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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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Gene architecture of ovine SCF gene in reference to human, mouse and dog.(a) Schematic representation of human SCF (huSCF) gene is shown. It consists of 10 exons (open boxes) intervened by 9 introns (linear black lines). Regular splicing and polyadenylation generates the full length huSCF mRNA transcript variant-b, a longer (+) form (5460 bp) encoding for a soluble product (273 aa; see Figure 5a); (b) The 84 bp exon 6 encoding for the 28 aa proteolytic site is skipped by an AS event of the huSCF gene is shown. The resultant full length huSCF mRNA transcript variant-a, known as (−) form (5376 bp) encodes for a membrane-bound product (245 aa; see Figure 5b); (c) Schematic representation of ovine SCF (oSCF) gene is shown. Regular splicing of exons 1–9/10(?) generate the full length oSCF (+) mRNA transcript (1519 bp) that encodes for a soluble product (274 aa; see Figure 5a); (d) Conversely, the possible AS events on intron-5 (Ref. human, mouse and dog) resulted in an alternative ORF with a premature termination (red symbol, PTC; see key to symbols). This resulted in retaining of 161 bp intronic sequence and completely eliminating (skipping of) the involvement of exon 6–9/10(?). The deduced protein sequence of this novel, shorter splice transcript variant (835 bp) resulted in 181 aa (see Figure 5b), a membrane-bound product of oSCF (−). In the above illustration, the open square or rectangle box symbolize exon and inverted triangle box symbolize intron. The open and shaded ‘black sparkle’ symbol on exon 10 (in 3a,b), exon ‘?’ (in 3c) and intron-5 (in 3d) all indicate the posssible position of predicted polyadenylation signal (PAS) sites. The two different sizes of the opened ‘black sparkle’ symbol (in 3a,b) denote the frequency of the common PAS such as ‘AAUAAA’ (8–12 times) and ‘AUUAAA’ (4–6 times) in the longer 3′ UTR of human, goat, mouse and rat. In contrast, the shaded ‘black sparkle’ symbol (in 3c,d) represents the other single basse ‘variants’ of PAS (see text in Results). Exon ‘?’ symbol (in 3c) represents the uncertainity of exon 10 position for oSCF (+). A ‘black hook’ symbolize the capped 5′ end and p(A) represents the polyA stretch on the preRNA, mRNA, respectively. The point of transcription termination (TAA) is symbolized as ‘red’ mark on intron-5 of oSCF (−) followed by the illustration of two possible mechanisms that resulted in a PTC of oSCF (−) (in dotted lines). The ASSP predicted constitutive and/or alternative splice donor (GT) and splice acceptor (AG) site(s) are labeled in blue and red letters respectively; (e) Schematic representation of the soluble oSCF (+) gene structure is shown. The exons/introns and the location of non-coding regions are determined in comparison to the mouse (chr 10) and dog (chr 15) SCF gene. The ‘intron (?)’ labeled in blue on the oSCF (+) in reference to mouse chr 10 indicates that the corresponding intron-7 is incomplete at that point i.e., it doesn’t show appropriate 5′ and/or 3′ splice sites; (f) Figure shows the gene stucture of membrane-bound oSCF (−). The ‘black line’ at the end of exon 5 of oSCF (−) in reference to mouse and dog indicate ‘gap’ i.e., coding sequence not found on the respective contig. The ‘vertical red lines’ over the exons indicate ‘mismatch’ of the oSCF (+) and (−) protein with dog (27 aa and 22 aa) and mouse (53 aa and 39 aa) SCF gene.
