High-Throughput Screening for Functional Adenosine to Inosine RNA

Adenosine to Inosine RNA Editing Systems. Subhash Pokharel and Peter A. Beal*. Department of Chemistry, University of Utah, 315 South 1400 East, Salt ...
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LETTER

High-Throughput Screening for Functional Adenosine to Inosine RNA Editing Systems Subhash Pokharel and Peter A. Beal* Department of Chemistry, University of Utah, 315 South 1400 East, Salt Lake City, Utah 84112-0850

D

eamination of adenosine (A) in RNA is an example of base-modification RNA editing (1). This transformation generates inosine (I) at the corresponding nucleotide position. Because l is decoded as guanosine (G) during translation, the reaction can lead to codon changes in messenger RNA (mRNA) and the introduction of amino acids into a gene product not encoded in the gene (2, 3). Translation of the different coding strands created by this process leads to protein structural diversity. Indeed, A to I RNA editing appears to be necessary to create the structural diversity required for properly functioning central nervous systems in metazoa (4, 5). Two multidomain human proteins have been shown to carry out A deamination in mRNA and have been given the name A deaminases that act on RNA (ADARs) (6). A related family of A deaminases that act on transfer RNA are referred to as ADATs (7). Each ADAR enzyme is made up of identifiable RNA-binding domains containing double-stranded RNAbinding motifs (dsRBMs) and a deaminase domain. Editing site selectivity arises in part by selective binding to double-helical RNA structures found in substrate transcripts mediated by the dsRBMs (8, 9). However, published results suggest the deaminase domain also plays an important role in controlling site selectivity (10, 11). How this takes place is not well understood at the structural–biochemical level. Genetic strategies are effective for rapidly defining structure–activity relationships in enzyme reactions and for the discovery of

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mutant enzymes with new properties. Application of this approach to the study of ADARs requires coupling the generation of libraries of mutant ADARs or mutant substrates with simple screens for editing activity. ADARs are not naturally found in bacteria or yeast, so simple genetic manipulation of these organisms to study A to I RNA editing is not possible. However, active ADARs can be isolated from yeast overexpression systems (12, 13). Therefore, we developed a screening strategy in the yeast Saccharomyces cerevisiae that is useful for the rapid identification of active ADAR mutants and new editing substrates capable of supporting the ADAR reaction. Our screen is based on the known ability of ADAR2 to deaminate within a stop codon, converting the sequence to a tryptophan codon (14). Lazinski et al. (14) had previously shown that editing of a stop codon found in the hepatitis D virus (HDV) antigenomic RNA could be used as a reporter of RNA editing efficiency by measuring the ratio of long form vs short form of the HDV antigen expressed. This observation suggested to us that a simple ADAR2 substrate upstream of sequence encoding a reporter enzyme could be translated directly and the conversion of a stop codon to a tryptophan codon could control reporter enzyme expression (Figure 1). Thus, we inserted RNA secondary structure known to support ADAR2 editing (human glutamate receptor-B (GluR-B) R/G site hairpin stem) into an mRNA transcript and made the necessary sequence changes to maintain an open reading frame and create a stop codon

A B S T R A C T Deamination of adenosines within messenger RNAs catalyzed by adenosine deaminases that act on RNA (ADAR) enzymes generates inosines at the corresponding nucleotide positions. Because inosine is decoded as guanosine, this reaction can lead to codon changes and the introduction of amino acids into a gene product not encoded in the gene. Translation of the different coding strands created by this process leads to protein structural diversity in the parent organism and is necessary for nervous system function in metazoa. The basis for selective editing of adenosines within certain codons is not well understood at the structural/ biochemical level. Here we describe a highthroughput screen for ADAR/substrate combinations capable of RNA editing that can be carried out in the yeast Saccharomyces cerevisiae growing on agar plates. Results from the screening of libraries of human ADAR2 mutants and libraries of RNA substrates shed light on structure– activity relationships in the ADAR-catalyzed adenosine to inosine RNA editing reaction.

*Corresponding author, [email protected].

