Bolch Susan N, Dugger Donald R, Chong Timothy, McDowell J Hugh, Smith W Clay
Department of Ophthalmology, University of Florida, Gainesville, Florida, United States of America.
PLoS One. 2016 Feb 11;11(2):e0148773. doi: 10.1371/journal.pone.0148773. eCollection 2016.
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.
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.
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.
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.
巴德-比德尔综合征是一种复杂的纤毛病，通常表现为某种形式的视网膜变性以及其他与纤毛相关的缺陷。该综合征的一个遗传原因是巴德-比德尔综合征5（BBS5）蛋白存在缺陷。BBS5是BBSome的一个组成部分，BBSome是一种调节纤毛中蛋白质组成的蛋白质复合物。在本研究中，我们鉴定出一种分子量较小的BBS5形式，它是由可变剪接形成的变体，并表明这种剪接变体的表达仅限于视网膜。
利用RNA进行逆转录PCR，以分离和鉴定Bbs5的潜在可变转录本。合成了一种BBS5剪接变体C末端特有的肽，并用于制备能选择性识别BBS5剪接变体的抗体。这些抗体用于组织提取物的免疫印迹，以确定可变转录本的表达程度，并用于组织切片，以确定表达蛋白的定位。通过对BBS5剪接变体进行免疫沉淀来下拉荧光标记的抑制蛋白1，以评估这两种蛋白之间的功能相互作用。
使用Bbs5特异性引物对小鼠视网膜cDNA进行PCR扩增，得到一个独特的cDNA，它被证明是BBS5的剪接变体，是由于内含子7中隐蔽剪接位点的使用而产生的。所得转录本编码一种截短形式的BBS5蛋白，其C末端有一个独特的24个氨基酸，预测分子量为26.5 kD。对从各种纤毛组织分离的RNA进行PCR筛选，以及对这些相同组织的蛋白质提取物进行免疫印迹分析，结果表明这种剪接变体在视网膜中表达，但在脑、心脏、肾脏或睾丸中不表达。定量PCR显示，剪接变体转录本的丰度比全长转录本低8.9倍（±1.1倍）。在视网膜中，BBS5的剪接变体似乎在光感受器的连接纤毛中最为丰富，BBS5也定位于此处。与BBS5一样，BBS5L与抑制蛋白1的结合可通过蛋白激酶C的磷酸化作用进行调节。
在本研究中，我们鉴定出一种新的BBS5剪接变体，它似乎仅在视网膜中表达。BBS5剪接变体的表达水平约为全长BBS5水平的10%。与BBS5相比，未发现该剪接变体有独特的功能或定位特性。
