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Mapping and functional characterization of the murine smoothelin-like 1 promoter.

Ulke-Lemée A, Turner SR, Mughal SH, Borman MA, Winkfein RJ, MacDonald JA - BMC Mol. Biol. (2011)

Bottom Line: Smtnl1 transcript was identified in all smooth muscle tissues examined to date, albeit at much lower levels than found in skeletal muscle.It is unlikely that multiple SMTNL1 isoforms exist since a single Smtnl1 transcription start site was identified in both skeletal and intestinal smooth muscle.Promoter studies suggest restrictive control of Smtnl1 expression in non-muscle cells.

View Article: PubMed Central - HTML - PubMed

Affiliation: Smooth Muscle Research Group, Department of Biochemistry & Molecular Biology, University of Calgary, Alberta, Canada.

ABSTRACT

Background: Smoothelin-like 1 (SMTNL1, also known as CHASM) plays a role in promoting relaxation as well as adaptive responses to exercise, pregnancy and sexual development in smooth and skeletal muscle. Investigations of Smtnl1 transcriptional regulation are still lacking. Thus, in this study, we identify and characterize key regulatory elements of the mouse Smtnl1 gene.

Results: We mapped the key regulatory elements of the Smtnl1 promoter region: the transcriptional start site (TSS) lays -44 bp from the translational start codon and a TATA-box motif at -75 bp was conserved amongst all mammalian Smtnl1 promoters investigated. The Smtnl1 proximal promoter enhances expression up to 8-fold in smooth muscle cells and a second activating region lays 500 bp further upstream. Two repressing motifs were present (-118 to -218 bp and -1637 to -1869 bp). The proximal promoter is highly conserved in mammals and contains a mirror repeat sequence. In silico analysis suggests many transcription factors (notably MyoD) could potentially bind within the Smtnl1 proximal promoter sequence.

Conclusion: Smtnl1 transcript was identified in all smooth muscle tissues examined to date, albeit at much lower levels than found in skeletal muscle. It is unlikely that multiple SMTNL1 isoforms exist since a single Smtnl1 transcription start site was identified in both skeletal and intestinal smooth muscle. Promoter studies suggest restrictive control of Smtnl1 expression in non-muscle cells.

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Identification of the transcription start site of Smtnl1 by rapid amplification of cDNA 5'-ends (5'-RACE) and ribonuclease protection assay (RPA). 5'-RACE reactions were performed on cDNA synthesized from smooth (A) or skeletal (B) muscle mRNA with primers specific for Smtnl1. Representative ethidium bromide-stained agarose gels of PCR products are shown. A 5'-RACE CDS primer (Stratagene) and three Smtnl1 gene specific primers (GSPs) were used with expected product sizes: GSP1 (1479 bp), GSP2 (176 bp) and GSP3 (83 bp). The 5'-RACE product from each reaction was used as template in a subsequent nested PCR reaction. The expected sizes of the nested PCR products were: NGSP1 (63 bp) and NGSP2 (823 bp). The arrowhead in (B) indicates the PCR product visible during the primary amplification when skeletal muscle cDNA was used as template for 5'-RACE with GSP1. The DNA ladder markers are indicated on the left. In (C), ribonuclease protection assay (RPA) analysis was performed on 10 μg of total RNA from smooth (SM) and skeletal (SKM) muscle. A biotin-labeled Smtnl1 riboprobe was generated to span -112 bp to the TSS, joined to exon 1 and exon 2 from the Smtnl1 mRNA, up to + 319 bp. Total RNA from S. cerevisiae (10 μg) was used instead of muscle RNA as a negative control. The band appearing with skeletal muscle mRNA is marked by an arrow. The DNA ladder markers are indicated on the left. In (D), the promoter region, eight exons (E1-E8) and 7 intronic DNA sections are shown for the mouse Smtnl1 gene. The positions of the start codon (ATG), transcriptional start site (TSS) defined by 5'-RACE, the TATA box and initiator sequence (Inr) are also indicated. The nucleotides identified by sequencing the cDNA clones (n = 5) obtained from 5'-RACE are underlined.
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Figure 2: Identification of the transcription start site of Smtnl1 by rapid amplification of cDNA 5'-ends (5'-RACE) and ribonuclease protection assay (RPA). 5'-RACE reactions were performed on cDNA synthesized from smooth (A) or skeletal (B) muscle mRNA with primers specific for Smtnl1. Representative ethidium bromide-stained agarose gels of PCR products are shown. A 5'-RACE CDS primer (Stratagene) and three Smtnl1 gene specific primers (GSPs) were used with expected product sizes: GSP1 (1479 bp), GSP2 (176 bp) and GSP3 (83 bp). The 5'-RACE product from each reaction was used as template in a subsequent nested PCR reaction. The expected sizes of the nested PCR products were: NGSP1 (63 bp) and NGSP2 (823 bp). The arrowhead in (B) indicates the PCR product visible during the primary amplification when skeletal muscle cDNA was used as template for 5'-RACE with GSP1. The DNA ladder markers are indicated on the left. In (C), ribonuclease protection assay (RPA) analysis was performed on 10 μg of total RNA from smooth (SM) and skeletal (SKM) muscle. A biotin-labeled Smtnl1 riboprobe was generated to span -112 bp to the TSS, joined to exon 1 and exon 2 from the Smtnl1 mRNA, up to + 319 bp. Total RNA from S. cerevisiae (10 μg) was used instead of muscle RNA as a negative control. The band appearing with skeletal muscle mRNA is marked by an arrow. The DNA ladder markers are indicated on the left. In (D), the promoter region, eight exons (E1-E8) and 7 intronic DNA sections are shown for the mouse Smtnl1 gene. The positions of the start codon (ATG), transcriptional start site (TSS) defined by 5'-RACE, the TATA box and initiator sequence (Inr) are also indicated. The nucleotides identified by sequencing the cDNA clones (n = 5) obtained from 5'-RACE are underlined.

