Proc. Natl. Acad. Sci. USA Vol. 91, pp. 105%-10600, October 1994 Cell Biology Purification and properties of double-stranded RNA-specific adenosine deaminase from calf thymus (Inosine/RNA modiflcation/RNA edltlng/RNA-protein interaction) MARY A. O'CONNELL AND WALTER KELLER Department of Cell Biology, Biozentrum, University of Basel, CH4056 Basel, Switzerland Communicated by John Abelson, July 22, 1994 ABSTRACT A double-stranded RNA-specific adenosine dsRNA adenosine deaminase may also be responsible for deaminase, which converts adenosine to inosine, has been editing the mRNA ofglutamate-gated ion-channel proteins in purified to homogeneity from calf thymus. The enzyme was the brain (12). This alteration changes the permeability ofthe purified -340,000-fold by a series of column chromatogra- ion channels to calcium (12). In the case of the GluR-B phy steps. The enzyme consists of a single polypeptide with a ion-channel subunit a glutamine codon (CAG) is converted to molecular mass of 116 kDa as determined by electrophoresis an arginine codon (CGG), and this editing is dependent on the on a SDS/polyacrylamide gel. The native protein sediments formation of a short intramolecular duplex between exonic at 4.2 s in glycerol gradients and has a Stokes radius of 42 A and intronic sequences at the site of editing (13). The con- upon gel-filtration chromatography. This leads to an estimate version of CAG to CIG by dsRNA adenosine deaminase of =74,100 for the native molecular weight, suggesting that would appear as CGG in the cDNA derived from edited the enzyme exists as a monomer in solution. Enzyme activity mRNA. is optimal at 0.1 M KCI and 37°C. Divalent metal ions or ATP We have purified dsRNA adenosine deaminase to homo- is not required for activity. The K. for double-stranded RNA geneity from calf thymus by chromatography over seven substrate is =7 x 10-11 M. The V. is 10-9' mol of inosine columns. Ion-exchange chromatography was the principal produced per min per mg and the Ket is 0.13 min-'. method used, and the final purification was achieved by chromatography on a dsRNA affinity column. Enzyme ac- Double-stranded RNA-specific adenosine deaminase tivity copurifies with a single polypeptide of 116 kDa. (dsRNA adenosine deaminase) was first discovered in ex- tracts from Xenopus laevis (1, 2). The enzyme was originally MATERIALS AND METHODS thought to be an RNA helicase and was called dsRNA unwinding/modifying enzyme. Subsequent studies have Preparation ofdsRNA Substrate. The dsRNA substrate was shown that the enzyme does not actively unwind dsRNAs but prepared by in vitro transcription of both the sense and destabilizes them by converting adenosine (A) to inosine (I), antisense RNA of a shortened form of chloramphenicol which results in inosine'uracil (IMU) base pairing (3, 4). The acetlytransferase (1). The plasmid pSP65 was digested with IFU base is less stable than the Watson-Crick A'U BamHI and EcoRI and the 548-nucleotide insert was sub- pairing cloned into the polylinker of Bluescript KS. The sense RNA base pairing; therefore, the RNA duplex becomes increas- was transcribed with 17 RNA polymerase (Stratagene) after ingly unstable as the enzyme modifies it. The mechanism for linearizing the plasmid with HindIII to give a 605-nucleotide converting A to I is hydrolytic deamination (5). The enzyme transcript. The antisense RNA was transcribed with T3 RNA is ubiquitous in metazoans; it is found in mammalian tissues polymerase (Stratagene) after linearizing the plasmid with as well as in tissue culture cells (6) and in the silkmoth BamHI to give a 594-nucleotide transcript. Either the sense Bombyx mori (7). The other enzymes that can convert A to or antisense transcript was internally labeled with [a-32P]ATP I, adenosine deaminase and 5'-adenylic acid deaminase, (3000 Ci/mmol; 1 Ci = 37 GBq) (Amersham), which was cannot use dsRNA as substrate for the reaction (4). diluted with 0.4 mM unlabeled ATP as described in ref. 1. The The physiological function ofdsRNA adenosine deaminase amount of dsRNA substrate routinely used in an assay was is not known. The enzyme has been implicated in modifying RNA containing 45 fmol of labeled adenosine, with only one a specific single A residue in the transactivation response strand of the duplex being labeled. Therefore, the inosine element ofhuman immunodeficiency virus 1 (HIV-1) RNA at measured came only from the labeled RNA strand, so in position +27, where Tat binds to RNA folded into a stem- theory probably twice the amount of inosine was produced. loop structure (8, 9). It is not certain whether this modifica- Enzyme Assay. The enzyme was assayed by thin-layer