Received for review September 1, 2006 and accepted November 15, 2006. Published online December 8, 2006 10.1021/cb6003838 CCC: $33.50 © 2006 by American Chemical Society

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a

b

d W

ADAR substrate

N −3′

5′− Secretion signal

α-Galactosidase

− − −

V

STOP V G G I V Y H A A G U GGUGGGUGG A U A U A U A C C A U S C C A U C C A C C U A U A U AU GGU C C C G G Q T P P I S Y W

5′ − − − . C G U U U 3′ − − − . G C A G A C

− − −

T

ADAR2 wild-type

ADAR2 E396A

5′ U U U GGG U

5′ U U U A GG U

c Secretion signal A U G A G A G C U U U C U U G U U U C U C A C C G C A U G C A U C A G U U U G C C A GG C G U U U U U GGG U C C G U U M R A F L F L T A C I S L P G V F G S V ADAR substrate α-Galactosidase U A G G U G GG U GG A A U A G U A U A C C A U U C G U G G U A U A G U A U C C C A C C U A C C C A G A C G G U G U C U C C G... Stop V G G I V Y H S W Y S I P P T Q T V S P

Figure 1. ␣-Galactosidase reporter for evaluating A to I RNA editing activity in S. cerevisiae. a) Schematic of reporter mRNA (17, 18). b) Sequence of ADAR substrate. The loop and boxed nucleotides have been altered from the original GluR-B pre-mRNA sequence. c) Full sequence of reporter mRNA near editing site with the encoded protein sequence shown. d) Secreted ␣-galactosidase activity (top) and reporter mRNA editing (bottom) in yeast expressing wild-type ADAR2 or the inactive mutant E396A.

at the editing site (15). We also reduced the length of the duplex and the size of the loop relative to that naturally found in the GluR-B pre-mRNA. Downstream of this ADAR substrate structure is the sequence encoding ␣-galactosidase, which is a secreted enzyme readily assayed directly on agar plates containing the colorimetric substrate 5-bromo-4-chloro-3-indolyl-␣-galactopyranoside (X-␣-Gal) (16). When wild-type human ADAR2 is expressed along with this reporter substrate, yeast grown on X-␣-Gal plates appears green, indicating expression and secretion of ␣-galactosidase (Figure 1) (16). This correlates with editing of the in-frame stop codon as evidenced by sequencing of a reverse transcriptase (RT)-polymerase chain reaction (PCR) product generated from total RNA isolated from this yeast using primers specific for the reporter transcript (Figure 1). No color and no editing are apparent when a known inactive mutant of ADAR2 (E396A) is coexpressed with the reporter mRNA (Figure 1). To test this system as a method for screening for editing activity, we chose to generate ADAR2 mutants that varied in the identity of two active site residues for which no structure–activity data were previously available. Thr375 is believed to be proximal to the 2=-hydroxyl of the edited nucleotide based on docking AMP into the structure of the deaminase domain (Figure 2) (19). However, this residue is not conserved in the ADAR family (e.g., asparagine in ADAR1) (Figure 2, panel a). In addition, we chose to vary the identity of the amino acid residue at position 376. This residue is a lysine in ADAR2 and a positively charged residue in the known ADARs and ADATs (7). 762

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Its position in the protein suggests a possible interaction with the 3=-phosphodiester of the edited nucleotide (Figure 2, panel b). Plasmid libraries were created by saturation mutagenesis at the codons for these residues. Yeast expressing the ␣-galactosidase reporter was transformed with the resulting libraries. Colored colonies growing on X-␣-Gal plates were then identified (Figure 2, panel c). Plasmid DNA encoding ADAR2 was isolated from yeast colonies appearing either green or white on X-␣-Gal plates. Sequencing revealed the identity of codons leading to active or inactive editing enzyme. In a screen where only the Thr375 codon was varied, plasmid DNA from 21 green and 13 white colonies was isolated and sequenced (Table 1). Common among the clones from green colonies were codons for small hydrophobic residues (8 for alanine, 6 for valine, and 2 for glycine), with wild-type threonine observed once. White colonies typically had position 375 codons encoding large residues (tyrosine, arginine, and lysine), with stop codons and a codon for proline also observed. The green color phenotype was confirmed and shown to correlate with editing of the reporter mRNA by sequencing of the RT-PCR product from yeast expressing either the T375A or T375Y mutants (Supplementary Figure 1). Interestingly, the T375A mutant is less active in this assay than wild-type ADAR2, since a lower level of editing is observed in the sequenced RT-PCR product. The purified ADAR2 T375A mutant also displayed a lower deamination rate than wild-type in in vitro deamination assays with a model substrate (data not shown). Thus, although T375A mutant scored as a hit in the screen, this mutation POKHAREL AND BEAL

