k.) 1991 Oxford University Press

Nucleic Acids Research, Vol. 19, No. 6 1197

Site-specific creation of uridine from cytidine in apolipoprotein B mRNA editing Peter E.Hodges+, Naveenan Navaratnam, Jobst C.Greeve and James Scott* Division of Molecular Medicine, MRC Clinical Research Centre, Watford Road, Harrow, Middlesex HAl 3UJ, UK Received January 22, 1991; Accepted February 21, 1991

ABSTRACT Human apolipoprotein (apo) B mRNA is edited in a tissue specific reaction, to convert glutamine codon 2153 (CAA) to a stop translation codon. The RNA editing product templates and hybridises as uridine, but the chemical nature of this reaction and the physical identity of the product are unknown. After editing in vitro of [32p] labelled RNA, we are able to demonstrate the production of uridine from cytidine; [a32p] cytidine triphosphate incorporated into RNA gave rise to [32p] uridine monophosphate after editing in vitro, hydrolysis with nuclease P1 and thin layer chromatography using two separation systems. By cleaving the RNA into ribonuclease Ti fragments, we show that uridine is produced only at the authentic editing site and is produced in quantities that parallel an independent primer extension assay for editing. We conclude that apo B mRNA editing specifically creates a uridine from a cytidine. These observations are inconsistent with the incorporation of a uridine nucleotide by any polymerase, which would replace the a-phosphate and so rule out a model of endonucleolytic excision and repair as the mechanism for the production of uridine. Although transamination and transglycosylation remain to be formally excluded as reaction mechanisms our results argue strongly in favour of the apo B mRNA editing enzyme as a site-specific cytidine deaminase. INTRODUCTION The sequence of mRNA can be modified posttranscriptionally in a process described as RNA editing. Included in this process are several observed RNA modifications that bear little mechanistic similarity. The best characterised example occurs in the mitochondria of kinetoplastid protozoa (Leishmania, Trypanosoma and Crithidia) (1-4). Reiterative cycles of specific endonucleolytic cleavage, polymerisation of additional uridine residues and religation change the mRNAs to optimise base pairing of discrete target segments to guide RNA sequences. Thereby the edited RNAs are rendered translatable by the correction of internal reading frameshifts and the creation of AUG *

initiation codons. Recognition of RNA duplexes in vertebrate cells leads to extensive adenosyl residue deamination which weakens base pairing (5). Interconversion of cytidine and uridine residues has been observed in both plant mitochondrial mRNA (6-8) and at a specific site in the mRNA encoding apolipoprotein (apo) B (9, 10). The apo B editing reaction creates a uridine-like base from the genomically-encoded cytidine. This nucleotide templates the incorporation of adenosine by reverse transcriptase and produces a codon that terminates translation. The simplest possible mechanism of RNA modification would be the creation of a uridine by hydrolytic deamination of the specific cytosine at position 4, as has been proposed by Driscoll et al and Bostrom et al (11, 12). A similar site-specific cytidine deaminase has been proposed to explain the multiple C to U transitions in plant mitochondrial mRNA editing where the reaction may be reversible to give cytidine from uridine (6-8, 13, 14). However, the exact mechanism (and the specific product) of these editing reactions is not known. From the study of tRNA modifications, it is apparent that the base-pairing ability of nucleotides can be changed by simple or complex modifications. The base pairing potential of a pyrimidine can be changed simply by tautomerisation, inverting the proton-donating or accepting potential of the base's substituents. For example attachment of a lysine amino acid at the 2 position of cytosine creates lysidine, which is read as uridine, and changes the base pairing of the anticodon of a minor isoleucine tRNA (15, 16). Similarly, proline tRNA from a number of species contains 5-carbamoylmethyl uridine apparently derived from cytidine, which must be both deaminated and hypermodified (17, 18). Selenocysteine tRNA from vertebrates undergoes two different patterns of C to U and U to C conversions (19, 20). The RNA products have been chemically identified by tRNA sequencing, and are indistinguishable from natural uridine or cytidine. Therefore an enzyme must exist in vertebrate cells which can interconvert cytidine and uridine in RNA in a site-specific, post-tanscriptional reaction. We have established a system in vitro for the editing of apo B RNA and have identified cytosolic S100 extracts from rat enterocytes as a potent source of the editing extract (12 and

To whom correspondence should be addressed

'Present address: Institute of Cell and Molecular Biology, University of Edinburgh, King's Buildings, Mayfield Road, Edinburgh EH9 3JR, UK

1198 Nucleic Acids Research, Vol. 19, No. 6 Materials and Methods). The availability of an in vitro system allows the direct chemical investigation of the products of apo B RNA editing. Full investigation of the mechanism of apo B mRNA modification requires: 1) identification of the exact chemical nature of the nucleotide product; 2) determination of whether or not this nucleotide is produced while maintaining the phosphodiester backbone of the RNA; 3) determination of whether the new base results from chemical modification or base exchange. Our results provide strong evidence that the editing enzyme is a site-specific cytidine deaminase.