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pone-0038657-g003: Gene architecture of ovine SCF gene in reference to human, mouse and dog.(a) Schematic representation of human SCF (huSCF) gene is shown. It consists of 10 exons (open boxes) intervened by 9 introns (linear black lines). Regular splicing and polyadenylation generates the full length huSCF mRNA transcript variant-b, a longer (+) form (5460 bp) encoding for a soluble product (273 aa; see Figure 5a); (b) The 84 bp exon 6 encoding for the 28 aa proteolytic site is skipped by an AS event of the huSCF gene is shown. The resultant full length huSCF mRNA transcript variant-a, known as (−) form (5376 bp) encodes for a membrane-bound product (245 aa; see Figure 5b); (c) Schematic representation of ovine SCF (oSCF) gene is shown. Regular splicing of exons 1–9/10(?) generate the full length oSCF (+) mRNA transcript (1519 bp) that encodes for a soluble product (274 aa; see Figure 5a); (d) Conversely, the possible AS events on intron-5 (Ref. human, mouse and dog) resulted in an alternative ORF with a premature termination (red symbol, PTC; see key to symbols). This resulted in retaining of 161 bp intronic sequence and completely eliminating (skipping of) the involvement of exon 6–9/10(?). The deduced protein sequence of this novel, shorter splice transcript variant (835 bp) resulted in 181 aa (see Figure 5b), a membrane-bound product of oSCF (−). In the above illustration, the open square or rectangle box symbolize exon and inverted triangle box symbolize intron. The open and shaded ‘black sparkle’ symbol on exon 10 (in 3a,b), exon ‘?’ (in 3c) and intron-5 (in 3d) all indicate the posssible position of predicted polyadenylation signal (PAS) sites. The two different sizes of the opened ‘black sparkle’ symbol (in 3a,b) denote the frequency of the common PAS such as ‘AAUAAA’ (8–12 times) and ‘AUUAAA’ (4–6 times) in the longer 3′ UTR of human, goat, mouse and rat. In contrast, the shaded ‘black sparkle’ symbol (in 3c,d) represents the other single basse ‘variants’ of PAS (see text in Results). Exon ‘?’ symbol (in 3c) represents the uncertainity of exon 10 position for oSCF (+). A ‘black hook’ symbolize the capped 5′ end and p(A) represents the polyA stretch on the preRNA, mRNA, respectively. The point of transcription termination (TAA) is symbolized as ‘red’ mark on intron-5 of oSCF (−) followed by the illustration of two possible mechanisms that resulted in a PTC of oSCF (−) (in dotted lines). The ASSP predicted constitutive and/or alternative splice donor (GT) and splice acceptor (AG) site(s) are labeled in blue and red letters respectively; (e) Schematic representation of the soluble oSCF (+) gene structure is shown. The exons/introns and the location of non-coding regions are determined in comparison to the mouse (chr 10) and dog (chr 15) SCF gene. The ‘intron (?)’ labeled in blue on the oSCF (+) in reference to mouse chr 10 indicates that the corresponding intron-7 is incomplete at that point i.e., it doesn’t show appropriate 5′ and/or 3′ splice sites; (f) Figure shows the gene stucture of membrane-bound oSCF (−). The ‘black line’ at the end of exon 5 of oSCF (−) in reference to mouse and dog indicate ‘gap’ i.e., coding sequence not found on the respective contig. The ‘vertical red lines’ over the exons indicate ‘mismatch’ of the oSCF (+) and (−) protein with dog (27 aa and 22 aa) and mouse (53 aa and 39 aa) SCF gene.

Mentions: To verify the alternative splicing (AS) event that resulted in the shorter mRNA transcript i.e., ovine m-SCF (−) form, we amplified the intervening sequence between two exons. The sequenced chromatogram from the cDNA and gDNA of oSCF illustrating a PTC followed by the p(A)11/18 tail signal is shown in Figure 2(a,b), respectively. The reference SCF genomic locus at the exon 5-intron(5)-exon 6 splice junction was determined in comparison to the orthologous SCF gene assembly of human, mouse, rat, cow, horse and dog (source: Ensembl). The genomic DNA (gDNA) was obtained from the blood of white merino sheep. A expected amplicon size of 948 bp amplicon (Figure 2(d)) was amplified using an exon-5 (common CDS) specific forward primer and exon 6 specific reverse primer (+ form, proteolytic site; Table S2) as shown in Figure 2(c). Sequence analyses and orthologous comparison of the oSCF gene product (948 bp) with other mammals revealed that the first 136 bp corresponds to exon 5, followed by an intron-5 of 729 bp (Figure S4(b)) and an exon 6 containing 83 bp which encodes for the primary proteolytic site. This result was compared with the shorter cDNA transcript. The first 161 nt including a 11 bp polyA (pA) stretch of the intron-5 exhibited 100% identity to the nt pos. 668–835 of the shorter cDNA (Figure S4(c)). However, careful annotation of the 161 nt unveil a premature stop codon at nt pos. 21–23 of the 729 bp intronic sequnce. Figure 3 shows the oSCF gene structure(s) in reference to mouse, dog and human SCF gene (see also Figure S2 for the humanSCF alternative forms). The overall similarity for this 948 bp DNA splice region in other vertebrates was found to be highest with goat and cow SCF (99 and 94%) where as the lowest was detected with chicken and zebra finch SCF (62%).


