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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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Ovine SCF RNA fold model explaining the observed results.(a) Secondary structure predicted for the major 1519 nt s-SCF (+) mRNA. The m-fold circle diagram, generated by minimal free energy (dG = −437.3 kcal/mol) indicate G–C, A–U and G–U base pairs in red, blue and green arc lines, respectively. It also differentiates the 825 nt coding and 505 nt non-coding region fold by the vertical black dotted line which divides the circle diagram. The 189 nt GC-rich segment of 5′ UTR which forms a dense secondary structure and the presumptive splice site of 84 nt proteolytic segment are highlighted and labelled in cyan and dark red dotted line respectively. The start (ATG) and stop (UAA) codons are labeled in bold blue and red letters respectively. The numbers present outside the circle diagram indicates the nucleotide position in base pairs (bp) at every 100 bp intervals; (b) Stemloop secondary structure representation of 1519 nt s-SCF isoform-1 (+), 825 nt m-SCF isoform-2a (−) and the partial image depicting the 5′ UTR segment fold of 725 nt m-SCF isoform-2b (−). The major structural features in illustration (a) and (b) are labeled alike. Except the GC-rich 5′ UTR segments where in I (189 nt), II (144 nt) and III (34 nt) represent the difference in 5′ UTR fold (light blue/cyan dotted arrows pointing to the corresponding dotted oval shape; see also Figure S4(a1,2,3). Similarly, IV (+84 nt) and V (−84 nt) represents potential fold difference for the proteolytic segments (dotted dark red arrows directd to the corresponding expanded structures).
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pone-0038657-g008: Ovine SCF RNA fold model explaining the observed results.(a) Secondary structure predicted for the major 1519 nt s-SCF (+) mRNA. The m-fold circle diagram, generated by minimal free energy (dG = −437.3 kcal/mol) indicate G–C, A–U and G–U base pairs in red, blue and green arc lines, respectively. It also differentiates the 825 nt coding and 505 nt non-coding region fold by the vertical black dotted line which divides the circle diagram. The 189 nt GC-rich segment of 5′ UTR which forms a dense secondary structure and the presumptive splice site of 84 nt proteolytic segment are highlighted and labelled in cyan and dark red dotted line respectively. The start (ATG) and stop (UAA) codons are labeled in bold blue and red letters respectively. The numbers present outside the circle diagram indicates the nucleotide position in base pairs (bp) at every 100 bp intervals; (b) Stemloop secondary structure representation of 1519 nt s-SCF isoform-1 (+), 825 nt m-SCF isoform-2a (−) and the partial image depicting the 5′ UTR segment fold of 725 nt m-SCF isoform-2b (−). The major structural features in illustration (a) and (b) are labeled alike. Except the GC-rich 5′ UTR segments where in I (189 nt), II (144 nt) and III (34 nt) represent the difference in 5′ UTR fold (light blue/cyan dotted arrows pointing to the corresponding dotted oval shape; see also Figure S4(a1,2,3). Similarly, IV (+84 nt) and V (−84 nt) represents potential fold difference for the proteolytic segments (dotted dark red arrows directd to the corresponding expanded structures).

Mentions: In addition to the coding region (+/−84 bp proteolytic site), SCF mRNA(s) has four notable features relevant to its secondary structure (Figure 8(a,b)). First, the 5′ UTR is enriched in G+C nucleotides (Figure S4(a3)), with 64% and 60% or 56% (G+C content) in the 189 nt and 144 nt segment for the (+) and (−) form respectively. Second, the 5′ UTR segment has specific trinucleotide elements (Py-G-C; Figure S4(a1)), in our case ‘CGC’ at nt pos. 4, 16/18, 33, 36, 68, 100, 154, 176 and and ‘TGC’ at nt pos. 66, 81, 94, 103, 109, 116, 146, 157, 179 that are known to cause DNA polymerase pausing [111]. These trinucleotides (CGC, TGC) which accounts for 9.6% of the ovine s-SCF (+) 5′ UTR segment (189 bp), could attribute to the smaller 5′ RACE cDNA product(s) for example, the one of oSCF isoform-2a (−) (Acc. No. GU386374; Figure S4(a1)). Third, sheep SCF mRNA contains a frequent hexamer direct repeats (DRs) i.e., ‘CGCTGC’ (1.6%) at nt pos. 100, 154, 176 located in the 5′ UTR of (+) form (also present in (−) form but nt pos. differs; see Figure S4(a1)). This repeat is highly conserved among mammalian SCF mRNAs (Figure S4(a2)). Fourth, the 3′ UTR has a DRs containing a consecutive hepatamer ‘GTGGGGG’ at nt pos. 1461, 1468 in the (+) form which is highly conserved only in goat (Figure S4(d)). In contrast, a perfect dinucleotide repeats (GT)5 at nt pos. 775 is present in between the hepatamer tandem repeats ‘CAAATAT’ at nt pos. 748, 801 in the (−) form, are also highly conserved with goat but varies only in the dinucleotide repeats with ‘AT’ for cow, dog and horse as shown in Figure S4(c). In between this feature, there exists a putative alternative isoform/cryptic splice donor (GT) at nt pos. 117 of 729 bp intron-5 of oSCF (Figure S4(b)) with 92.5% score as predicted by the ASSP [91] classification.