MATERIALS AND METHODS Preparation of editing extract Rat intestinal epithelial cells were isolated as described (21). Cytosolic S100 extracts were prepared according to the method of Dignam et al (22), dialysed against Dignam buffer D containing 20% glycerol and stored at -20°C.

Preparation of RNA substrates Human apo B cDNA was PCR amplified with oligonucleotides PCR 5 (10) and PCR 12 (12), digested with Pst I and Hind HI and ligated into similarly digested pGem2 (Promega). The sequence of spGO-1, was confirmed throughout its apo B sequence. From human apo B mRNA, a cDNA spSTOP-1, was isolated, which contained as its only difference a T for the conversion site C. To further truncate the 3' end of these templates, plasmid DNA was PCR amplified with the SP6 promoter primer (sequence from New England Biolabs) and oligonucleotide DD3 (12). The products of these amplifications were used as templates for in vitro transcription with SP6 polymerase (Boehringer Mannheim) according to manufacturer's instruction. The RNA transcripts are 106 bases in length. The sequence shown is in Figure 1. It contains 19 cytidines per RNA molecule. To obtain RNA of maximal specific activity, 100 ItCi [&32P]CTP at 3000 Ci/mmole was dried and redissolved in a transcription reaction mixture of 5 1l giving a final concentration of CTP of 6.7 ,uM. This nucleotide concentration limited the final yield to typically 1 ng (- 30 fmole; 4 x 106 dpm). All RNAs were evaluated on a denaturing polyacrylamide gel to ensure that most of the RNA was of full length. RNAs were isolated by phenol:chloroform extractions, spin column chromatography and ethanol precipitation. -

Conversion of RNA in vitro Two aliquots of [32P]RNA (each 10 finoles) were incubated in the presence or absence of rat enterocyte cytosolic S100 extract in 10 mM Hepes (pH 7.9), 50 mM KCI, 50 mM EDTA, 0.25 mM DTT, 10 units RNase inhibitor (RNAguard, Pharmacia) in a final volume of 20 /d. 10-30 /g of S100 protein was used per assay. After 3 hours at 30°C, the RNA was reisolated and redissolved in 6 IAI water. 2 Id were assayed for editing by the standard conditions for primer extension (12) except that the reverse transcriptase extension was terminated by heating to 85°C for 5 minutes, and the [32P] RNA removed by adding RNAse A (Sigma) to 1 mg/ml final concentration, and RNAse H (Boehringer Mannheim) to 100 units/ml and incubating for 15 minutes at 37°C. The extension products were separated on a 7% polyacrylamide, 7 M urea gel, and after autoradiography the corresponding areas were cut out. Liquid scintillation counting with a quench programme with external standard was performed on a Packard TriCarb 2200 CA liquid scintillation analyser up

.

to a counting error of less than 2%. As a standard for quantitation, primer extension was performed on defined mixtures of GO and

STOP RNA. RNA hydrolysis and thin layer chromatography (TLC) 2 /Al (- 106 dpm) of edited or mock incubated RNA was made up to 50 mM sodium acetate (pH 5.3) and 0.67 units 1 d41 nuclease P1 (BRL) in 4 I1 and incubated at 370 for 3 hours. 2-4 td was spotted onto a cellulose TLC plate (Sigmacell Type 100, Sigma), with 1 yd each of 100 mM solutions of cytidine-, uridineadenosine- and guanosine- 5'-mono-phosphates (Sigma). Chromatography in the first dimension was in isobutyric acid/NH4OH/water (66:1:33 by volume) and in the second dimension in isopropanol/HCl/water (70:15:15 by volume) according to Nishimura (23) or in 0.1 M sodium phosphate (pH 6.8)/ammonium sulphate/n-propanol 100:60:2, v/w/v) according to Silberklang et al (24).