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)

Gene architecture of ovine SCF gene in reference to human, mouse and dog.(a) Schematic representation of human SCF (huSCF) gene is shown. It consists of 10 exons (open boxes) intervened by 9 introns (linear black lines). Regular splicing and polyadenylation generates the full length huSCF mRNA transcript variant-b, a longer (+) form (5460 bp) encoding for a soluble product (273 aa; see Figure 5a); (b) The 84 bp exon 6 encoding for the 28 aa proteolytic site is skipped by an AS event of the huSCF gene is shown. The resultant full length huSCF mRNA transcript variant-a, known as (−) form (5376 bp) encodes for a membrane-bound product (245 aa; see Figure 5b); (c) Schematic representation of ovine SCF (oSCF) gene is shown. Regular splicing of exons 1–9/10(?) generate the full length oSCF (+) mRNA transcript (1519 bp) that encodes for a soluble product (274 aa; see Figure 5a); (d) Conversely, the possible AS events on intron-5 (Ref. human, mouse and dog) resulted in an alternative ORF with a premature termination (red symbol, PTC; see key to symbols). This resulted in retaining of 161 bp intronic sequence and completely eliminating (skipping of) the involvement of exon 6–9/10(?). The deduced protein sequence of this novel, shorter splice transcript variant (835 bp) resulted in 181 aa (see Figure 5b), a membrane-bound product of oSCF (−). In the above illustration, the open square or rectangle box symbolize exon and inverted triangle box symbolize intron. The open and shaded ‘black sparkle’ symbol on exon 10 (in 3a,b), exon ‘?’ (in 3c) and intron-5 (in 3d) all indicate the posssible position of predicted polyadenylation signal (PAS) sites. The two different sizes of the opened ‘black sparkle’ symbol (in 3a,b) denote the frequency of the common PAS such as ‘AAUAAA’ (8–12 times) and ‘AUUAAA’ (4–6 times) in the longer 3′ UTR of human, goat, mouse and rat. In contrast, the shaded ‘black sparkle’ symbol (in 3c,d) represents the other single basse ‘variants’ of PAS (see text in Results). Exon ‘?’ symbol (in 3c) represents the uncertainity of exon 10 position for oSCF (+). A ‘black hook’ symbolize the capped 5′ end and p(A) represents the polyA stretch on the preRNA, mRNA, respectively. The point of transcription termination (TAA) is symbolized as ‘red’ mark on intron-5 of oSCF (−) followed by the illustration of two possible mechanisms that resulted in a PTC of oSCF (−) (in dotted lines). The ASSP predicted constitutive and/or alternative splice donor (GT) and splice acceptor (AG) site(s) are labeled in blue and red letters respectively; (e) Schematic representation of the soluble oSCF (+) gene structure is shown. The exons/introns and the location of non-coding regions are determined in comparison to the mouse (chr 10) and dog (chr 15) SCF gene. The ‘intron (?)’ labeled in blue on the oSCF (+) in reference to mouse chr 10 indicates that the corresponding intron-7 is incomplete at that point i.e., it doesn’t show appropriate 5′ and/or 3′ splice sites; (f) Figure shows the gene stucture of membrane-bound oSCF (−). The ‘black line’ at the end of exon 5 of oSCF (−) in reference to mouse and dog indicate ‘gap’ i.e., coding sequence not found on the respective contig. The ‘vertical red lines’ over the exons indicate ‘mismatch’ of the oSCF (+) and (−) protein with dog (27 aa and 22 aa) and mouse (53 aa and 39 aa) SCF gene.
