Molecular characterization of coding and untranslated regions of rat cortex lithium-sensitive myo-inositol monophosphatase cDNA.
Lithium sensitive myo-inositol monophosphatase (IMPase) is a pivotal enzyme which controls the levels of brain inositol within the inositol-based signaling system. Its capacity to release free myo-inositol from inositol monophosphates generated from receptor-linked and de novo pathways is crucial to the maintenance of appropriate amounts of intracellular myo-inositol, which is essential for both inositol-based cell signaling and cell volume control. We present here the full length cDNA encompassing the coding and untranslated regions (5'- and 3'-UTRs) of rat brain IMPase. This cDNA was derived from rat cortex mRNA by the RT-PCR technique. Analysis of this cDNA revealed several interesting features which include a short 5'-untranslated region (5'-UTR) of 68 nucleotides followed by coding region of approximately 0.8 kb and a long 3'-untranslated region (3'-UTR) of 1.2 kb. Both 5'-rapid amplification of cDNA ends (5'-RACE) and 3'-RACE techniques were carried out to isolate both UTRs and double stranded sequencing was carried out to its entirety (approximately 2.1 kb) by 'gene walking' using several oligonucleotide primers. All nucleotides were sequenced unambiguously using the sense and antisense strands of DNA. PCR analysis for the coding region and the deduced amino acid sequence demonstrated a DNA fragment of 831 bp and 277 amino acids, respectively, which are strikingly similar to human hippocampal IMPase. The 5'-UTR demonstrated distinct CpG doublets, characteristic of 'housekeeping' genes. The sequence around the initiator methionine, AAGATGG, conforms well to the Kozak consensus sequence for mammalian protein biosynthesis and the 3'-UTR demonstrated three canonical (AATAAT, AATTAA, AATACA) and one unusual polyadenylation signals (ATTAAA) followed by a 31 base poly(A) tail. The presence of a CCTGTG in the 3'-UTR (putative carbohydrate response element) links IMPase mRNA to brain carbohydrate metabolic pathways. Computer analyses demonstrated several unique features of this mRNA, including the potential formation of hairpin loops which might be important in its intracellular regulation and turn-over. In summary, this lithium-sensitive brain IMPase mRNA has the following characteristics: a 5'-CpG-rich short untranslated segment, a highly conserved coding region, and a long 3'-untranslated region with several polyadenylation signals.
Parthasarathy
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Molecular Neuroscience Laboratory, Mental Health and Behavioral Science Service, Department of Veterans Affairs Medical Center, Louisville, KY 40206, USA.
Parthasarathy
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Vadnal
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D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002540:Cerebral Cortex / Q000378:metabolism*; D018076:DNA, Complementary; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005091:Exons; D006801:Humans; D007438:Introns; D008094:Lithium / Q000494:pharmacology; D008969:Molecular Sequence Data; D010744:Phosphoric Monoester Hydrolases / Q000187:drug effects / Q000235:genetics*; D011061:Poly A; D051381:Rats; D017207:Rats, Sprague-Dawley; D017386:Sequence Homology, Amino Acid
D018076:DNA, Complementary; D004791:Enzyme Inhibitors; D011061:Poly A; D008094:Lithium; D010744:Phosphoric Monoester Hydrolases; C021822:myo-inositol-1 (or 4)-monophosphatase