Isolation of RNase Ti fragments To isolate an oligoribonucleotide containing [32P]cytidine only at the RNA editing site, the edited and mock-incubated RNAs (2 1, 3 fmoles) were made to 50 mM Tris HCI (pH 7.5) 2 mM EDTA, and 33 units/ml ribonuclease TI in 4 d1, and incubated for 3 hours at 37°C. The fragments were separated by electrophoresis on 20% polyacrylamide-urea gel and exposed to film undried. Bands corresponding to the decamer (the largest fragment), the nonamers and an octamer were excised and isolated after diffusion from the gel as described (25) except without crushing the gel slice and without addition of SDS. The oligoribonucleotides could be conveniendy purified by absorption and elution from a Sep-Pak C18 column (Waters). The individual TI oligoribonucleotides were hydrolysed with nuclease P1 and resolved on TLC. -

RESULTS Editing in vitro and ribonuclease Ti fragments Evidence concerning the modification of apo B mRNA has been inferential. The nucleotide product templates adenosine for reverse transcriptase, can be part of an efficient stop translation

;A v e

-

1m

Ak\ -

_0

Figure 1. RNA substrate used for editing in vitro. a) The sequence of the 106 base RNA used in the editing in vitro is shown with colons after G residues to denote ribonuclease TI cleavage sites. The 61 base apo B sequence is to the right of the arrow, and pGem2 polylinker sequence to the left. The editing site is boxed. Decanucleotide, nonanucleotide, and octanucleotide ribonuclease Ti fragments are bracketed. b) Denaturing polyacrylamide gel separation of [32P] RNA-derived ribonuclease TI fragment from edited (+) or unedited (-) RNA.

Nucleic Acids Research, Vol. 19, No. 6 1199 codon, and base pairs more efficiently to oligodeoxynucleotides containing a base pairing adenosine than ones with guanosine (9-12). With the establishment of an in vitro editing system (12 and Materials and Methods) we can analyse the products of the editing directly. In this impure system, we are limited by low levels editing of exogenous templates (1-5%). Our analysis of the modified nucleotide is therefore limited to the most sensitive detection systems. We have chosen a RNA substrate for editing, which is 106 bases containing 61 bases of apo B sequences surrounding the editing site, and 45 bases of pGem2 polylinker sequence. The sequence of the RNA is presented in Figure 1. This RNA contains 19 cytidine residues, diluting the signal strength available for analysis. However, the RNA has the advantage of producing ribonuclease Tl fragments of which the largest (a decamer) contains the editing site as its sole cytidine, and which is convenient to isolate (Figure 1). RNA was synthesised at 3000 Ci/mmole of cytidine incorporated. Our typical assay conditions (3 fmoles RNA/20 jl reaction with 5 gl of rat intestinal S100 cytosolic extract (12 and Materials and Methods) was scaled-up 5-fold, to allow primer extension assay and several TLC assays on each experiment. The RNA was incubated in vitro with and without rat enterocyte editing activity. One fifth of the repurified RNA was tested for editing by primer extension (Figure 2). From one experiment, 3.3% of the RNA allowed reverse transcriptase to read through the editing site in the presence of dideoxy GTP. In the second independent experiment only 1.4% of the RNA was edited. This analysis tells us how much of a modified nucleotide we would predict to detect by physical separation techniques. Thin layer chromatography Aliquots of the RNAs were hydrolysed with nuclease P1 to 5'-monophosphates. Separation of the nucleotides should give [CaP32]CMP and any products derived from it. The samples, from the incubation with extract and from the mock incubation, were resolved on a two dimensional TLC system according to Nishimura (23). This system gave the best separation of CMP and UMP, based on the detection of marker nucleotides by

ultraviolet (UV) light absorption (shadowing the fluorescence of the TLC plate). The RNA from control incubations gave predominantly pC (5'-CMP), and several other spots (abelled X, Y and Pi in Figure 3). These three extraneous spots were detected in fresh RNA never incubated at 30°C (results not shown) with editing extract, so they are not the products of an aberrant RNA modification. They are not non-nucleotide contaminants of the CTP (such as phosphosugars or proteins) since they can be produced from isolated oligoribonucleotides. They do not comigrate with pU, pC or pA. However, one spot is in the position predicted of free phosphate (Pi). The recovery of Pi varied, but could be produced from isolated oligoribonucleotides, implicating a phosphatase contaminating the P1 nuclease at a low level. Spot X has the same mobility as that published for the dinucleotides pCpA or pApC, and so could represent partial digestion products. Spot Y had a mobility similar to pCp (5'-,3'-cytidine diphosphate), but pCp was efficiently cleaved when mixed with nuclease P1 incubations. The nature of these products X and Y is uncertain, but they are relatively constant in proportion and not specific to apo B mRNA editing. The edited RNA shows an additional spot on the TLC, which exacdy comigrates with unlabelled pU detected by UV shadowing (Figure 3). The uridine produced is in low quantities (consistent with 3.3% editing of one of the 19 cytidines) and was specific for adding enterocyte S100 conversion extract to the incubation. To examine the specificity of this uridine production, the RNA was digested with ribonuclease TI, and the decamer, nonamers and the octamer eluted from a 20% polyacrylamide-urea gel (Figure 1). The decamer contains only one cytidine, at the editing site. There is also an unlabelled decamer. The nonamers are a mixture of three fragments containing 5 cytidines. The octamer is unique, but contains two cytidines. The uridine production was

