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Identification of novel Kirrel3 gene splice variants in adult human skeletal muscle.

Durcan PJ, Conradie JD, Van deVyver M, Myburgh KH - BMC Physiol. (2014)

Bottom Line: Several anti Kirrel3 immunoreactive proteins were detected in all adult human skeletal muscle samples analysed and results suggest the presence of different isoforms or posttranslational modification, or both.Importantly, mRNA of all splice variants was not always present, a finding with potential physiological relevance.These initial discoveries highlight the need for more molecular and functional studies to understand the role of Kirrel3 in human skeletal muscle.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiological Science, Stellenbosch University, Private Bag X1 Matieland, 7602, Stellenbosch, South Africa. pdurcan@sun.ac.za.

ABSTRACT

Background: Multiple cell types including trophoblasts, osteoclasts and myoblasts require somatic cell fusion events as part of their physiological functions. In Drosophila Melanogaster the paralogus type 1 transmembrane receptors and members of the immunoglobulin superfamily Kin of Irre (Kirre) and roughest (Rst) regulate myoblast fusion during embryonic development. Present within the human genome are three homologs to Kirre termed Kin of Irre like (Kirrel) 1, 2 and 3. Currently it is unknown if Kirrel3 is expressed in adult human skeletal muscle.

Results: We investigated (using PCR and Western blot) Kirrel3 in adult human skeletal muscle samples taken at rest and after mild exercise induced muscle damage. Kirrel3 mRNA expression was verified by sequencing and protein presence via blotting with 2 different anti-Kirrel3 protein antibodies. Evidence for three alternatively spliced Kirrel3 mRNA transcripts in adult human skeletal muscle was obtained. Kirrel3 mRNA in adult human skeletal muscle was detected at low or moderate levels, or not at all. This sporadic expression suggests that Kirrel3 is expressed in a pulsatile manner. Several anti Kirrel3 immunoreactive proteins were detected in all adult human skeletal muscle samples analysed and results suggest the presence of different isoforms or posttranslational modification, or both.

Conclusion: The results presented here demonstrate for the first time that there are at least 3 splice variants of Kirrel3 expressed in adult human skeletal muscle, two of which have never previously been identified in human muscle. Importantly, mRNA of all splice variants was not always present, a finding with potential physiological relevance. These initial discoveries highlight the need for more molecular and functional studies to understand the role of Kirrel3 in human skeletal muscle.

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Schematic of the exon structure of Kirrel3 A and B mRNA transcripts. A – Schematic of the exon structure of Kirrel3 A and B mRNA transcripts. Start codon is highlighted as ATG and stop codon as TAA. The unusually large first intron is highlighted. The exons to which primers are targeted are also shown. PS1F – Primer set 1 forward primer, PS2F – Primer set 2 forward primer, PS1R – Primer set 1 reverse primer, PS3R – Primer set 3 reverse primer. B – Schematic of the predicted protein domains and putative N-linked glycosylation sites of Kirrel3 A and B.
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Fig1: Schematic of the exon structure of Kirrel3 A and B mRNA transcripts. A – Schematic of the exon structure of Kirrel3 A and B mRNA transcripts. Start codon is highlighted as ATG and stop codon as TAA. The unusually large first intron is highlighted. The exons to which primers are targeted are also shown. PS1F – Primer set 1 forward primer, PS2F – Primer set 2 forward primer, PS1R – Primer set 1 reverse primer, PS3R – Primer set 3 reverse primer. B – Schematic of the predicted protein domains and putative N-linked glycosylation sites of Kirrel3 A and B.

Mentions: Analysis of the National Centre for Biotechnology (NCBI) gene database highlighted the presence of two human Kirrel3 reference sequences NM_032531.3 (hereafter referred to as Kirrel3 A) containing 3777 nucleotides and NM_001161707.1 (hereafter referred to as Kirrel3 B) containing 2534 nucleotides. Aligning Kirrel3 A and B mRNA transcripts to the human genome via DNA sequence present in the genomic contig NT_033899.8, demonstrated that Kirrel3 A has 17 exons while B has 14 exons (see Figure 1A for schematic). Of particular note in both transcripts was the very large first intron which spanned approximately 438 kilo bases (KB). Both transcripts are predicted to start their translation initiation in their first exons. The first 2057 nucleotides of Kirrel3 A and B are identical. This region spans exons 1-14 of Kirrel3 A. Subsequently Kirrel3 A has a splice site that is not present in B thus resulting in exon 14 of Kirrel3 B containing an additional 477 nucleotides. The predicted stop codon (TAA) in Kirrel3 B is 107 nucleotides 3′ to the missed splice site. In comparison to Kirrel3 B, Kirrel3 A has an additional 3 exons totalling 1720 nucleotides 3′ to the missed spliced site. The stop codon (TAA) for Kirrel3 A is located in exon its 17th exon 641 nucleotides 3′ to the missed spliced site in B. The 3′ untranslated regions of Kirrel3 A and B are 1079 and 370 nucleotides respectively.Figure 1


Identification of novel Kirrel3 gene splice variants in adult human skeletal muscle.

