 bp amplicon with some non-specific amplicons. The purified fragment of 793 bp (Figure 1A(b2)) was then cloned and sequenced. Sequence analyses by BLASTN and BLASTP confirmed the oSCF and named as ‘SCF isoform-1’, hereafter referred as (+) form, which is the counterpart of previously reported ‘soluble’ SCF (s-SCF) sequences in other vertebrate species [99]–[103] (source: GenBank, NCBI). Overlapping and editing of the 793 bp 3′ UTR fragment with the 621 bp CDS fragment, we obtained a total length of 1330 bp (without the 5′ UTR). The ORF of 825 bp corresponding to the deduced amino acid sequence of 274 aa revealed it as the s-SCF (+) form, indicating that this cDNA encodes the ‘soluble’ form of oSCF. This ovine s-SCF (+) form included the stretch of 28 aa recognized as a putative primary proteolytic site (Figure S1B) right after the D175 at its C-terminus as observed in the previously reported sequences [99]–[103]. The remaining long 505 bp (after removing the adapter sequences from the oligo(dT)18 modified primer) including the polyA nucleotides belong to the 3′ UTR of ovine s-SCF (+) form. The other two amplicons (data not shown) were found to be non-specific and omitted from further characterization.

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