®K k

Experiments

IStop I

Test dilutions Go I t:10 1:2011:40

2__

- |

+

-

1

+

Extract 2

10,

+

0 72

0.1 6.4

2.7 1.5 0.5

3.3

0.4

1.4

%STOP

Figure 2. Analysis of apo B mRNA conversion by primer extension. STOP and GO RNAs and analysis of test mixtures of RNA at 1:10, 1:20 and 1:40 GO: STOP RNA are shown on the left. Experiments 1 and 2 are independent editing reactions carried out in the absence (-) and presence (+) of editing S100 extract.

Figure 3. Separation by TLC of nucleotide monophosphates after hydrolysis of substrate RNA. a) Diagrammatic representation of spots on the TLC plate after separation according to the system of Nishimura (23). pA, pC, pG and pU denote 5' nucleotide monophosphates. Pi denotes free phosphate. Two unknown spots X and Y are shown. b) Autoradiograph of TLC separation of hydrolysed substrate RNA after treatment (+) with apo B RNA editing extract. c) Autoradiograph of hydrolysed RNA from a mock incubation (-). The position of cochromatographed pU is circled.

1200 Nucleic Acids Research, Vol. 19, No. 6 10.

DISCUSSION The modification in vitro of apo B mRNA specifically converts a cytidine into a uridine. When substrate RNA, labelled in [32P]CMP residues, is incubated with an active rat S100 enterocyte extract and then hydrolysed to mononucleotides, the only novel [32P]product comigrates with 5'-UMP in two TLC separation systems. Uridine production depends on the protein extract; mock incubation does not give rise to it. By separating oligoribonucleotide fragments, we have shown that uridine production is unique to the in vivo editing site. Uridine is produced in quantities that correlate well with the independent assay by primer extension. In one experiment editing of 3.3% of the input RNA (assayed by primer extension) resulted in 3.8% uridine. In the second, 1.4% primer extension correlates with 1.0% uridine production. It is evident from both experiments

10

Figure 4. Separation by TLC of monophosphates derived from iribonuclease TI fragments. Ribonuclease Ti decanucleotide (containing the natu ral editing site) after editing (10+), and after mock incubation (10-), as well as inonanucleotides (9+) and an octanucleotide (8+) from edited RNA, but whici h are elsewhere in the RNA. pU is circled. The TLC solvent system of Nishimuraa (23) was used. A

f

,t

;DC te

.

t,

s

--N. ,__

A

pU

.

u

-~~~~ j~~~P 2..

-jc

Figure 5. Separation of nucleotides by TLC in the solvent systen (24). a) Diagramnmatic representation showing the positioIn of nucleotide monophosphates. pC and pU are CMP and UMP respectively. Pi isnfre phosphate X and Y are unknown spots. b) Autoradiograph of TLC separatioon of hydrolysed substrate RNA after treatment with the editing extract (+). plU is circled. c) Hydrolysed RNA substrate that has undergone mock conversiion without the presence of the editing extract (-). et al

specific for the decamer fragment that had been in(cubated with enterocyte extract (Figure 4). Decamer from mo ck incubated RNA, and nonamers or octamer from extract tireated RNA produced no uridine. To confirm that the modified nucleotide was uridirie, an aliquot of the total RNA hydrolysate was resolved in another TLC system according to Silberklang et al (24). The plates werre dried, and exposed to film. The positions of the unlabelled miarkers were detected by UV illumination. In the hydrolysate off the extracttreated RNA a new spot was detected that co-migrated with uridine (Figure 5).