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Related In: Results  -  Collection

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

pone-0038657-g003: Gene architecture of ovine SCF gene in reference to human, mouse and dog.(a) Schematic representation of human SCF (huSCF) gene is shown. It consists of 10 exons (open boxes) intervened by 9 introns (linear black lines). Regular splicing and polyadenylation generates the full length huSCF mRNA transcript variant-b, a longer (+) form (5460 bp) encoding for a soluble product (273 aa; see Figure 5a); (b) The 84 bp exon 6 encoding for the 28 aa proteolytic site is skipped by an AS event of the huSCF gene is shown. The resultant full length huSCF mRNA transcript variant-a, known as (−) form (5376 bp) encodes for a membrane-bound product (245 aa; see Figure 5b); (c) Schematic representation of ovine SCF (oSCF) gene is shown. Regular splicing of exons 1–9/10(?) generate the full length oSCF (+) mRNA transcript (1519 bp) that encodes for a soluble product (274 aa; see Figure 5a); (d) Conversely, the possible AS events on intron-5 (Ref. human, mouse and dog) resulted in an alternative ORF with a premature termination (red symbol, PTC; see key to symbols). This resulted in retaining of 161 bp intronic sequence and completely eliminating (skipping of) the involvement of exon 6–9/10(?). The deduced protein sequence of this novel, shorter splice transcript variant (835 bp) resulted in 181 aa (see Figure 5b), a membrane-bound product of oSCF (−). In the above illustration, the open square or rectangle box symbolize exon and inverted triangle box symbolize intron. The open and shaded ‘black sparkle’ symbol on exon 10 (in 3a,b), exon ‘?’ (in 3c) and intron-5 (in 3d) all indicate the posssible position of predicted polyadenylation signal (PAS) sites. The two different sizes of the opened ‘black sparkle’ symbol (in 3a,b) denote the frequency of the common PAS such as ‘AAUAAA’ (8–12 times) and ‘AUUAAA’ (4–6 times) in the longer 3′ UTR of human, goat, mouse and rat. In contrast, the shaded ‘black sparkle’ symbol (in 3c,d) represents the other single basse ‘variants’ of PAS (see text in Results). Exon ‘?’ symbol (in 3c) represents the uncertainity of exon 10 position for oSCF (+). A ‘black hook’ symbolize the capped 5′ end and p(A) represents the polyA stretch on the preRNA, mRNA, respectively. The point of transcription termination (TAA) is symbolized as ‘red’ mark on intron-5 of oSCF (−) followed by the illustration of two possible mechanisms that resulted in a PTC of oSCF (−) (in dotted lines). The ASSP predicted constitutive and/or alternative splice donor (GT) and splice acceptor (AG) site(s) are labeled in blue and red letters respectively; (e) Schematic representation of the soluble oSCF (+) gene structure is shown. The exons/introns and the location of non-coding regions are determined in comparison to the mouse (chr 10) and dog (chr 15) SCF gene. The ‘intron (?)’ labeled in blue on the oSCF (+) in reference to mouse chr 10 indicates that the corresponding intron-7 is incomplete at that point i.e., it doesn’t show appropriate 5′ and/or 3′ splice sites; (f) Figure shows the gene stucture of membrane-bound oSCF (−). The ‘black line’ at the end of exon 5 of oSCF (−) in reference to mouse and dog indicate ‘gap’ i.e., coding sequence not found on the respective contig. The ‘vertical red lines’ over the exons indicate ‘mismatch’ of the oSCF (+) and (−) protein with dog (27 aa and 22 aa) and mouse (53 aa and 39 aa) SCF gene.
Mentions: To verify the alternative splicing (AS) event that resulted in the shorter mRNA transcript i.e., ovine m-SCF (−) form, we amplified the intervening sequence between two exons. The sequenced chromatogram from the cDNA and gDNA of oSCF illustrating a PTC followed by the p(A)11/18 tail signal is shown in Figure 2(a,b), respectively. The reference SCF genomic locus at the exon 5-intron(5)-exon 6 splice junction was determined in comparison to the orthologous SCF gene assembly of human, mouse, rat, cow, horse and dog (source: Ensembl). The genomic DNA (gDNA) was obtained from the blood of white merino sheep. A expected amplicon size of 948 bp amplicon (Figure 2(d)) was amplified using an exon-5 (common CDS) specific forward primer and exon 6 specific reverse primer (+ form, proteolytic site; Table S2) as shown in Figure 2(c). Sequence analyses and orthologous comparison of the oSCF gene product (948 bp) with other mammals revealed that the first 136 bp corresponds to exon 5, followed by an intron-5 of 729 bp (Figure S4(b)) and an exon 6 containing 83 bp which encodes for the primary proteolytic site. This result was compared with the shorter cDNA transcript. The first 161 nt including a 11 bp polyA (pA) stretch of the intron-5 exhibited 100% identity to the nt pos. 668–835 of the shorter cDNA (Figure S4(c)). However, careful annotation of the 161 nt unveil a premature stop codon at nt pos. 21–23 of the 729 bp intronic sequnce. Figure 3 shows the oSCF gene structure(s) in reference to mouse, dog and human SCF gene (see also Figure S2 for the humanSCF alternative forms). The overall similarity for this 948 bp DNA splice region in other vertebrates was found to be highest with goat and cow SCF (99 and 94%) where as the lowest was detected with chicken and zebra finch SCF (62%).

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