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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

Schematic illustration of primer walking strategy for the ovine SCF (oSCF) mRNA/cDNA transcripts from skin. Gel pictures show subsequent RT-PCR and RACE amplification of the resultant full-length structural coverage of s-SCF (+) and m-SCF (−).The (+) and (−) product is indicated with two different symbols (see key to symbols). In the above figure, the arrows indicate the corresponding position of fwd and rev primers and the split regions of 5′ UTRs, CDS and 3′ UTRs are labeled with respective positions and base pairs (bp). The start and stop codon is labeled in ‘black’ (ATG) and ‘red’ (TAA) letters repectively. (A) Illustration for the full-length cDNA coverage of ovine s-SCF (+) isofom-1. (a) Amplification of isoform specific coding region (CDS) of ovine s-SCF (+) cDNA fragment (621 bp). Individual animal of merino sheep such as Black, Brown, White and the PCR negative control is indicated as Bl, Br, Wh1,2 (two individuals) and (−)ve, respectively; (b) 3′ RACE first round amplification of oSCF common region (+/−) showing three different sizes of amplicon ranging from ∼700 bp to 1300 bp; (b1) Isoform specific second round (Nested 1) 3′ RACE of ovine s-SCF (+) cDNA fragment (855 bp); (b2) Gel picture shows the purified 3′ RACE product of 793 bp (Nested 2 amplification); (c) 5′ RACE proteolytic site specific amplification (364 bp) of ovine s-SCF (+). (B) Illustration for the full-length cDNA coverage of ovine m-SCF (−) isoform-2. A premature termination codon (PTC) is indicated in red symbol and the resultant alternative open reading frame (ORF) responsible for the shorter truncated product is highlighted in black open box symbol; (b3) 3′ RACE amplification (Nested-1) from (b) indicates a 597 bp ovine m-SCF (−) amplicon and the other non-specific products (∼0.7/1.2 kb); (b4) Further, Nested 2 amplification yielded a 389 bp ovine m-SCF (−) amplicon; (b5) Gel picture shows the Nested 3 amplification of a 336 bp ovine m-SCF (−) product amplified from either (b3) or (b4) or (b) or directly from the oligo(dT)18 modified primed cDNA; (d) 5′ RACE amplification of the common region (+/−) showing two oSCF cDNA products (325 and 215 bp) characterized as ovine m-SCF (−) isoform2a/ab, respectively. DNA size markers are indicated as, M1– λ-DNA EcoRI/HindIII digest; and M2 - 1 kb Gene Ruler. In the above figure, the arrow marks indicate the appropriate size(s) of amplicon of the respective (RT)-PCR amplification. Note: For simplification we removed the tag ‘scf’ from the primer notation (see. Table S2).
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pone-0038657-g001: Schematic illustration of primer walking strategy for the ovine SCF (oSCF) mRNA/cDNA transcripts from skin. Gel pictures show subsequent RT-PCR and RACE amplification of the resultant full-length structural coverage of s-SCF (+) and m-SCF (−).The (+) and (−) product is indicated with two different symbols (see key to symbols). In the above figure, the arrows indicate the corresponding position of fwd and rev primers and the split regions of 5′ UTRs, CDS and 3′ UTRs are labeled with respective positions and base pairs (bp). The start and stop codon is labeled in ‘black’ (ATG) and ‘red’ (TAA) letters repectively. (A) Illustration for the full-length cDNA coverage of ovine s-SCF (+) isofom-1. (a) Amplification of isoform specific coding region (CDS) of ovine s-SCF (+) cDNA fragment (621 bp). Individual animal of merino sheep such as Black, Brown, White and the PCR negative control is indicated as Bl, Br, Wh1,2 (two individuals) and (−)ve, respectively; (b) 3′ RACE first round amplification of oSCF common region (+/−) showing three different sizes of amplicon ranging from ∼700 bp to 1300 bp; (b1) Isoform specific second round (Nested 1) 3′ RACE of ovine s-SCF (+) cDNA fragment (855 bp); (b2) Gel picture shows the purified 3′ RACE product of 793 bp (Nested 2 amplification); (c) 5′ RACE proteolytic site specific amplification (364 bp) of ovine s-SCF (+). (B) Illustration for the full-length cDNA coverage of ovine m-SCF (−) isoform-2. A premature termination codon (PTC) is indicated in red symbol and the resultant alternative open reading frame (ORF) responsible for the shorter truncated product is highlighted in black open box symbol; (b3) 3′ RACE amplification (Nested-1) from (b) indicates a 597 bp ovine m-SCF (−) amplicon and the other non-specific products (∼0.7/1.2 kb); (b4) Further, Nested 2 amplification yielded a 389 bp ovine m-SCF (−) amplicon; (b5) Gel picture shows the Nested 3 amplification of a 336 bp ovine m-SCF (−) product amplified from either (b3) or (b4) or (b) or directly from the oligo(dT)18 modified primed cDNA; (d) 5′ RACE amplification of the common region (+/−) showing two oSCF cDNA products (325 and 215 bp) characterized as ovine m-SCF (−) isoform2a/ab, respectively. DNA size markers are indicated as, M1– λ-DNA EcoRI/HindIII digest; and M2 - 1 kb Gene Ruler. In the above figure, the arrow marks indicate the appropriate size(s) of amplicon of the respective (RT)-PCR amplification. Note: For simplification we removed the tag ‘scf’ from the primer notation (see. Table S2).