Durcan PJ, Conradie JD, Van deVyver M, Myburgh KH - BMC Physiol. (2014)

Schematic of the exon structure of Kirrel3 A and B mRNA transcripts. A – Schematic of the exon structure of Kirrel3 A and B mRNA transcripts. Start codon is highlighted as ATG and stop codon as TAA. The unusually large first intron is highlighted. The exons to which primers are targeted are also shown. PS1F – Primer set 1 forward primer, PS2F – Primer set 2 forward primer, PS1R – Primer set 1 reverse primer, PS3R – Primer set 3 reverse primer. B – Schematic of the predicted protein domains and putative N-linked glycosylation sites of Kirrel3 A and B.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4269076&req=5

Fig1: Schematic of the exon structure of Kirrel3 A and B mRNA transcripts. A – Schematic of the exon structure of Kirrel3 A and B mRNA transcripts. Start codon is highlighted as ATG and stop codon as TAA. The unusually large first intron is highlighted. The exons to which primers are targeted are also shown. PS1F – Primer set 1 forward primer, PS2F – Primer set 2 forward primer, PS1R – Primer set 1 reverse primer, PS3R – Primer set 3 reverse primer. B – Schematic of the predicted protein domains and putative N-linked glycosylation sites of Kirrel3 A and B.
Mentions: Analysis of the National Centre for Biotechnology (NCBI) gene database highlighted the presence of two human Kirrel3 reference sequences NM_032531.3 (hereafter referred to as Kirrel3 A) containing 3777 nucleotides and NM_001161707.1 (hereafter referred to as Kirrel3 B) containing 2534 nucleotides. Aligning Kirrel3 A and B mRNA transcripts to the human genome via DNA sequence present in the genomic contig NT_033899.8, demonstrated that Kirrel3 A has 17 exons while B has 14 exons (see Figure 1A for schematic). Of particular note in both transcripts was the very large first intron which spanned approximately 438 kilo bases (KB). Both transcripts are predicted to start their translation initiation in their first exons. The first 2057 nucleotides of Kirrel3 A and B are identical. This region spans exons 1-14 of Kirrel3 A. Subsequently Kirrel3 A has a splice site that is not present in B thus resulting in exon 14 of Kirrel3 B containing an additional 477 nucleotides. The predicted stop codon (TAA) in Kirrel3 B is 107 nucleotides 3′ to the missed splice site. In comparison to Kirrel3 B, Kirrel3 A has an additional 3 exons totalling 1720 nucleotides 3′ to the missed spliced site. The stop codon (TAA) for Kirrel3 A is located in exon its 17th exon 641 nucleotides 3′ to the missed spliced site in B. The 3′ untranslated regions of Kirrel3 A and B are 1079 and 370 nucleotides respectively.Figure 1

Bottom Line: Several anti Kirrel3 immunoreactive proteins were detected in all adult human skeletal muscle samples analysed and results suggest the presence of different isoforms or posttranslational modification, or both.Importantly, mRNA of all splice variants was not always present, a finding with potential physiological relevance.These initial discoveries highlight the need for more molecular and functional studies to understand the role of Kirrel3 in human skeletal muscle.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiological Science, Stellenbosch University, Private Bag X1 Matieland, 7602, Stellenbosch, South Africa. pdurcan@sun.ac.za.

ABSTRACT

Background: Multiple cell types including trophoblasts, osteoclasts and myoblasts require somatic cell fusion events as part of their physiological functions. In Drosophila Melanogaster the paralogus type 1 transmembrane receptors and members of the immunoglobulin superfamily Kin of Irre (Kirre) and roughest (Rst) regulate myoblast fusion during embryonic development. Present within the human genome are three homologs to Kirre termed Kin of Irre like (Kirrel) 1, 2 and 3. Currently it is unknown if Kirrel3 is expressed in adult human skeletal muscle.

Results: We investigated (using PCR and Western blot) Kirrel3 in adult human skeletal muscle samples taken at rest and after mild exercise induced muscle damage. Kirrel3 mRNA expression was verified by sequencing and protein presence via blotting with 2 different anti-Kirrel3 protein antibodies. Evidence for three alternatively spliced Kirrel3 mRNA transcripts in adult human skeletal muscle was obtained. Kirrel3 mRNA in adult human skeletal muscle was detected at low or moderate levels, or not at all. This sporadic expression suggests that Kirrel3 is expressed in a pulsatile manner. Several anti Kirrel3 immunoreactive proteins were detected in all adult human skeletal muscle samples analysed and results suggest the presence of different isoforms or posttranslational modification, or both.

Conclusion: The results presented here demonstrate for the first time that there are at least 3 splice variants of Kirrel3 expressed in adult human skeletal muscle, two of which have never previously been identified in human muscle. Importantly, mRNA of all splice variants was not always present, a finding with potential physiological relevance. These initial discoveries highlight the need for more molecular and functional studies to understand the role of Kirrel3 in human skeletal muscle.

Show MeSH
Related in: MedlinePlus