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Susan Bolch, Donald Dugger, Timothy Chong, J. Hugh McDowell, W Clay Smith; A Splice Variant of Bardet-Biedl Syndrome 5 (BBS5) Protein that Is Selectively Expressed in Retina. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):4627.
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© ARVO (1962-2015); The Authors (2016-present)
Bardet-Biedl syndrome is a complex ciliopathy that usually manifests with some form of retinal degeneration, amongst other ciliary-related deficiencies. One of the genetic causes of this syndrome results from a defect in Bardet-Biedl Syndrome 5 (BBS5). BBS5 protein is one component of the BBSome, a complex of proteins that regulates protein access to the cilium. In this study, we identify a novel splice variant of BBS5 and show that expression of this splice variant is restricted to the retina.
RNA was isolated from mouse retinas, reverse transcribed, and used as the template for PCR amplification of portions of the BBS5 cDNA. PCR products were sequenced to identify potential splice variants. Quantitative reverse-transcription PCR was performed with primers specific to the splice variant and to the full-length BBS5 to determine relative expression levels of the splice variant. Reverse-transcription PCR was performed on RNA isolated from various mouse tissues to determine tissue expression specificity. Peptides unique to the BBS5 splice variant were synthesized and used to prepare antibodies that selectively recognized the BBS5 splice variant. Immunoblots of tissue protein extracts were performed to validate the expression pattern.
PCR from mouse retinal cDNA using BBS5-specific primers amplified a unique cDNA that upon sequencing was shown to be a splice variant of BBS5 that resulted from the use of an occult splicing site in Intron 7. The resulting message codes for a truncated form of the BBS5 protein with a unique 24 amino acid C-terminus, and predicted 26.5 kD molecular mass. PCR screening of RNA isolated from various ciliated tissues showed that this splice variant was expressed in retina, but not brain, heart, kidney, or testes. This expression pattern was confirmed on immunoblots of protein extracts from these tissues using a monoclonal antibody specific to the unique C-terminus of the splice variant. Quantitative PCR showed that the splice variant transcript is 8.9-fold less abundant than the full-length transcript.
We have identified a novel splice variant of BBS5 that appears to be expressed only in the retina. Expression levels at ~10% of full-length BBS5 are sufficiently high to suggest a unique function of this splice variant. We are currently exploring the cellular expression pattern to develop information about its potential function.
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