title: A Splice Variant of Bardet-Biedl Syndrome 5 (BBS5) Protein that Is Selectively Expressed in Retina.
abstract: OBJECTIVE
  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) protein. BBS5 is one component of the BBSome, a complex of proteins that regulates the protein composition in cilia. In this study, we identify a smaller molecular mass form of BBS5 as a variant formed by alternative splicing and show that expression of this splice variant is restricted to the retina.
  
  
  METHODS
  Reverse transcription PCR from RNA was used to isolate and identify potential alternative transcripts of Bbs5. A peptide unique to the C-terminus of the BBS5 splice variant was synthesized and used to prepare antibodies that selectively recognized the BBS5 splice variant. These antibodies were used on immunoblots of tissue extracts to determine the extent of expression of the alternative transcript and on tissue slices to determine the localization of expressed protein. Pull-down of fluorescently labeled arrestin1 by immunoprecipitation of the BBS5 splice variant was performed to assess functional interaction between the two proteins.
  
  
  RESULTS
  PCR from mouse retinal cDNA using Bbs5-specific primers amplified a unique cDNA that was shown to be a splice variant of BBS5 resulting from the use of cryptic splicing sites in Intron 7. The resulting transcript 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 and immunoblots of protein extracts from these same tissues showed that this splice variant was expressed in retina, but not brain, heart, kidney, or testes. Quantitative PCR showed that the splice variant transcript is 8.9-fold (+/- 1.1-fold) less abundant than the full-length transcript. In the retina, the splice variant of BBS5 appears to be most abundant in the connecting cilium of photoreceptors, where BBS5 is also localized. Like BBS5, the binding of BBS5L to arrestin1 can be modulated by phosphorylation through protein kinase C.
  
  
  CONCLUSIONS
  In this study we have identified a novel splice variant of BBS5 that appears to be expressed only in the retina. The BBS5 splice variant is expressed at approximately 10% of full-length BBS5 level. No unique functional or localization properties could be identified for the splice variant compared to BBS5.
authors: Department of Ophthalmology, University of Florida, Gainesville, Florida, United States of America.
authors: Susan N
authors: SN
authors: Bolch
authors: Department of Ophthalmology, University of Florida, Gainesville, Florida, United States of America.
authors: Donald R
authors: DR
authors: Dugger
authors: Department of Ophthalmology, University of Florida, Gainesville, Florida, United States of America.
authors: Timothy
authors: T
authors: Chong
authors: Department of Ophthalmology, University of Florida, Gainesville, Florida, United States of America.
authors: J Hugh
authors: JH
authors: McDowell
authors: Department of Ophthalmology, University of Florida, Gainesville, Florida, United States of America.
authors: W Clay
authors: WC
authors: Smith
mesh_terms: D017398:Alternative Splicing; D000595:Amino Acid Sequence; D000818:Animals; D020788:Bardet-Biedl Syndrome / Q000235:genetics*; D001483:Base Sequence; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D002417:Cattle; D002923:Cilia / Q000378:metabolism; D003598:Cytoskeletal Proteins; D018076:DNA, Complementary / Q000378:metabolism; D005786:Gene Expression Regulation; D006801:Humans; D047468:Immunoprecipitation; D051379:Mice; D008810:Mice, Inbred C57BL; D008969:Molecular Sequence Data; D009474:Neurons / Q000378:metabolism; D028044:Phosphate-Binding Proteins; D010766:Phosphorylation; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011506:Proteins / Q000235:genetics / Q000378:metabolism*; D011817:Rabbits; D012160:Retina / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D034421:Sus scrofa; D014018:Tissue Distribution; D014982:Xenopus laevis
chemical_list: C487434:BBS5 protein, human; C561645:BBS5 protein, mouse; D002352:Carrier Proteins; D003598:Cytoskeletal Proteins; D018076:DNA, Complementary; D028044:Phosphate-Binding Proteins; D011506:Proteins