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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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Related in: MedlinePlus

NCBI Map Viewer of ovine SCF (oSCF) aligned to the bovine SCF gene.The comparative map depicts unknown map region (in red dots) of the oSCF gene to the counter-part of bovine SCF (KITLG) on chr_5:Btau_5.2 displaying regions of 20,582,700–20,618,400 bp. Arrows indicate mapped GenBank records (Acc. No.) with description, respectively.
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pone-0038657-g004: NCBI Map Viewer of ovine SCF (oSCF) aligned to the bovine SCF gene.The comparative map depicts unknown map region (in red dots) of the oSCF gene to the counter-part of bovine SCF (KITLG) on chr_5:Btau_5.2 displaying regions of 20,582,700–20,618,400 bp. Arrows indicate mapped GenBank records (Acc. No.) with description, respectively.

Mentions: Upon scanning through the sheep genome Oarv2.0 (March 2011 – till date) covering position from 124,495,129 to 124,515,933 of Ovine (Texel) Version 2.0 (current) Genome Assembly we obtained the mere size of OAR3: 20.8 kbp (data not shown). It represents only 19% of the known SCF gene size when compared to human (108.74 kbp), mouse (104.78 kbp), cow (122.28 kbp) and dog (100.21 kbp) (source: Ensembl). The gene encoding the ovine SCF (NCBI gene ID: 443371) is located within a syntenic group on chromosome 3 [105], corresponding to the Sl or kitlg gene locus. This portion of ovine chr 3 is homologous to cattle chr 5. Hence, a comparative chromosomal mapping (Figure 4) was performed at the NCBI Map Viewer [84] of the sheep SCF to the cow SCF i.e., O.ari chr 5 to B.tau chr 5 and O.ari chr 3 to B.tau chr 5. Genomic DNA and cDNA sequence comparison and prediction [63] revealed that the oSCF gene consists of 9 exons interrupted by 8 introns to the dog (Figure 3(e)), pig, horse SCF gene where as in comparison to human, chimpanzee, marmoset, mouse (Figure 3(e)) and rat including the unfinished alpaca genome (source: Ensembl), oSCF gene has been characterized by 10 exons and 9 introns. Comparative analyses of oSCF (+) protein to the dog and mouse SCF gene assembly exhibited 96/90.1 and 93/80.6 match ratio and % identity, respectively. Similarly, oSCF (−) protein showed the match ratio and % identity of 91/87.2 and 90/77.7 to dog and mouse SCF gene assembly, respectively. Among the 9/10 exons, it is predicted by gene annotation (source: Ensembl) that the exon 5 and exon 6 has its importance in determining the final protein product through AS event(s) and the longer exon 10 (9) corresponds to ∼4.4 kb 3′ UTR in human, chimpanzee, mouse, rat and goat in contrast to the shorter 3′ UTR in sheep (reported in this study), cow, pig, horse, dog, cat and panda (source: Ensembl).


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)

NCBI Map Viewer of ovine SCF (oSCF) aligned to the bovine SCF gene.The comparative map depicts unknown map region (in red dots) of the oSCF gene to the counter-part of bovine SCF (KITLG) on chr_5:Btau_5.2 displaying regions of 20,582,700–20,618,400 bp. Arrows indicate mapped GenBank records (Acc. No.) with description, respectively.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0038657-g004: NCBI Map Viewer of ovine SCF (oSCF) aligned to the bovine SCF gene.The comparative map depicts unknown map region (in red dots) of the oSCF gene to the counter-part of bovine SCF (KITLG) on chr_5:Btau_5.2 displaying regions of 20,582,700–20,618,400 bp. Arrows indicate mapped GenBank records (Acc. No.) with description, respectively.
Mentions: Upon scanning through the sheep genome Oarv2.0 (March 2011 – till date) covering position from 124,495,129 to 124,515,933 of Ovine (Texel) Version 2.0 (current) Genome Assembly we obtained the mere size of OAR3: 20.8 kbp (data not shown). It represents only 19% of the known SCF gene size when compared to human (108.74 kbp), mouse (104.78 kbp), cow (122.28 kbp) and dog (100.21 kbp) (source: Ensembl). The gene encoding the ovine SCF (NCBI gene ID: 443371) is located within a syntenic group on chromosome 3 [105], corresponding to the Sl or kitlg gene locus. This portion of ovine chr 3 is homologous to cattle chr 5. Hence, a comparative chromosomal mapping (Figure 4) was performed at the NCBI Map Viewer [84] of the sheep SCF to the cow SCF i.e., O.ari chr 5 to B.tau chr 5 and O.ari chr 3 to B.tau chr 5. Genomic DNA and cDNA sequence comparison and prediction [63] revealed that the oSCF gene consists of 9 exons interrupted by 8 introns to the dog (Figure 3(e)), pig, horse SCF gene where as in comparison to human, chimpanzee, marmoset, mouse (Figure 3(e)) and rat including the unfinished alpaca genome (source: Ensembl), oSCF gene has been characterized by 10 exons and 9 introns. Comparative analyses of oSCF (+) protein to the dog and mouse SCF gene assembly exhibited 96/90.1 and 93/80.6 match ratio and % identity, respectively. Similarly, oSCF (−) protein showed the match ratio and % identity of 91/87.2 and 90/77.7 to dog and mouse SCF gene assembly, respectively. Among the 9/10 exons, it is predicted by gene annotation (source: Ensembl) that the exon 5 and exon 6 has its importance in determining the final protein product through AS event(s) and the longer exon 10 (9) corresponds to ∼4.4 kb 3′ UTR in human, chimpanzee, mouse, rat and goat in contrast to the shorter 3′ UTR in sheep (reported in this study), cow, pig, horse, dog, cat and panda (source: Ensembl).

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