Oligoribonuclease is a common downstream target of lithium-induced pAp accumulation in Escherichia coli and human cells

Abstract

We identified Oligoribonuclease (Orn), an essential Escherichia coli protein and the only exonuclease degrading small ribonucleotides (5mer to 2mer) and its human homologue, small fragment nuclease (Sfn), in a screen for proteins that are potentially regulated by 3'-phosphoadenosine 5'-phosphate (pAp). We show that both enzymes are sensitive to micromolar amounts of pAp in vitro. We also demonstrate that Orn can degrade short DNA oligos in addition to its activity on RNA oligos, similar to what was documented for Sfn. pAp was shown to accumulate as a result of inhibition of the pAp-degrading enzyme by lithium, widely used to treat bipolar disorder, thus its regulatory targets are of significant medical interest. CysQ, the E.coli pAp-phosphatase is strongly inhibited by lithium and calcium in vitro and is a main target of lithium toxicity in vivo. Our findings point to remarkable conservation of the connection between sulfur- and RNA metabolism between E.coli and humans.

Figure 1

pAp-binding proteins from E.coli. Shown is colloidal coomassie stained SDS–PAA gel separating the pAp-binding fraction of E.coli extract. M, marker; lane 1, fraction binding to blocked agarose beads (control); lane 2, pAp-binding fraction.

Figure 2

Inhibition of recombinant CysQ by Lithium (1 mM) and Calcium (2 mM). Reactions contained 5.5 mM pAp and 0.5 µg of CysQ. Closed circles, no inhibitor; gray circles, LiCl; open squares, CaCl₂.

Figure 3

Lithium induced growth inhibition in E.coli MG1655 containing pBAD18 (vector control) or pUM404 (arabinose-inducible cysQ). Strains were grown on MOPS-minimal plates containing all amino acids but cysteine and methionine, 0.4% glycerol and 0.0002% arabinose.

Figure 4

Orn is sensitive to micromolar amounts of pAp. Shown is the quantification of the distribution of reaction products that were separated on 22% PAA gels. Reactions included 0.07 µg Orn and 3 µM of RNA oligo 5mer (5′Cy5-CCCCC3′). Reactions were started by the addition of substrate. pAp (20 µM) was present where indicated. Closed circle, 5mer; open circle, 4mer; closed triangle, 3mer; open triangle, 2mer; square, 1mer.

Figure 5

pAp-binding proteins from humans. Shown is colloidal coomassie stained SDS–PAA gel separating the pAp-binding fraction of HeLa cytoplasmic extract. M, marker; lane 1, control; lane 2, pAp-binding fraction.

Figure 6

Sensitivity of Sfn to pAp. Shown is the quantification of the distribution of reaction products that were separated on 22% PAA gels. Reactions included 0.5 µg Sfn and and 3 µM of RNA oligo 5mer (5′Cy5-CCCCC3′). Reactions were started by the addition of enzyme. pAp (20 or 50 µM) was present where indicated. Closed circle, 5mer; open circle, 4mer; closed triangle, 3mer; open triangle, 2mer; square, 1mer.

Figure 7

Orn is active on DNA oligos. Reactions contained 3 µM of DNA oligo 5mer (5′Cy5-CCCCC3′) and were started by the addition of 1 µg of Orn. Dash labels a control lacking enzyme incubated for 20 min. Samples were taken as indicated. The DNA monomer cannot enter the gel. The different gel mobility as compared with RNA is caused by the one missing charge of DNA. Varying the pH of the electrophoresis buffer could not change this effect.