A Phenotypic Screen for Functional Mutants of Human Adenosine Deaminase acting on RNA 1

Transcription

1 Supporting Information A Phenotypic Screen for Functional Mutants of Human Adenosine Deaminase acting on RNA 1 Yuru Wang, Jocelyn Havel, and Peter A. Beal* Department of Chemistry, University of California, Davis, One Shields Ave, Davis, California (USA) Contents: Table S1 Sequences of primers used in this study.s2-s3 General Methods..S4 Construction of the BDF2 reporter plasmid...s4-s5 Figure S1 Sequence of gene in BDF2 reporter plasmid..s5-s6 Site-directed mutagenesis to generate hadar1-d libraries and hadar-d mutants.s6-s7 Coexpression of reporter plasmid and hadar1-d; evaluation of editing level on reporter mrna.s7-s8 Overexpression and purification of hadar-d and derived mutants... S8 In vitro RNA transcription and purification...s8-s9 Figure S2 Alignment of sequences of ADARs from different organisms..s9 Figure S3 Off target editing in BDF2 RNA in vitro....s10 Figure S4 Comparison of rate constants for reactions of hadar2-d and hadar2-d E488H..S10 References......S11 S1

2 Table S1. Primers Purpose Primers BDF2 reporter plasmid ADAR1-D E912A inactive mutant E1008X library G1007X library RT-PCR on the BDF2 substrate within the reporter plasmid PCR to amplify the region in ADAR1-D surrounding the mutagenesis sites 5 -AGCTTATGAGAGCTTTCTTGTTTCTCACC GCATGCATCAGTTTGCCAGGCGTTTTTGGG TTCCCCACTTGCCATTAGACGTTCAGTAAGT ACCACCAATGGCAATATTGGGGAG-3 5 -GATCCTCCCCAATATTGCCATTGGTGGTA CTTACTGAACGTCTAATGGCAATGGGGGAA CCCAAAAACGCCTGGCAAACTGATGCATGC GGTGAGAAACAAGAAAGCTCTCATA-3 5 -GTCAATGACTGCCATGCAGCCATAATCTC CCGGAGAGGC-3 5 -GCCTCTCCGGGAGATTATGGCTGCATGGC AGTCATTGAC-3 5 -AAGGTGGAGAACGGANNSGGCACAATC CCTGTG-3 5 -CACAGGGATTGTGCCSNNTCCGTTCTCC ACCTT-3 5 -ACCAAGGTGGAGAACNNSGAAGGCACA ATCCCT-3 5 -AGGGATTGTGCCTTCSNNGTTCTCCACCT TGGT-3 5 -AATTGGCAGTAACCTGGCC AGGTGGTCTGCAACATGG-3 5 -TCTCACTGGCAGCACCTTCCAT-3 S2

3 E1008Q mutant plasmid E1008H mutant plasmid E1008R mutant plasmid RT-PCR on Bdf200 RNA RT-PCR on GluR B RNA C->G mutation in BDF2 reporter RT-PCR on BDF2(A G) RNA 5 -AGGGATGTCGTAGTCCATCCT-3 5 -AAGGTGGAGAACGGACAGGGCACAATC CCTGTG-3 5 -CACAGGGATTGTGCCCTGTCCGTTCTCC ACCTT-3 5 -AAGGTGGAGAACGGACACGGCACAATC CCTGTG-3 5 -CACAGGGATTGTGCCGTGTCCGTTCTCC ACCTT-3 5 -AAGGTGGAGAACGGAAGGGGCACAATC CCTGTG-3 5 -CACAGGGATTGTGCCCCTTCCGTTCTCCA CCTT-3 5 -GGATCCGCAATGCCACCAAG-3 5 -CCACCACTGACGCCTTCTC-3 5 -GGGAATATTAAGCTTATGAGAGC-3 5 -CGTCTGGGTAGGTGGGATA-3 5 -CGTTCAGTAAGTACCACGAATGGCAATAT TGGGGA 5 -TCCCCAATATTGCCATTCGTGGTACTTACT GAACG-3 5 -GGGAATATTAAGCTTATGAGAGC-3 5 -TTCCCCAATATTGCCATTC-3 General Methods S3

4 Restriction enzymes, T4 ligase and other cloning enzymes were purchased from New England Biolabs. Phusion hot start DNA polymerase was purchased from Thermo Scientific for high fidelity DNA amplification. Oligonucleotides were synthesized by Integrated DNA Technology. Components for yeast culture media were purchased from: BTS: biological grade peptone, yeast extracts, biological grade agar; BD: yeast nitrogen base w/o amino acid; Sigma-Aldrich: yeast synthetic dropout supplement medium without uracil, yeast synthetic dropout medium without tryptophan, glucose, glycerol, lactate, galactose, raffinose and amino acids. Reagents for in vitro transcription, in vitro editing, and PCR amplification were purchased from: GE Healthcare: MicroSpin G-25 columns; Promega: yeast trna Phe, RNasin, ribonucleotides; New England Biolabs: bovine serum albumin, RNase Inhibitor; Sigma Aldrich: glycerol and phenol:chloroform; Ni-NTA agaorse beads were purchased from Qiagen for enzyme purification. SYPRO Orange was purchased from Life Technologies for protein staining. SDS page gels stained with SYPRO Orange were imaged using a Typhoon FLA 9000 from GE healthcare. Data were analyzed using Molecular Dynamics ImageQuant 5.2 software, Chromas (Technelysium, Australia), KaleidaGraph (synergy Software, PA, USA) and ImageJ software (NIH, Bethesda, MD, USA). Sequences for all the primers used are listed in Table S1. Construction of the BDF2 reporter plasmid. The BDF2 reporter plasmid was generated based on the GluR B substrate/α-galactosidase reporter plasmid pr/gαgal (GluR B reporter plasmid) (1). To facilitate cloning, a restriction site was introduced between the sequence of GluR S4