Mentions: To examine the SCF variant(s) expressed in the skin of white merino sheep, 1–1.5 µg of total RNAs from the skin were reverse transcribed and the synthesized single strand cDNAs were amplified by PCR. We initially carried out the cDNA coding (CDS) region amplification using the primer pair scffwd1 and scfrev1 (Table S2). Primer walking and the mRNA/cDNA structural coverage of the longer and shorter cDNA amplification strategies from the ovine total RNA (skin) are shown in Figure 1A and 1B. RT-PCR primers were selected based on the mammalian nucleotide (nt) sequence alignment of the soluble-SCF (s-SCF) cDNA encompassed to the open reading frame (ORF) of 606 bp of the 621 bp amplicon (Figure 1A(a)) commonly known as ‘soluble or secreted form’. The purified RT-PCR amplification product was then cloned and sequenced. Sequencing results revealed no differences among white, black and brown clones of the 621 bp (Figure S1A), which additionally appear to be identical (99%) with two of the previously submitted NCBI GenBank mRNA (partial) sequences of ovine s-SCF (U89874.1 in 2002 and Z50743.1 in 2005; see Figure S1B) from ovarian follicles and keratinocytes, respectively. An exception of transition at T54C in U89874.1 was observed among the 621 bp sequences. Similarly, a transversion at C81G was observed (see the chromatogram of Figure S1B) in 2 out of 5 clones sequenced in white animal. The possible allelic variant at this position will elucidate its true identity. Nevertheless, these substitutions do not result in an amino acid substitution change.


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)

Schematic illustration of primer walking strategy for the ovine SCF (oSCF) mRNA/cDNA transcripts from skin. Gel pictures show subsequent RT-PCR and RACE amplification of the resultant full-length structural coverage of s-SCF (+) and m-SCF (−).The (+) and (−) product is indicated with two different symbols (see key to symbols). In the above figure, the arrows indicate the corresponding position of fwd and rev primers and the split regions of 5′ UTRs, CDS and 3′ UTRs are labeled with respective positions and base pairs (bp). The start and stop codon is labeled in ‘black’ (ATG) and ‘red’ (TAA) letters repectively. (A) Illustration for the full-length cDNA coverage of ovine s-SCF (+) isofom-1. (a) Amplification of isoform specific coding region (CDS) of ovine s-SCF (+) cDNA fragment (621 bp). Individual animal of merino sheep such as Black, Brown, White and the PCR negative control is indicated as Bl, Br, Wh1,2 (two individuals) and (−)ve, respectively; (b) 3′ RACE first round amplification of oSCF common region (+/−) showing three different sizes of amplicon ranging from ∼700 bp to 1300 bp; (b1) Isoform specific second round (Nested 1) 3′ RACE of ovine s-SCF (+) cDNA fragment (855 bp); (b2) Gel picture shows the purified 3′ RACE product of 793 bp (Nested 2 amplification); (c) 5′ RACE proteolytic site specific amplification (364 bp) of ovine s-SCF (+). (B) Illustration for the full-length cDNA coverage of ovine m-SCF (−) isoform-2. A premature termination codon (PTC) is indicated in red symbol and the resultant alternative open reading frame (ORF) responsible for the shorter truncated product is highlighted in black open box symbol; (b3) 3′ RACE amplification (Nested-1) from (b) indicates a 597 bp ovine m-SCF (−) amplicon and the other non-specific products (∼0.7/1.2 kb); (b4) Further, Nested 2 amplification yielded a 389 bp ovine m-SCF (−) amplicon; (b5) Gel picture shows the Nested 3 amplification of a 336 bp ovine m-SCF (−) product amplified from either (b3) or (b4) or (b) or directly from the oligo(dT)18 modified primed cDNA; (d) 5′ RACE amplification of the common region (+/−) showing two oSCF cDNA products (325 and 215 bp) characterized as ovine m-SCF (−) isoform2a/ab, respectively. DNA size markers are indicated as, M1– λ-DNA EcoRI/HindIII digest; and M2 - 1 kb Gene Ruler. In the above figure, the arrow marks indicate the appropriate size(s) of amplicon of the respective (RT)-PCR amplification. Note: For simplification we removed the tag ‘scf’ from the primer notation (see. Table S2).
© Copyright Policy
Related In: Results  -  Collection

