Identification of myo-Inositol-3-phosphate Synthase Isoforms

Abstract

Myo-inositol is an important constituent of membrane phospholipids and is a precursor for the phosphoinositide signaling pathway. It is synthesized from glucose 6-phosphate by myo-inositol-3-phosphate synthase (IP synthase), a homotrimer composed of a 68-kDa polypeptide in most mammalian tissues. It is a putative target for mood-stabilizing drugs such as lithium and valproate. Here, we show that the rat gene (Isyna1) encoding this enzyme generates a number of alternatively spliced transcripts in addition to the fully spliced form that encodes the 68-kDa subunit (the alpha isoform). Specifically, we identify a small 16-kDa subunit (the gamma(c) isoform) derived by an intron retention mechanism and provide evidence for its existence in rat tissues. The gamma(c) isoform is highly conserved in mammals, but it lacks the catalytic domain while retaining the NAD(+) binding domain. Both alpha and gamma(c) isoforms are predominantly expressed in many rat tissues and display apparent stoichiometry in purified enzyme preparations. An IP synthase polyclonal antibody not only detects the alpha and gamma(c) isoforms but also several other isoforms in pancreas, intestine, and testis suggesting that the holoenzyme is composed of unique subunits in various tissues. Interestingly, the alpha isoform is not expressed in the intestine. IP synthase activity assays using purified alpha and gamma(c) isoforms indicate that the latter negatively modulates alpha isoform activity, possibly by competing for NAD(+) molecules. Our findings have important ramifications for understanding the mood stabilization process and suggest that inositol biosynthesis is a highly regulated and dynamic process.

Differential methylation of the gene encoding myo -inositol 3-phosphate synthase ( Isyna1 ) in rat tissues

“…Conversely, in testis and pancreas, the digested bands were comparatively more intense than the 3.0-kb band suggesting that the internal Sma I sites are poorly methylated. The observation with the testis is consistent with this tissue expressing high levels of IP synthase [2]. In the heart, all three bands appeared equally pronounced with the 3.0-kb band slightly more intense than the two digested bands.…”

“…We have previously demonstrated that ISYNA1 is upregulated by E2F1 [20], an observation that has been confirmed in yeast [21]. More recently, we demonstrated that Isyna1 generates a number of alternatively spliced isoforms, one of which was shown to negatively modulate enzyme activity [2]. Studies presented here clearly implicate epigenetic mechanisms (DNA methylation) in the tissue-specific expression of Isyna1 .…”

“…While the total number of significantly methylated (≥30%) sites in region 3 of the Isyna1 gene was highest in DNA from spleen (four CpG sites), followed by brain cortex (one CpG site), no significant methylated residue in region 3 of the gene was observed in DNA from the remaining tissues. The CpG methylation profiles in region 3 of the gene in DNA from spleen, testis and brain cortex appear to be inversely correlated to Isyna1 mRNA expression levels in both rat and human tissues [2,29], which suggests that methylation in this region of the gene may dictate tissue-specific expression. Northern blot analysis of Isyna1 mRNA indicates that expression is highest in testis [2], moderate in brain, and poor in spleen, a pattern also seen in humans [29], which correlates extremely well with increasing MMI values of 1.0, 5.5 and 18.2, respectively, for the gene in these tissues.…”

“…In rats, these subunits arise by translation of a full-length mRNA, spliced from 11 exons, encoded by the Isyna1 gene ( ISYNA1 in human). Recently, we and others have identified and characterized a plethora of shorter isoforms [2,3], arising by alternative splicing and intron retention mechanisms. The identification of novel isoforms in different tissues, in addition to the full-length isoform (now termed the α-isoform), suggests that the regulation of IP synthase gene expression is complex.…”

Direct Ionic Regulation of the Activity of Myo-Inositol Biosynthesis Enzymes in Mozambique Tilapia

“…For example, in rat, at least eight MIPS variants have been detected either at the mRNA or protein level [23]. In humans, at least four MIPS variants have been observed at mRNA level [43].…”