Limits...
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.

Show MeSH

Related in: MedlinePlus

Detection of Kirrel3 splice variants with primer set 2. A – PCR amplicons obtained with Kirrel3 primer set 3 that was specific for Kirrel3 B. mRNA was isolated from cultured human astrocytes (2 amplicons present) and adult human skeletal muscle biopsies obtained at rest and 4 and 24 hr post performance of a plyometric jumping exercise (amplicons not evident). B – Schematic of the exon structure of both amplicons detected in A based on sequence information.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig3: Detection of Kirrel3 splice variants with primer set 2. A – PCR amplicons obtained with Kirrel3 primer set 3 that was specific for Kirrel3 B. mRNA was isolated from cultured human astrocytes (2 amplicons present) and adult human skeletal muscle biopsies obtained at rest and 4 and 24 hr post performance of a plyometric jumping exercise (amplicons not evident). B – Schematic of the exon structure of both amplicons detected in A based on sequence information.

Mentions: Subsequently it was of interest to ascertain whether Kirrel3 B mRNA was present in adult human skeletal muscle and astrocytes. The same samples as those from the plyometric jumping exercise intervention were assessed for Kirrel3 B mRNA using primer set 3 that contained the same forward primer as primer set 2 and a reverse primer targeted towards a Kirrel3 B specific sequence present in its 14th exon. The predicted amplicon was 607 nucleotides. No amplicon was detectable in adult human skeletal muscle samples from either the plyometric jumping protocol (Figure 3B) or the downhill running samples (Data not shown), however, two amplicons migrating at approximately 610 and 590 nucleotides were detected in astrocytes (Figure 3B). Both amplicons were excised from the gel and sequenced. The amplicon migrating at approximately 610 nucleotide corresponded to Kirrel3 B while the amplicon that migrated at approximately 590 nucleotides lacked the 13th exon of Kirrel3 B which contains 37 nucleotides (see Table 2 for raw sequence data obtained).Figure 3


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

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

Detection of Kirrel3 splice variants with primer set 2. A – PCR amplicons obtained with Kirrel3 primer set 3 that was specific for Kirrel3 B. mRNA was isolated from cultured human astrocytes (2 amplicons present) and adult human skeletal muscle biopsies obtained at rest and 4 and 24 hr post performance of a plyometric jumping exercise (amplicons not evident). B – Schematic of the exon structure of both amplicons detected in A based on sequence information.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig3: Detection of Kirrel3 splice variants with primer set 2. A – PCR amplicons obtained with Kirrel3 primer set 3 that was specific for Kirrel3 B. mRNA was isolated from cultured human astrocytes (2 amplicons present) and adult human skeletal muscle biopsies obtained at rest and 4 and 24 hr post performance of a plyometric jumping exercise (amplicons not evident). B – Schematic of the exon structure of both amplicons detected in A based on sequence information.
Mentions: Subsequently it was of interest to ascertain whether Kirrel3 B mRNA was present in adult human skeletal muscle and astrocytes. The same samples as those from the plyometric jumping exercise intervention were assessed for Kirrel3 B mRNA using primer set 3 that contained the same forward primer as primer set 2 and a reverse primer targeted towards a Kirrel3 B specific sequence present in its 14th exon. The predicted amplicon was 607 nucleotides. No amplicon was detectable in adult human skeletal muscle samples from either the plyometric jumping protocol (Figure 3B) or the downhill running samples (Data not shown), however, two amplicons migrating at approximately 610 and 590 nucleotides were detected in astrocytes (Figure 3B). Both amplicons were excised from the gel and sequenced. The amplicon migrating at approximately 610 nucleotide corresponded to Kirrel3 B while the amplicon that migrated at approximately 590 nucleotides lacked the 13th exon of Kirrel3 B which contains 37 nucleotides (see Table 2 for raw sequence data obtained).Figure 3

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