does reduce ADAR2 deaminase activity. It is clear that under the current screening conditions, it is only possible to distinguish ADAR mutants with large differences in editing activity. Additional studies will be necessary to identify conditions that allow for ranking among ADAR mutants with similar activities. We also carried out a screen of a library of mutants that varied the identity of residues at both positions 375 and 376 of ADAR2. From the 14 green colonies isolated, we found 10 clones encoding the T375C, K376H mutant (Table 1). In addition, although a positively charged residue is conserved at position 376 in the known ADARs and ADATs, several clones were identified with hydrophobic amino acids encoded for this residue, including the K376I mutant (7). The phenotype and reporter RNA editing were confirmed for the T375C, K376H, and K376I mutants (Supplementary Figure 1). Important structure–activity relationships for the ADAR2 reaction can be gleaned from analysis of the amino acids accommodated at positions 375 and 376 in the active mutants identified in this study. Small hydrophobic amino acids at position 375 lead to activity and large residues are excluded, consistent with the proposed proximity of Thr375 with the editing site nucleoside (Figure 2, panel b) (19). Interestingly, when the amino acids at positions 375 and 376 were varied simultaneously, only threonine or cysteine was selected at position 375 in active mutants. These residues are capable of hydrogen bonding to the 2=-hydroxyl of the edited A. Threonine was selected in this screen encoded by three different codons, highlighting the www.acschemicalbiology.org

LETTER a

b

375 H. sapiens H. sapiens H. sapiens D. rerio G. gallus R. norvegicus M. musculus M. musculus C. elegans D. melanogaster H. sapiens M. fascicularis

ADAR2: ADAR1: ADAR3: ADAR: ADAR: ADAR: ADAR1: ADAR2: ADAR: ADAT1: ADAT1: ADAT1:

V V V V V V V V I L V V

I V V V V V V I I V V V

S S A S S S S S A S S S

V S LG L S LG I G LG LG V S L S LG MG MG

T T S T T T T T T C T T

G G G G G G G G G G G G

T N T N N N N T N T T T

K R K R R R R K K K K K

C C C C C C C C G C C C

I V I V V V V I L I I I

N K S K K K K N R G G G

G G G G G G G G G E Q Q

E D E E E D D E D S S S

Y S H E E S S Y K K K K

M L L L L L L M I L M M

Lys376

3′OH Thr375 2′OH Arg455 His394 Zn Cys451 Cys516 Glu396

c − − − S T G T K C I − − − 5′ − − − TCT ACA GGA ACA AAA TGT AT T − − − 3′

Screen for colored colonies

5′ − − − TCT ACA GGA NNN AAA TGT AT T − − − 3′

Figure 2. The ADAR active site. a) Sequence alignment for ADARs and ADATs. b) ADAR2 active site residues with docked AMP (19). c) Saturation mutagenesis was carried out at the T375 codon, and the resulting plasmids were screened for the generation of differently colored colonies.

importance of this amino acid at the 375 position of ADAR2. The selection system described here allows one to vary the sequence of either the editing enzyme or the editing substrate, since both are encoded on plasmids. To evaluate the effect of varying the editing substrate, we prepared two different plasmid libraries with different parts of the substrate sequence randomized (Table 2). In one library, six nucleotides were varied in the sequence complementary to the editing site (ECS library). In a different library, the four-nucleotide loop and two nucleotides of the loop-closing base pair were randomized. Interestingly, ⬍1% of the colonies observed during selections with the ECS library appeared green, indicating a strong dependence on the local sequence environment of the edited A (Supplementary Figure 2). When plasmids encoding the ADAR substrate from six green colonies observed in this screen were sequenced, the original sequence (CCCAGA) appeared four times along with two other sequences (CGGUAG and CGGUGA) (Table 2). The latter two related sequences are predicted to place the www.acschemicalbiology.org

edited A in a six-nucleotide, purine-rich symmetrical loop. Sequencing of an RT-PCR product from yeast expressing the CGGUGA mutant confirms efficient editing within this structure (⬃50% conversion to I) (Supplementary Figure 3). Activity with such a structure would not have been predicted given our current understanding of the role of the base pairing partner for the reactive A and the preference for As within duplex secondary structures (11, 15). This particular loop sequence must form a unique structure that properly positions the editing site for reaction with ADAR2. Importantly, a small change to this sequence has a dramatic effect on the screening results. Among the RNAs identified in the white colonies from this screen was the sequence AUCUGA, differing from an active sequence by only three nucleotides. However, folding of this RNA sequence in silico predicts an entirely different secondary structure (20). In this case, the edited A is predicted to exist in an A-U pair surrounded by four other stable base pairs. Furthermore, this structure is predicted to have single nucleotide bulges in locations where they do not exist naturally

