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Dror Sharon, Alexey Obolensky, Elia Shevach, Eyal Banin, Shahar Frenkel, Adi Inbal, Dikla Bandah-Rozenfeld; FAM161A Produces Two Protein Isoforms with a Differential Retinal Expression Pattern. Invest. Ophthalmol. Vis. Sci. 2012;53(14):1618.
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We have previously reported that mutations in FAM161A are the major cause of autosomal recessive retinitis pigmentosa in the Israeli population. In addition, we reported that FAM161A produces two alternatively-spliced transcripts in the retina. The more common transcript (#1) does not contain exon 4 while the less common transcript (#2) contains all known exons. The purpose of the current study is to verify the expression of the two transcripts at the protein level, to determine the expression pattern of each isoform, and to characterize the effect of blocking or reducing the expression of these isoforms in the zebrafish retina.
Polyclonal antibodies were raised against the C-terminal and the alternatively-spliced and highly conserved exon 4 of FAM161A, using rabbit as the host species. Immunohistochemistry was performed on mouse and human retinal sections using a commercial antibody (HPA032119) as well as two costume-made antibodies. Morpholino oligonucleotides that were designed to block translation or splicing of FAM161A were injected into 1-4-cell zebrafish embryos.
We raised antibodies against the C-terminal and the alternatively-spliced and highly conserved exon 4 of FAM161A. In the mouse retina, all antibodies yielded a similar expression pattern with a major signal in photoreceptor inner-segments as well as a weaker staining in the outer plexiform layer. In the human retina, a similar pattern of expression was obtained with antibodies that were designed to recognize both isoforms, while the antibody designed to recognize isoform #2 showed a cone-enriched staining. Double staining with a FAM161A antibody and either PNA or S-cone opsin supported the cone-enriched expression of this isoform. Aiming to gain a better understanding on the function of each isoform, morpholinos were designed to block the expression of either all isoforms or isoform #2 specifically. RT-PCR analysis of injected embryos indicated that the splicing morpholino inhibit the expression of both transcripts. The effect of the different morpholinos on retinal structure and function is currently being evaluated and will be reported at the meeting.
We report here a unique expression pattern in which the same gene produces two isoforms with a differential expression pattern in either rods or cones. This phenomenon might be produced by retinal splicing factors that are expressed exclusively in one photoreceptor type.
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