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

pone-0038657-g001: Schematic illustration of primer walking strategy for the ovine SCF (oSCF) mRNA/cDNA transcripts from skin. Gel pictures show subsequent RT-PCR and RACE amplification of the resultant full-length structural coverage of s-SCF (+) and m-SCF (−).The (+) and (−) product is indicated with two different symbols (see key to symbols). In the above figure, the arrows indicate the corresponding position of fwd and rev primers and the split regions of 5′ UTRs, CDS and 3′ UTRs are labeled with respective positions and base pairs (bp). The start and stop codon is labeled in ‘black’ (ATG) and ‘red’ (TAA) letters repectively. (A) Illustration for the full-length cDNA coverage of ovine s-SCF (+) isofom-1. (a) Amplification of isoform specific coding region (CDS) of ovine s-SCF (+) cDNA fragment (621 bp). Individual animal of merino sheep such as Black, Brown, White and the PCR negative control is indicated as Bl, Br, Wh1,2 (two individuals) and (−)ve, respectively; (b) 3′ RACE first round amplification of oSCF common region (+/−) showing three different sizes of amplicon ranging from ∼700 bp to 1300 bp; (b1) Isoform specific second round (Nested 1) 3′ RACE of ovine s-SCF (+) cDNA fragment (855 bp); (b2) Gel picture shows the purified 3′ RACE product of 793 bp (Nested 2 amplification); (c) 5′ RACE proteolytic site specific amplification (364 bp) of ovine s-SCF (+). (B) Illustration for the full-length cDNA coverage of ovine m-SCF (−) isoform-2. A premature termination codon (PTC) is indicated in red symbol and the resultant alternative open reading frame (ORF) responsible for the shorter truncated product is highlighted in black open box symbol; (b3) 3′ RACE amplification (Nested-1) from (b) indicates a 597 bp ovine m-SCF (−) amplicon and the other non-specific products (∼0.7/1.2 kb); (b4) Further, Nested 2 amplification yielded a 389 bp ovine m-SCF (−) amplicon; (b5) Gel picture shows the Nested 3 amplification of a 336 bp ovine m-SCF (−) product amplified from either (b3) or (b4) or (b) or directly from the oligo(dT)18 modified primed cDNA; (d) 5′ RACE amplification of the common region (+/−) showing two oSCF cDNA products (325 and 215 bp) characterized as ovine m-SCF (−) isoform2a/ab, respectively. DNA size markers are indicated as, M1– λ-DNA EcoRI/HindIII digest; and M2 - 1 kb Gene Ruler. In the above figure, the arrow marks indicate the appropriate size(s) of amplicon of the respective (RT)-PCR amplification. Note: For simplification we removed the tag ‘scf’ from the primer notation (see. Table S2).
Mentions: To examine the SCF variant(s) expressed in the skin of white merino sheep, 1–1.5 µg of total RNAs from the skin were reverse transcribed and the synthesized single strand cDNAs were amplified by PCR. We initially carried out the cDNA coding (CDS) region amplification using the primer pair scffwd1 and scfrev1 (Table S2). Primer walking and the mRNA/cDNA structural coverage of the longer and shorter cDNA amplification strategies from the ovine total RNA (skin) are shown in Figure 1A and 1B. RT-PCR primers were selected based on the mammalian nucleotide (nt) sequence alignment of the soluble-SCF (s-SCF) cDNA encompassed to the open reading frame (ORF) of 606 bp of the 621 bp amplicon (Figure 1A(a)) commonly known as ‘soluble or secreted form’. The purified RT-PCR amplification product was then cloned and sequenced. Sequencing results revealed no differences among white, black and brown clones of the 621 bp (Figure S1A), which additionally appear to be identical (99%) with two of the previously submitted NCBI GenBank mRNA (partial) sequences of ovine s-SCF (U89874.1 in 2002 and Z50743.1 in 2005; see Figure S1B) from ovarian follicles and keratinocytes, respectively. An exception of transition at T54C in U89874.1 was observed among the 621 bp sequences. Similarly, a transversion at C81G was observed (see the chromatogram of Figure S1B) in 2 out of 5 clones sequenced in white animal. The possible allelic variant at this position will elucidate its true identity. Nevertheless, these substitutions do not result in an amino acid substitution change.

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