Table 1.
Oligonucleotide primers used for cDNA synthesis and PCR.
Table 2.
Alternative transcripts for murine Bbs5 predicted in the Ensembl and MGI databases, and the oligonucleotide primers designed to identify these potential transcripts.
Fig 1.
A smaller molecular mass form of BBS5 is detected in retinal extracts from a variety of vertebrates.
(A) Immunoblots of extracts of aqueous soluble proteins prepared from whole retinas from the indicated species probed with anti-BBS5 #7–15 monoclonal antibody described in [19]. Arrow indicates the expected molecular size of BBS5; arrowhead indicates a smaller immunoreactive band at 26 kD. (B) A replicate blot was probed with the same antibody that was pre-incubated with heterologously-expressed recombinant murine BBS5. Inset, at the bottom of the blot shows a replicate blot stained for beta-tubulin as a loading control (beta-tubulin indicated with an open arrow).
Fig 2.
The mouse Bbs5 gene and alternative transcripts.
(A) Structure of the murine Bbs5 gene; locations for sense (S) and anti-sense (A) primers used to test for alternative transcripts are indicated above the gene structure, along with oligo(dT) and 5’RACE primers (5’R). (B) PCR amplification of reverse-transcribed murine retinal poly(A)+ RNA with S2 and A5 primers separated by agarose electrophoresis; arrowhead indicates a DNA product larger than the expected 216 bp product. (C) DNA sequence of intron 7 of Bbs5 with the alternatively spliced region indicated in red (potential splice donor/acceptor sites are bold faced). (D) Sequence chromatogram of the alternative Bbs5 transcript (BBS5L) with the beginning of the cryptic exon 7a (arrow) and stop codon (asterisk) indicated. (E) Gene structure of exons 7, 7a, and 8 showing the two splicing patterns detected for the Bbs5 transcript (the location of the stop codon in Exon 7a is indicated with an asterisk).
Fig 3.
Reverse transcription-PCR amplification of Bbs5 and Bbs5L from various murine tissues.
(A) cDNA prepared from poly(A)+ RNA isolated from the five indicated tissues was amplified with primers specific for Bbs5 (upper panel; arrowhead indicates expected 215 bp product), Bbs5L (middle panel; arrow indicates expected 126 bp product), or alpha-tubulin for quality control (lower panel). (B) Quantitative RT-PCR was performed for Bbs5 and Bbs5L using cDNA prepared from retinal poly(A)+ RNA and quantified relative to levels of β-actin cDNA (n = 3; error bars indicate SEM; ρ<0.05).
Fig 4.
RT-PCR of predicted Bbs5 transcripts from retinal cDNA.
The alternative transcripts for mouse Bbs5 predicted in the Ensembl (Bbs5-002 through -008) and MGI (Bbs5-X1) were amplified with primers that would selectively identify the alternative transcript. The predicted sizes (in bp) for each transcript are indicated below each lane for the full-length transcript and the alternative splice variant transcript [two alternative transcripts are possible in lane 1, corresponding to the deletion of Exon 8 (152 bp) or the inclusion of Exon 7A (379 bp)]. Lower panel shows amplification reactions with primers against alpha-tubulin to control for cDNA quality.
Fig 5.
Characterization of a polyclonal antibody specific for BBS5L.
(A) Conceptual translation of the alternative Bbs5 transcript (Bbs5L) in comparison with Bbs5 (the unique C-terminus is colored in red). (B) Immunoblots of murine retinal extracts and heterologously expressed BBS5L (rBBS5L) and BBS5 (rBBS5) were probed with either anti-BBS5#7–15 monoclonal antibody (left half) or with anti-BBS5L polyclonal serum (right half); arrowhead indicates BBS5 or rBBS5, arrow indicates BBS5L or rBBS5L. (C) Immunoblot of aqueous-soluble extracts prepared from the indicated mouse tissue probed with anti-BBS5L polyclonal serum identifies anti-BBS5L immunoreactivity only in the retinal extract (upper panel; arrow); an antibody that recognizes both the BBS5 and BBS5L reacts with BBS5 in all extracts, and BBS5L only in retinal extract (middle panel); inset below the blot shows a replicate blot of the tissue extracts probed with anti-beta-tubulin as a loading control.
Fig 6.
BBS5L localizes to the axonemal structure of the photoreceptors.
(A) Lower magnification view of indirect immunofluorescence of antibodies against BBS5L (green), BBS5 (red), RP1 (white), and DAPI (blue) on cryofixed mouse retinal tissue (see methods for specific details on antibodies). (B-E) Enlargement of region indicated in (A) showing localization of BBS5L (B) and BBS5 (C). BBS5L and BBS5 reactivity are highly overlapping as seen in the merge (D). Staining with the axonemal marker RP1 (E) shows that BBS5L co-localizes with RP1 in the distal axoneme (F), but also stains the proximal transition zone of the axoneme where RP1 is absent (white arrowheads). G-L shows parallel staining with pre-absorption controls, using anti-BBS5L antiserum preabsorbed against BBS5L protein (H) or anti-BBS5 antibody preabsorbed against BBS5 protein (I). OS, outer segments, IS, inner segments, ONL, outer nuclear layer; scale bars are 10 μm (A) and 2.5 μm (B-F).
Fig 7.
Binding of arrestin1 by BBS5L relative to BBS5, comparing unphosphorylated and phosphorylated conditions.
ARR1-Alexa546 was co-immunoprecipitated with BBS5/GST or BBS5L/GST fusion protein with anti-GST antibody and precipitated arrestin1 measured fluorimetrically. ARR1-Alexa546 was pulled down by both BBS5L and BBS5, although the precipitation by BBS5L/GST was significantly less than by BBS5 (n = 6; ρ<0.05). Phosphorylation significantly reduced the pulldown of arrestin1 by both BBS5 and BBS5L (n = 6; ρ<0.05); error bars indicate SEM. Inset shows an immunoblot of the BBS5 and BBS5L proteins probed with anti-phosphoserine antibody after phosphorylation with (+) or without (-) PKC; arrowheads indicate the expected molecular mass for BBS5L-GST (open arrowhead) and BBS5-GST (black arrowhead).