Abstract
Turnover of mRNA releases, in addition to the four regular nucleoside monophosphates, the methylated cap nucleotide in the form of 7-methylguanosine monophosphate (m(7)GMP) or diphosphate (m(7)GDP). The existence of pathways to eliminate the modified nucleotide seems likely, as its incorporation into nucleic acids is undesirable. Here we describe a novel 5' nucleotidase from Drosophila that cleaves m(7)GMP to 7-methylguanosine and inorganic phosphate. The enzyme, encoded by the predicted gene CG3362, also efficiently dephosphorylates CMP, although with lower apparent affinity; UMP and the purine nucleotides are poor substrates. The enzyme is inhibited by elevated concentrations of AMP and also cleaves m(7)GDP to the nucleoside and two inorganic phosphates, albeit less efficiently. CG3362 has equivalent sequence similarity to two human enzymes, cytosolic nucleotidase III (cNIII) and the previously uncharacterized cytosolic nucleotidase III-like (cNIII-like). We show that cNIII-like also displays 5' nucleotidase activity with a high affinity for m(7)GMP. CMP is a slightly better substrate but again with a higher K(m). The activity of cNIII-like is stimulated by phosphate. In contrast to cNIII-like, cNIII and human cytosolic nucleotidase II do not accept m(7)GMP as a substrate. We suggest that the m(7)G-specific nucleotidases protect cells against undesired salvage of m(7)GMP and its incorporation into nucleic acids.
Highlights
MRNA decay releases, in addition to the regular nucleotides, 7-methyl GMP derived from the 5Ј cap
The decapping enzyme Dcp1/Dcp2 is active in mRNA decay in Drosophila embryos at 2– 4 h of development (46), a time window bordering on the one from which our extracts were derived, but Dcp2 activity was not apparent in the extract
As there is no known reaction by which m7G nucleotides released during mRNA decay could be recycled into new caps, these nucleotides are likely to be disposed of
Summary
Enzymes, and Other Reagents—Extract was prepared from 0.5–2.5-h-old embryos of wild type Drosophila melanogaster as described (35, 36). An ATP regenerating system (80 g/ml creatine kinase (rabbit muscle; Roche Applied Science), 30 mM creatine phosphate) or an ATP depleting system (20 mM glucose, 0.1 units/l of hexokinase; Sigma) was added In these cases the extract was preincubated for 10 min under ATP depleting or regenerating conditions, and the ATP status was checked by TLC analysis of a control reaction containing a trace of [␣-32P]ATP. When unlabeled nucleotides were used as substrates, a colorimetric assay for orthophosphate was used as described (40) except that Tween 20 was left out In this case the reaction (20 l per time point) was stopped by the addition of the malachite green oxalate/ammonium molybdate reagent to the reaction mixture. Each experiment was carried out at least twice with R values
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