Searching for Alternatively Spliced Variants of Phospholipase Domain-Containing 2 (Pnpla2), a Novel Gene in the Retina.
OBJECTIVE
Ensembl and other expressed sequence tag (EST) databases reveal putative alternative splice variants in mouse and rat forPnpla2, the gene encoding pigment epithelium-derived factor-receptor (PEDF-R). The purpose of this study was to obtain experimental evidence forPnpla2splice variants in mouse.


METHODS
Cultures of a mouse cell line derived from photoreceptors (661W cells) and mouse eye, heart, adipose, kidney, and liver tissues were used. Messenger RNA (mRNA) was isolated from cells and tissues, and complementary DNA (cDNA) was synthesized. Polymerase chain reaction (PCR) primer pairs were designed to flank the putative splice sites. Exon exclusion real time PCR was used to reduce amplification of the full-lengthPnpla2transcript and enhance amplification of low abundant splice variants. PCR products were resolved by agarose gel electrophoresis and detected with a UV transilluminator. Recombinant plasmids containing a human full-lengthPNPLA2cDNA or aPNPLA2cDNA lacking exon 5b (E5b) were controls to validate the techniques. Total cell lysates from 661W cells were prepared. PEDF-R protein detection was performed using western blots.


RESULTS
PCR products forPnpla2transcripts obtained from 661W cells or various mouse tissues resolved into a single band following amplification with multiple primer pairs. Simultaneous amplification of twoPNPLA2cDNAs at various molar ratios prevented the detection of lower abundant transcripts. However, even when the cDNA for the full-lengthPnpla2transcript was significantly excluded using the exon exclusion method, no bands corresponding toPnpla2splice variants were detectable. Nonetheless, western blots of total 661W cell lysates with two different antibodies revealed isoforms for the PEDF-R protein.


CONCLUSIONS
The data provide evidence for the existence of a single, full-lengthPnpla2transcript that could give rise to a single protein product that undergoes posttranslational processing.
Section of Protein Structure and Function, Laboratory of Retinal Cell and Molecular Biology, National Eye Institute, National Institutes of Health, Bethesda, MD 20892-0608, USA.
Jacqueline Talea
JT
Desjardin
Section of Protein Structure and Function, Laboratory of Retinal Cell and Molecular Biology, National Eye Institute, National Institutes of Health, Bethesda, MD 20892-0608, USA.
S Patricia
SP
Becerra
Section of Protein Structure and Function, Laboratory of Retinal Cell and Molecular Biology, National Eye Institute, National Institutes of Health, Bethesda, MD 20892-0608, USA.
Preeti
P
Subramanian
Alternative splicing; PEDF; PEDF-R; Pnpla2; Retina