Alternative splicing generates novel Fads3 transcript in mice

Abstract

Fads3 is the third member of the fatty acid desaturase gene cluster; with at least eight evolutionarily conserved alternative transcripts (AT), having no clearly established function as are known for FADS2 and FADS1. Here we present identification of a novel Fads3 transcript in mice (Fads3AT9), characterize Fads3AT9 expression in mouse tissues and evaluate correlations with metabolite profiles. Total RNA obtained from mouse tissues is reverse-transcribed into cDNA and used as template for PCR reactions. Tissue fatty acids were extracted and quantified by gas chromatography. Sequencing analysis revealed complete absence of exon 2 resulting in an open reading frame of 1239 bp, encoding a putative protein of 412 aa with loss of 37 aa compared to classical Fads3 (Fads3CS). FADS3AT9 retains all the conserved regions characteristic of front end desaturase (cytochrome b5 domain and three histidine repeats). Both Fads3CS and Fads3AT9 are ubiquitously expressed in 11 mouse tissues. Fads3AT9 abundance was greater than Fads3CS in pancreas, liver, spleen, brown adipose tissue and thymus. Fads3CS expression is low in pancreas while Fads3AT9 is over ten-fold greater abundance. The eicosanoid precursor fatty acid 20:4n - 6, the immediate desaturation product of the Fads1 coded Δ5-desaturase, was highest in pancreas where Fads3CS is low. Changes in expression patterns and fatty acid profiles suggest that Fads3AT9 may play a role in the regulation and/or biosynthesis of long chain polyunsaturated fatty acids from precursors.

Keywords: Alternative splicing; Alternative transcript; Fatty acid desaturase; Long chain polyunsaturated fatty acids.

Fig. 1

Amplicons of Fads3CS and Fads3AT9 and cartoon image of Fads3CS and Fads3AT9 gene structure Top: Fads3CS–Classical splicing, Fads3AT9–alternative transcript. The products were separated on 2 % agarose gel and visualized under UV using ethidium bromide. Lane 1: 100 base pair molecular weight marker and Lane 2: PCR products amplified by RT-PCR. Bottom: Fads3CS–Classical splicing, Fads3AT9 with missing exon 2 is shown. Numbers 1–12 are exons

Fig. 2

Determination of mRNA expression of Fads3AT9 in eleven tissues using Image J software. Relative mRNA levels of Fads3CS and Fads3AT9 were normalized to reference gene 18S. mRNA expression of Fads3CS was regarded as “1” (dash line) and the change between CS and AT9 was measured by ratio of AT9/CS. Pancreases, liver, spleen, brown adipose tissue and thymus have higher levels of Fads3AT9 compared to Fads3CS. Data is expressed as mean ± SD (n = 4) and asterisk represents P < 0.05