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)

Ovine SCF RNA fold model explaining the observed results.(a) Secondary structure predicted for the major 1519 nt s-SCF (+) mRNA. The m-fold circle diagram, generated by minimal free energy (dG = −437.3 kcal/mol) indicate G–C, A–U and G–U base pairs in red, blue and green arc lines, respectively. It also differentiates the 825 nt coding and 505 nt non-coding region fold by the vertical black dotted line which divides the circle diagram. The 189 nt GC-rich segment of 5′ UTR which forms a dense secondary structure and the presumptive splice site of 84 nt proteolytic segment are highlighted and labelled in cyan and dark red dotted line respectively. The start (ATG) and stop (UAA) codons are labeled in bold blue and red letters respectively. The numbers present outside the circle diagram indicates the nucleotide position in base pairs (bp) at every 100 bp intervals; (b) Stemloop secondary structure representation of 1519 nt s-SCF isoform-1 (+), 825 nt m-SCF isoform-2a (−) and the partial image depicting the 5′ UTR segment fold of 725 nt m-SCF isoform-2b (−). The major structural features in illustration (a) and (b) are labeled alike. Except the GC-rich 5′ UTR segments where in I (189 nt), II (144 nt) and III (34 nt) represent the difference in 5′ UTR fold (light blue/cyan dotted arrows pointing to the corresponding dotted oval shape; see also Figure S4(a1,2,3). Similarly, IV (+84 nt) and V (−84 nt) represents potential fold difference for the proteolytic segments (dotted dark red arrows directd to the corresponding expanded structures).
© Copyright Policy
Related In: Results  -  Collection

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

pone-0038657-g008: Ovine SCF RNA fold model explaining the observed results.(a) Secondary structure predicted for the major 1519 nt s-SCF (+) mRNA. The m-fold circle diagram, generated by minimal free energy (dG = −437.3 kcal/mol) indicate G–C, A–U and G–U base pairs in red, blue and green arc lines, respectively. It also differentiates the 825 nt coding and 505 nt non-coding region fold by the vertical black dotted line which divides the circle diagram. The 189 nt GC-rich segment of 5′ UTR which forms a dense secondary structure and the presumptive splice site of 84 nt proteolytic segment are highlighted and labelled in cyan and dark red dotted line respectively. The start (ATG) and stop (UAA) codons are labeled in bold blue and red letters respectively. The numbers present outside the circle diagram indicates the nucleotide position in base pairs (bp) at every 100 bp intervals; (b) Stemloop secondary structure representation of 1519 nt s-SCF isoform-1 (+), 825 nt m-SCF isoform-2a (−) and the partial image depicting the 5′ UTR segment fold of 725 nt m-SCF isoform-2b (−). The major structural features in illustration (a) and (b) are labeled alike. Except the GC-rich 5′ UTR segments where in I (189 nt), II (144 nt) and III (34 nt) represent the difference in 5′ UTR fold (light blue/cyan dotted arrows pointing to the corresponding dotted oval shape; see also Figure S4(a1,2,3). Similarly, IV (+84 nt) and V (−84 nt) represents potential fold difference for the proteolytic segments (dotted dark red arrows directd to the corresponding expanded structures).
Mentions: In addition to the coding region (+/−84 bp proteolytic site), SCF mRNA(s) has four notable features relevant to its secondary structure (Figure 8(a,b)). First, the 5′ UTR is enriched in G+C nucleotides (Figure S4(a3)), with 64% and 60% or 56% (G+C content) in the 189 nt and 144 nt segment for the (+) and (−) form respectively. Second, the 5′ UTR segment has specific trinucleotide elements (Py-G-C; Figure S4(a1)), in our case ‘CGC’ at nt pos. 4, 16/18, 33, 36, 68, 100, 154, 176 and and ‘TGC’ at nt pos. 66, 81, 94, 103, 109, 116, 146, 157, 179 that are known to cause DNA polymerase pausing [111]. These trinucleotides (CGC, TGC) which accounts for 9.6% of the ovine s-SCF (+) 5′ UTR segment (189 bp), could attribute to the smaller 5′ RACE cDNA product(s) for example, the one of oSCF isoform-2a (−) (Acc. No. GU386374; Figure S4(a1)). Third, sheep SCF mRNA contains a frequent hexamer direct repeats (DRs) i.e., ‘CGCTGC’ (1.6%) at nt pos. 100, 154, 176 located in the 5′ UTR of (+) form (also present in (−) form but nt pos. differs; see Figure S4(a1)). This repeat is highly conserved among mammalian SCF mRNAs (Figure S4(a2)). Fourth, the 3′ UTR has a DRs containing a consecutive hepatamer ‘GTGGGGG’ at nt pos. 1461, 1468 in the (+) form which is highly conserved only in goat (Figure S4(d)). In contrast, a perfect dinucleotide repeats (GT)5 at nt pos. 775 is present in between the hepatamer tandem repeats ‘CAAATAT’ at nt pos. 748, 801 in the (−) form, are also highly conserved with goat but varies only in the dinucleotide repeats with ‘AT’ for cow, dog and horse as shown in Figure S4(c). In between this feature, there exists a putative alternative isoform/cryptic splice donor (GT) at nt pos. 117 of 729 bp intron-5 of oSCF (Figure S4(b)) with 92.5% score as predicted by the ASSP [91] classification.

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