on the R/G stem. RT-PCR product sequencing confirms little editing within this RNA (Supplementary Figure 3). It is possible that this duplex is too stable to allow for efficient flipping of the reactive adenosine into the deaminase active site (21). However, the placement of bulged nucleotides may also be an important negative determinant for activity with this substrate (22). A recent report suggested that the fivenucleotide loop of the native R/G hairpin stem found in the GluR-B pre-mRNA is important for controlling editing efficiency at the R/G site (9). For the substrate studied here, most of the sequences in the library support editing since ⬃80% of transformants with the loop sequence library gave green colonies (Supplementary Figure 2). However, when 10 active clones were sequenced from this screen, the sequence ACUCAC was observed eight times (Table 2 and Supplementary Figure 3). The CUCA sequence contained within is remarkably similar to sequences found naturally in GluR-B R/G hairpin loops (human, CUAA; rat, CUCA; mouse, CUCA; tilapia D, CUAC) (23). Allain et al. (9) have presented NMR data indicating that dsRBM I of ADAR2 binds the loop of the native R/G hairpin stem. Our results provide additional evidence for an important role played by this particular loop sequence in facilitating editing at the R/G site. Furthermore, selection of a near wildtype GluR-B R/G editing site loop sequence previously implicated in direct binding to ADAR2 underscores the utility of screening RNA libraries for preferred substrates with this method. Sequencing of plasmids from white colonies in this screen revealed RNA sequences that could never lead to reporter enzyme expression (e.g., stop codons and frame shift mutations) (Table 2). Indeed, low reporter enzyme expression in these cases was the result of factors other than low editing efficiency. One sequence found in three different white colonies (AAGCAU) maintained an open reading frame and is VOL.1 NO.12 • 761–765 • 2006

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TABLE 1. Results of screening of ADAR2 T375X and T375X, K376X libraries Library

Colony color

T375X

Green (active)

White (inactive)

T375X, K376X

Green (active)

predicted to form a tetraloop with a loopclosing A-U pair. Sequencing of the RT-PCR product from this sample indicates that RNA editing is supported (data not shown), yet poor expression of ␣-galactosidase was observed. Therefore, one must be cautious in interpreting reduced ␣-galactosidase expression solely in terms of editing efficiency without independent confirmation, as other factors can play a role. Indeed, the predicted loop sequence (AGCA) and an adjacent long duplex are features of good substrates for Rnt1p, a S. cerevisiae RNase III (24). In summary, fusing a RNA-editing substrate containing a stop codon in frame with sequence encoding the reporter enzyme ␣-galactosidase allows for high-throughput screening in S. cerevisiae of different combinations of mutant ADARs and RNA substrates to identify combinations that support editing. Simple modifications to this screen should allow for the discovery of regulators of the RNA editing reaction or evolution of new ADARs with altered site selectivity. 764

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Codon(s) (Amino acid)

No. of clones

GCA (Ala) GCG (Ala) GCC (Ala) GCT (Ala) GTA (Val) GGA (Gly) GGG (Gly) ACA (Thr) CAG (Gln) ATG (Met) ATT (Ile) CAT (His) TAA (Stop) TAG (Stop) TAC (Tyr) AGG (Arg) CGG (Arg) AAG (Lys) CCG (Pro) CCT (Pro) TGT, CAT (Cys, His) ACA, ATA (Thr, Ile) ACG, CTT (Thr, Leu) TGC, TTG (Cys, Leu) ACT, AAC (Thr, Asn)