that only uridine is produced by the extract in sufficient quantity to explain the amount of editing. Identification of the product of RNA editing as uridine does not prove the mechanism of editing to be deamination. Modification of cytidine to uridine could occur by three general mechanisms: 1) direct modification by deamination or transamination; 2) by base exchange through transglycosylation; 3) or by nucleotide excision and replacement. Firstly, we cannot distinguish the mechanism of deamination with liberation of ammonia from transamination, where the amino leaving group is ligated to a second substrate, to another base on the RNA substrate, to an enzyme cofactor, or to the enzyme itself. Forward and backward reactions, coupled to the exchange of the amine, could for example explain the C to U and U to C conversions in Oenothera cytochrome b mRNA, wheat cytochrome oxidase subunit HI mRNA and selenocysteine tRNA (6 - 8, 13 - 14, 17 -20). No such conversion reaction has been reported in apo B mRNA. The apo B mRNA editing activity appears not to require any low molecular weight substrate or cofactor, as the activity tolerates dialysis and gel filtration (12; Greeve, Navaratnam and Scott, unpublished results). However, the known transaminases contain covalently bound pyridoxal phosphate which remains tightly associated as pyridoxamine phosphate after the first, deaminating half-reaction (26). Until the fate of leaving amino group is followed, experimentally (or a second recipient substrate is found) the exact mechanism of cytidine deamination cannot be asserted. Secondly, the introduction of a ready-formed uracil base into the RNA by trans-glycosylation has parallels in the modification of tRNA. A uridine residue may be created from a cytidine by exchange of a uracil base for the cytosine base. Base exchange occurs by breaking the ribosyl-base nitrogen bond and reformation of the glycosyl bond to the new base. Transglycosylation has been demonstrated to be responsible for the introduction of hypoxanthine (27) and queuosine (28, 29) into tRNA and pseudouridine in tRNA is created by breaking the glycosyl bond and reforming a carbon-carbon bond to the same, rotated uracil base (30, 31). A C to U modification by transglycosylation would require a donor base as substrate, but would not cleave the phosphodiester backbone of the RNA chain. We have failed to identify any cofactor or substrate requirement for apo B mRNA modification (12, Greeve, Navaratnam and Scott, unpublished results). Specifically, dialysed and filtered extracts retain conversion activity which is not stimulated by any added uridine nucleotide, nucleoside or base. This is in contrast to the tRNA base-inserting enzymes, which require exogenous base as substrate, but does not exclude a covalently bound

Nucleic Acids Research, Vol. 19, No. 6 1201 nucleotide donor. The RNA and DNA ligases, for example, form phosphoamide bonds to ATP before binding their polynucleotide substrate (32-34). Distinction of a direct modification mechanism from transglycosylation requires following a label in the cytosine base into a uracil base. Most directly, incorporation of ring-labelled [5-3H]CTP should lead to [5-3H]uridine in a deamination product, but would lead to no label in the product of transglycosylation. These experiments can only be attempted with more purified editing activity, since [3H]labelled CTP is available at much lower specific activity and is more difficult to detect than [32p] CTP. An indirect method of ring-labelling cytidine would be to incorporate a modified cytidine triphosphate (for instance 5-methyl cytidine) into [a32P]ATP-labelled RNA and nearest-neighbour analysis of the product nucleotide. Here, conservation of the cytosine ring would lead to 5-methyl uridine and transglycosylation would lead to unmodified uridine. While this scheme would allow more sensitive detection (with 32p), the commercially available CTP derivatives (5-mercuriCTP and 5-iodoCTP) prevent RNA modification (Hodges and Scott unpublished observations). Therefore the formal distinction of deamination and transamination from transglycosylation requires further experiments. Finally, these experiments demonstrate that the 5'-phosphate of the modified cytidine is maintained through the enzymatic editing reaction. No known nucleotide polymerase (including the ribozymes) introduce an exogenous nucleotide into a polynucleotide chain without incorporating the donor's 5' phosphate. This argues strongly against excision and nucleotide replacement and clearly differentiates apo B mRNA editing from the editing of kinetoplastid transcripts in trypanosomes. Kinetoplast mRNA editing appears to involve site-specific nucleolytic cleavage, polymerisation of exogenous nucleotide triphosphate and religation of the RNA (1-4). Although a modification mechanism for apo B mRNA similar to this editing reaction could have been proposed, it may now be dismissed. In summary, a small series of experiments have allowed us to exclude either a complex nucleotide modification or nucleotide excision and replacement as the reaction mechanism for apo B RNA editing. The apo B mRNA editing activity is dialysable and unstimulated by nucleotide supplementation. Together these results strongly argue that the mechanism of apo B mRNA editing is by site-specific cytidine deamination of an RNA target.

ACKNOWLEDGEMENTS The authors gratefully acknowledge Ms Sargeant and Mrs Haywood for preparing the manuscript and Ms Fallows for preparing the figures.

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