tion has any effect on the infection of mammalian cells by chromatography (TLC) of nuclease P1-digested RNA prod- HIV-1. In other instances, the enzyme appears to act non- ucts (3, 4) with minor modifications. The reaction volume specifically in that it will deaminate many A residues present was 25 ,4 and contained 0.15 mg of tRNA per ml, 5 mM in a stretch of duplex RNA sequence. This mechanism has EDTA, and dsRNA, which contained 45 fmpl of labeled been proposed to occur in persistent measles virus infection adenosine and 1-12.5 /4 ofenzyme faction; buffer A [50 mM (10), where 50% of the A residues in the viral matrix protein Tris HCl (pH 7.9)/50 mM KCI/5 mM EDTA/10o (vol/vol) mRNA are replaced by guanosine (G) residues in the viral glycerol/i mM dithiothreitol (DTT)/0.5 mM phenylmethyl- minus-strand RNA after replication (11). The matrix protein sulfonyl fluoride containing 0.7 gg ofpepstatin per ml and 0.4 is necessary for virus assembly and budding from the cell, pg of leupeptin per ml] was used to make up the remaining and, when it is not expressed in the brain, fatal neuropathic volume. If the final salt concentration in the dsRNA adeno- measles infection can result. In this case, the modification of sine deaminase assay would be higher than 150 mM, aliquots the mRNA prevents translation of functional protein (10). Abbreviations: dsRNA, double-stranded RNA; dsRNA adenosine The publication costs ofthis article were defrayed in part by page charge deaminase, dsRNA-specific adenosine deaminase; BSA, bovine payment. This article must therefore be hereby marked "advertisement" serum albumin; NP-40, Nonidet P-40; NEM, N-ethylmaleimide; in accordance with 18 U.S.C. §1734 solely to indicate this fact. DTT, dithiothreitol. 10596 Downloaded by guest on September 25, 2021 Cell Biology: O'Connell and Keller Proc. NatL. Acad. Sci. USA 91 (1994) 10597 of the column fractions were dialyzed before assaying. To active pool to increase protein stability. The active pool was increase the stability of the enzyme 0.2 mg of bovine serum loaded on a 1-ml Mono Q column. The column was washed albumin (BSA) per ml was added to the reaction mixture with 13 ml ofbuffer A after loading and the protein was eluted when assaying the pure column fractions. The assay was with a 30-ml gradient from 50 to 500 mM KC1 in buffer A. The performed at 300C for 1 hr (all values for purification in Table deaminase activity eluted at 150-230 mM KCl. The final 1 were determined at 300C). Pure protein was assayed at column was an affinity column ofpoly(G).poly(C) (see below 370C, as RNases present in the cruder fractions were more for details of preparation). The active pool was diluted to 50 active at the higher temperature. After incubation, 8.3 1ul of mM KCl and loaded on a 7-ml poly(G)-poly(C) column, 7.5 M ammonium acetate and 300 bd of ethanol were added washed with 2 column vol of buffer A at 1.5 column vol per and the samples were centrifuged for 30 min at 40C; the pellets hr, and then developed with a 63-ml gradient from 50 to 1000 were washed with 70%o ethanol and vacuum dried. The pellets mM KCl. The dsRNA adenosine deaminase eluted at =600 were resuspended in 10 p4 of P1 buffer (30 mM KOAc, pH mM KC1. 5.3/10 mM ZnSO4) and digested with 1.5 ug of nuclease P1 Protein concentration was determined by the Bradford (Boehringer Mannheim) for 1 hr at 500C. Unlabeled 5' inosine method (14) with BSA as the reference standard, except for monophosphate (pI) (Sigma) was added to the reaction mix- the pure protein, which was precipitated with trichloroacetic ture as an internal standard, and the digestion products were acid (final concentration, 15%), and electrophoresed on an separated on a cellulose NM 300 TLC plate (Macherey & SDS/8% polyacrylamide gel with different amounts of BSA Nagel) and dried. The chromatographic solvent was satu- as reference standard. rated (NH4)2SO4/100 mM NaOAc, pH 6/isopropanol dsRNA Affinity Column. One milligram of polycytidylic (79:19:2). The TLC plates were autoradiographed overnight acid [poly(C)] (Pharmacia) was annealed to 1 mg of short and then the spots corresponding to 5 pI and 5' adenosine polyguanylic acid [poly(G)] (Boehringer Mannheim) by heat- monophosphate (pA) were cut out and assayed in a scintil- ing to 85°C followed by slow cooling to 37°C. The lation counter. One unit of enzyme is defined as the amount poly(G)-poly(C) was coupled to 2 g of cyanogen bromide- required to produce 1 fmol of inosine in 1 min. Since only one activated Sepharose 4B (Pharmacia) in 0.1 M NaHCO3, pH strand of the dsRNA is labeled, the units indicate only the 5' 8.3/0.5 M NaCl for 2 hr at room temperature.