Mentions: The 5'-RACE PCR on smooth muscle (Figure 2A) and skeletal muscle (Figure 2B) total RNA produced low (< 250 bp) molecular weight cross-dimer products between the GSP and 5'-RACE UPM primers (as verified by sequencing). When skeletal muscle mRNA was used as template together with primer GSP1, a faint band was visible at approximately 1.5-kb that was close to the expected product size (Figure 2B). To increase specificity and sensitivity, a second round of PCR was performed using nested primers on the products from the 5'-RACE PCR. The expected amplicon sizes with the NCBI-predicted TSS were 63 bp for NGSP1 and 823 bp for NGSP2. A strong band of approximately 700 bp was observed for nested PCR reactions completed on both smooth and skeletal muscle GSP1 PCR products (Figure 2A &2B), somewhat smaller than the expected 823 bp product for the NGSP2 primer. Sequence analysis revealed a partial alignment of the 823 bp NGSP2 product with Smtnl1, starting within exon 1.


Mapping and functional characterization of the murine smoothelin-like 1 promoter.

Ulke-Lemée A, Turner SR, Mughal SH, Borman MA, Winkfein RJ, MacDonald JA - BMC Mol. Biol. (2011)

Identification of the transcription start site of Smtnl1 by rapid amplification of cDNA 5'-ends (5'-RACE) and ribonuclease protection assay (RPA). 5'-RACE reactions were performed on cDNA synthesized from smooth (A) or skeletal (B) muscle mRNA with primers specific for Smtnl1. Representative ethidium bromide-stained agarose gels of PCR products are shown. A 5'-RACE CDS primer (Stratagene) and three Smtnl1 gene specific primers (GSPs) were used with expected product sizes: GSP1 (1479 bp), GSP2 (176 bp) and GSP3 (83 bp). The 5'-RACE product from each reaction was used as template in a subsequent nested PCR reaction. The expected sizes of the nested PCR products were: NGSP1 (63 bp) and NGSP2 (823 bp). The arrowhead in (B) indicates the PCR product visible during the primary amplification when skeletal muscle cDNA was used as template for 5'-RACE with GSP1. The DNA ladder markers are indicated on the left. In (C), ribonuclease protection assay (RPA) analysis was performed on 10 μg of total RNA from smooth (SM) and skeletal (SKM) muscle. A biotin-labeled Smtnl1 riboprobe was generated to span -112 bp to the TSS, joined to exon 1 and exon 2 from the Smtnl1 mRNA, up to + 319 bp. Total RNA from S. cerevisiae (10 μg) was used instead of muscle RNA as a negative control. The band appearing with skeletal muscle mRNA is marked by an arrow. The DNA ladder markers are indicated on the left. In (D), the promoter region, eight exons (E1-E8) and 7 intronic DNA sections are shown for the mouse Smtnl1 gene. The positions of the start codon (ATG), transcriptional start site (TSS) defined by 5'-RACE, the TATA box and initiator sequence (Inr) are also indicated. The nucleotides identified by sequencing the cDNA clones (n = 5) obtained from 5'-RACE are underlined.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
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Figure 2: Identification of the transcription start site of Smtnl1 by rapid amplification of cDNA 5'-ends (5'-RACE) and ribonuclease protection assay (RPA). 5'-RACE reactions were performed on cDNA synthesized from smooth (A) or skeletal (B) muscle mRNA with primers specific for Smtnl1. Representative ethidium bromide-stained agarose gels of PCR products are shown. A 5'-RACE CDS primer (Stratagene) and three Smtnl1 gene specific primers (GSPs) were used with expected product sizes: GSP1 (1479 bp), GSP2 (176 bp) and GSP3 (83 bp). The 5'-RACE product from each reaction was used as template in a subsequent nested PCR reaction. The expected sizes of the nested PCR products were: NGSP1 (63 bp) and NGSP2 (823 bp). The arrowhead in (B) indicates the PCR product visible during the primary amplification when skeletal muscle cDNA was used as template for 5'-RACE with GSP1. The DNA ladder markers are