Illustration of the Rpgr and whirlin/DFNB31 gene structures, and analysis of whirlin expression in the mouse retina at the RNA level. (
A) Schematic representation of the
Rpgr gene structure. Alternative splicing leads to two groups
of Rpgr transcripts;
Rpgr ex1–19 includes exons 1–13 and exons 16–19, whereas
Rpgr ORF15 includes exons 1–13 plus a large, alternatively spliced ORF 14–15.
Orange: exons encoding an RCC1-like domain common to all Rpgr isoforms.
Green: remainder of exons common to all Rpgr isoforms.
Blue/purple: exon (ORF 14/15) unique to Rpgr
ORF15.
Purple: alternatively spliced region of ORF14/15 encoding glutamic acid-rich domain. (
B) Illustration of the Rpgr
ORF15 isoform. All colors correspond to their respective exons shown in
A.
Brackets indicate the location of domain used as bait in the yeast two-hybrid screen. (
C) Schematic representation of the
whirlin/DFNB31 gene structure. Whirlin is composed of 13 exons encoding three PDZ domains and a proline-rich region. Exons and encoded domains are drawn approximately to scale. PCR primers used for amplification of whirlin transcripts are shown as
red arrows. (
D) Amplification of whirlin N-terminal transcripts from C57BL/6 retinal cDNA.
Left/center: whirlin mRNA transcripts were reverse transcribed and amplified using primers shown in
Figure 2A. The whirlinNT1 transcript, which includes intron 3, was amplified by WiP1 and WiP14R, and the whirlinNT2 transcript, which includes intron 7, was amplified by WiP1 and Wi_intron7_P1R.
Right: whirlin N-terminal transcripts were also amplified by nested PCR of 3′RACE retinal cDNA. Transcripts were first amplified using WiP1 and GeneRacer 3′ Primer followed by WiP2 and GeneRacer 3′ Nested Primer. The regions excised and used to clone and sequence the whirlinNT1 and whirlinNT2 transcripts are indicated by the
red brackets.