Abstract
Ensembl and other expressed sequence tag (EST) databases reveal putative alternative splice variants in mouse and rat for Pnpla2, the gene encoding pigment epithelium-derived factor-receptor (PEDF-R). The purpose of this study was to obtain experimental evidence for Pnpla2 splice variants in mouse. 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-length Pnpla2 transcript 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-length PNPLA2 cDNA or a PNPLA2 cDNA 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. PCR products for Pnpla2 transcripts obtained from 661W cells or various mouse tissues resolved into a single band following amplification with multiple primer pairs. Simultaneous amplification of two PNPLA2 cDNAs at various molar ratios prevented the detection of lower abundant transcripts. However, even when the cDNA for the full-length Pnpla2 transcript was significantly excluded using the exon exclusion method, no bands corresponding to Pnpla2 splice variants were detectable. Nonetheless, western blots of total 661W cell lysates with two different antibodies revealed isoforms for the PEDF-R protein. The data provide evidence for the existence of a single, full-length Pnpla2 transcript that could give rise to a single protein product that undergoes posttranslational processing.
Highlights
In recent years, a group of genes encoding proteins with a common domain termed patatinlike phospholipase (PNPLA domain) has been discovered in the human genome
We have reported that the retina expresses PNPLA2 and its gene product pigment epithelium-derived factorreceptor (PEDF-R) throughout the retinal pigment epithelium, photoreceptors, and the ganglion cell layer [4,5,6,7]
We found that amplification using any of the three primer pairs in any of the five tissues yielded a distinct single band of Polymerase chain reaction (PCR) product that migrated as DNA of the size expected for Pnpla2 (Figure 3)
Summary
A group of genes encoding proteins with a common domain termed patatinlike phospholipase (PNPLA domain) has been discovered in the human genome. There are nine members of the PNPLA family, all of which display lipase, phospholipase and transacylase enzymatic activities and have major roles in adipocyte differentiation, lipid metabolism and signaling [1,2]. We have reported that the retina expresses PNPLA2 and its gene product PEDF-R throughout the retinal pigment epithelium, photoreceptors, and the ganglion cell layer [4,5,6,7]. PEDF binding stimulates the PLA2 activity of PEDF-R [4,5,8], and in turn its fatty acid products can act as bioactive lipid second messengers to trigger downstream antiapoptotic signaling in retina cells [12]. The PNPLA2 gene plays a crucial role in human embryonic stem cell self-renewal [13], human melanoma metastasis inhibition [14], and human prostate cell growth inhibition [15], all of which depend on PEDF
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