indicated on the left. In (C), ribonuclease protection assay (RPA) analysis was performed on 10 μg of total RNA from smooth (SM) and skeletal (SKM) muscle. A biotin-labeled Smtnl1 riboprobe was generated to span -112 bp to the TSS, joined to exon 1 and exon 2 from the Smtnl1 mRNA, up to + 319 bp. Total RNA from S. cerevisiae (10 μg) was used instead of muscle RNA as a negative control. The band appearing with skeletal muscle mRNA is marked by an arrow. The DNA ladder markers are indicated on the left. In (D), the promoter region, eight exons (E1-E8) and 7 intronic DNA sections are shown for the mouse Smtnl1 gene. The positions of the start codon (ATG), transcriptional start site (TSS) defined by 5'-RACE, the TATA box and initiator sequence (Inr) are also indicated. The nucleotides identified by sequencing the cDNA clones (n = 5) obtained from 5'-RACE are underlined.
Mentions: The 5'-RACE PCR on smooth muscle (Figure 2A) and skeletal muscle (Figure 2B) total RNA produced low (< 250 bp) molecular weight cross-dimer products between the GSP and 5'-RACE UPM primers (as verified by sequencing). When skeletal muscle mRNA was used as template together with primer GSP1, a faint band was visible at approximately 1.5-kb that was close to the expected product size (Figure 2B). To increase specificity and sensitivity, a second round of PCR was performed using nested primers on the products from the 5'-RACE PCR. The expected amplicon sizes with the NCBI-predicted TSS were 63 bp for NGSP1 and 823 bp for NGSP2. A strong band of approximately 700 bp was observed for nested PCR reactions completed on both smooth and skeletal muscle GSP1 PCR products (Figure 2A &2B), somewhat smaller than the expected 823 bp product for the NGSP2 primer. Sequence analysis revealed a partial alignment of the 823 bp NGSP2 product with Smtnl1, starting within exon 1.

Bottom Line: Smtnl1 transcript was identified in all smooth muscle tissues examined to date, albeit at much lower levels than found in skeletal muscle.It is unlikely that multiple SMTNL1 isoforms exist since a single Smtnl1 transcription start site was identified in both skeletal and intestinal smooth muscle.Promoter studies suggest restrictive control of Smtnl1 expression in non-muscle cells.

View Article: PubMed Central - HTML - PubMed

Affiliation: Smooth Muscle Research Group, Department of Biochemistry & Molecular Biology, University of Calgary, Alberta, Canada.

ABSTRACT

Background: Smoothelin-like 1 (SMTNL1, also known as CHASM) plays a role in promoting relaxation as well as adaptive responses to exercise, pregnancy and sexual development in smooth and skeletal muscle. Investigations of Smtnl1 transcriptional regulation are still lacking. Thus, in this study, we identify and characterize key regulatory elements of the mouse Smtnl1 gene.

Results: We mapped the key regulatory elements of the Smtnl1 promoter region: the transcriptional start site (TSS) lays -44 bp from the translational start codon and a TATA-box motif at -75 bp was conserved amongst all mammalian Smtnl1 promoters investigated. The Smtnl1 proximal promoter enhances expression up to 8-fold in smooth muscle cells and a second activating region lays 500 bp further upstream. Two repressing motifs were present (-118 to -218 bp and -1637 to -1869 bp). The proximal promoter is highly conserved in mammals and contains a mirror repeat sequence. In silico analysis suggests many transcription factors (notably MyoD) could potentially bind within the Smtnl1 proximal promoter sequence.

Conclusion: Smtnl1 transcript was identified in all smooth muscle tissues examined to date, albeit at much lower levels than found in skeletal muscle. It is unlikely that multiple SMTNL1 isoforms exist since a single Smtnl1 transcription start site was identified in both skeletal and intestinal smooth muscle. Promoter studies suggest restrictive control of Smtnl1 expression in non-muscle cells.

Show MeSH