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Edwin M. Stone, Robert F. Mullins, Budd A. Tucker, Alex H. Wagner, Terry A. Braun, Jade S. East, Todd E. Scheetz; Alternative Splicing of Exon 12 of the Male Germ Cell Associated Kinase Gene (MAK) Results in a Cone-specific Isoform. Invest. Ophthalmol. Vis. Sci. 2012;53(14):4594.
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The male germ cell associated kinase (MAK) is a 70 kDa protein present in many tissues. In the retina, MAK is involved in the regulation of cilia length. Mutations in the MAK gene were recently shown to cause autosomal recessive retinitis pigmentosa in humans (Tucker, et al., 2011 and Ozgül, et al., 2011). Investigation of MAK transcripts from mouse and human tissues revealed a retina specific exon (exon 12). This exon is highly conserved among all amniotes (reptiles, birds and mammals) but absent from earlier branches of phylogeny. The purpose of this experiment was to determine the fraction of retinal MAK transcripts that include exon 12 as well as the cellular distribution of the exon-12-bearing isoform.
Next-generation sequencing of poly-A RNA extracted from human donor retina was performed. mRNA sequences were mapped to the reference genome and exons were identified using the TopHat and Cufflinks analysis tools. Polyclonal antibodies directed against the retina specific isoform of MAK were generated by injecting rabbits with a 25mer peptide corresponding to the amino acids encoded by exon 12 (533-557). Immunofluorescence analysis was conducted on human and mouse retinas. Dual labeling with anti-exon-12-peptide antibody and either anti-rhodopsin antibody or peanut agglutinin was performed.
RNA sequence analysis demonstrated that 55.6% of MAK transcripts in human retina harbor exon 12. Immunohistochemical analysis using the anti-exon-12-peptide antibody revealed intense labeling of cone outer segments in both human and mouse retinas. In some human eyes, labeling was also observed in the inner segment, axon and pedicle of cone photoreceptors. Localization to cones was confirmed by dual labeling with peanut agglutinin and anti-rhodopsin antibodies.
The exon-12-bearing retina-specific MAK isoform appears to be restricted to cone photoreceptors in mammals. The functional difference between this isoform and the one lacking exon 12 has not yet been determined. At present, it is unclear which version of the transcript would be most appropriate for use in gene replacement therapy of human MAK-associated retinal degeneration.
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