4 2 1 1 6 1 1 1 1 1 1 1 1 1 2 2 2 3 1 1 10 1 1 1 1

METHODS Construction of Editing Substrate/ ␣-Galactosidase Reporter Plasmid pR/G␣Gal. The ␣-galactosidase expression plasmid pMEL␣ was obtained as a gift from EUROSCARF, Germany. The ␣-galactosidase coding sequence present on pMEL␣ was amplified by PCR and ligated into pYES3/CT (Invitrogen) using standard protocols to generate pR/G␣Gal. Sequences for all primers used in this study can be found in Supplementary Table 1. Co-expression of ␣-Galactosidase Reporter and Human ADAR2 in Yeast. INVSc1 S. cerevisiae strain (Invitrogen) was sequentially transformed with an ADAR2 expression plasmid (YEpTOP2PGAL1 (13) or YEpTOP2PGAL1-E396A) with URA3 selection followed by transformation with an editing reporter plasmid (pR/G␣Gal) with TRP1 selection using a lithium acetate protocol. Yeast colonies were harvested from these plates and transferred to agar plates containing complete minimal (CM) mediauracil, -tryptophan 2% raffinose, 3% galactose, and 60 ␮g mL⫺1 X-␣-Gal. The plates were incubated at 30 °C for 3–5 d until color was apparent. Sequencing of Reporter mRNA Isolated from Yeast Expressing ADAR2 Mutants. Single yeast colonies transformed with pR/G␣Gal and an ADAR2 expression plasmid were harvested and placed into 5 mL of CM-tryptophan, -uracil, 2% raffinose media for 48 h at 30 °C with shaking. Protein expression was induced by adding 3% galactose to the media, and growth was continued for another 48 h at 30 °C with shaking. mRNAs were isolated using the RiboPure-Yeast kit (Ambion) following the manufacturer’s protocol. Isolated mRNA POKHAREL AND BEAL

was reverse transcribed and amplified using the Access RT-PCR kit (Promega). The extent of RNA editing was assessed by sequencing the resulting RT-PCR product. Generation of Plasmid Libraries. The QuikChange XL site-directed mutagenesis kit (Stratagene) was used to generate plasmid libraries following the manufacturer’s protocol. Resulting PCR products were used to transform XL 10 gold Escherichia coli cells using ampicillin selection. Bacteria colonies were harvested, combined, and grown in LuriaBertani (LB) ampicillin media overnight at 37 °C with shaking. Plasmid DNA was isolated using a QIAprep Spin Miniprep kit (Qiagen). Sequence randomization at the desired site was confirmed by sequencing. Screening for Functional Editing Combinations. INVSc1 cells were transformed with either pR/G␣Gal or YEpTOP2PGAL1 (13) plasmids using a lithium acetate protocol. A second lithium acetate transformation was performed to introduce plasmid libraries. After the second transformation in screens for ADAR mutants, yeast were plated directly onto agar plates containing CM-tryptophan, -uracil, 2% raffinose, 3% galactose and X-␣-Gal. The plates were incubated at 30 °C for 5–10 d until color was apparent. After the second transformation in screens for mutant substrates, cells were plated onto agar plates containing CM-tryptophan, -uracil, 2% glucose. These plates were incubated at 30 °C for 3 days, after which they were replica plated onto agar plates containing CM-tryptophan, -uracil, 2% raffinose, 3% galactose, and X-␣-Gal. Within 72 h, green colored colonies were visualized. Yeast colonies appearing either green or white were harvested and lysed. Plasmid DNA was isolated from the lysate by ethanol precipitation and used to transform XL 10 gold E. coli cells (Stratagene) with selection on LB-ampicillin plates. Plasmid DNA was isolated from the resulting bacteria colonies and sequenced. Acknowledgment: P.B. acknowledges support from the National Institutes of Health (GM61115). Supporting Information Available: This material is free of charge via the Internet.

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LETTER TABLE 2. Results of screening of ADAR2 substrate libraries W

W

N − − − V STOP V G G I V Y H A A G U 5′ − CGUUU GGUGGGUGG AUA UAUACCA U S 3′ − GCNNN NNAUCCACC UAU AUAUGGU C N C G G C −−− X X X P P I S Y W

N − − − V STOP V G G I V Y X A A G N 5′ − CGUUU GGUGGGUGG AUA UAUACCN N X 3′ − GCAGA CCAUCCACC UAU AUAUGGN N C C G N C −−− T Q T P P I S Y X

ECS library

Loop library

Library

Colony color

RNA sequence (5= ¡ 3=)

No. of clones

Encoded sequence (N ¡ C)

ECS

Green

CCCAGA CGGUGA CGGUAG UAUAAU AUCUGA CCGCGC CCGGCA ACUCAC CAUCAA GCCGUA AAGCAU CCCAAUC CGUAAA

5 2 1 2 1 1 1 8 1 1 3 2 1

TQT TVT TVA I-Stop NLT TAP TGT HSR PSR RRR QAW Frame shift P-Stop

White

Loop

Green

White

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