5 B substrate and the sequence of α-galactosidase in the GluR B reporter plasmid by site-directed mutagenesis. The sequence composed of the secretion signal and the GluR B substrate in the GluR B plasmid is replaced by a sequence composed of the secretion signal and a modified BDF2 substrate by standard cloning. The final BDF2 reporter plasmid sequence was confirmed by Sanger sequencing. This plasmid has a GAL1 promotor, a TRP1 auxotrophic marker for selection of transformed yeast, and an ampicillin resistance marker for selection of transformed E. coli. Full sequence of the gene in the plasmid is found in Figure S1. 5 -CGGATTAGAAGCCGCCGAGCGGGTGACAGCCCTCCGAAGGAAGACTCTCCTCCGTGCGT CCTCGTCTTCACCGGTCGCGTTCCTGAAACGCAGATGTGCCTCGCGCCGCACTGCTCCGAACA ATAAAGATTCTACAATACTAGCTTTTATGGTTATGAAGAGGAAAAATTGGCAGTAACCTGGCCC CACAAACCTTCAAATGAACGAATCAAATTAACAACCATAGGATGATAATGCGATTAGTTTTTTA GCCTTATTTCTGGGGTAATTAATCAGCGAAGCGATGATTTTTGATCTATTAACAGATATATAAAT GCAAAAACTGCATAACCACTTTAACTAATACTTTCAACATTTTCGGTTTGTATTACTTCTTATTCA AATGTAATAAAAGTATCAACAAAAAATTGTTAATATACCTCTATACTTTAACGTCAAGGAGAAAA AACCCCGGATCGGACTACTAGCAGCTGTAATACGACTCACTATAGGGAATATTAAGCTTATGAG AGCTTTCTTGTTTCTCACCGCATGCATCAGTTTGCCAGGCGTTTTTGGGTTCCCCACTTGCCAT TAGACGTTCAGTAAGTACCACCAATGGCAATATTGGGGAGGATCCCCGAGTTACAATGGCCTT GGTCTCACTCCACAGATGGGTTGGGACAACTGGAATACGTTTGCCTGCGATGTCAGTGAACA GCTACTTCTAGACACCGCTGATAGAATTTCTGACTTGGGGCTAAAGGATATGGGTTACAAGTAT ATCATTCTGGATGACTGCTGGTCTAGCGGCAGAGATTCCGACGGTTTCCTCGTTGCAGATGAA CAAAAATTTCCCAATGGTATGGGCCATGTTGCAGACCACCTGCATAATAACAGCTTTCTTTTCG GTATGTATTCGTCTGCTGGTGAGTACACCTGTGCTGGATATCCTGGGTCTCTGGGTCGTGAGG AAGAAGATGCACAGTTCTTTGCAAATAACCGCGTTGACTACTTGAAGTACGATAATTGTTACAA TAAGGGTCAGTTTGGTACACCGGAAATTTCTTACCACCGTTACAAGGCCATGTCAGATGCTTT GAATAAAACTGGTAGGCCTATATTCTATTCTCTATGTAACTGGGGTCAGGATTTAACATTTTACT GGGGCTCTGGTATCGCCAATTCTTGGAGAATGAGTGGAGATGTTACTGCTGAGTTCACTCGTC CAGATAGCAGATGTCCCTGTGATGGCGATGAATACGATTGCAAGTACGCCGGTTTCCATTGTTC TATTATGAATATTCTTAACAAGGCAGCTCCAATGGGGCAAAATGCAGGTGTTGGTGGTTGGAA TGATCTGGACAATCTAGAGGTTGGTGTCGGGAATTTGACTGACGATGAGGAAAAGGCACATT TCTCTATGTGGGCAATGGTAAAGTCTCCACTTATCATTGGTGCCAATGTGAATAACTTAAAGGC ATCTTCGTACTCAATCTATAGTCAAGCCTCTGTCATCGCAATTAATCAAGATTCAAATGGTATTCC AGCAACAAGAGTCTGGAGATATTATGTTTCAGACACAGATGAATATGGACAAGGTGAAATTCA AATGTGGAGTGGTCCTCTTGACAATGGTGATCAAGTGGTTGCTTTATTGAATGGAGGAAGCGT ATCTAGACCAATGAACACGACCTTGGAAGAGATTTTTTTTGACAGCAATCTGGGTTCAAAGAA S5

6 ACTGACATCGACTTGGGATATCTACGACCTATGGGCCAACAGAGTTGACAACTCGACAGCGTC TGCTATCCTTGGACGGAATAAGACAGCCACCGGTATTCTCTACAATGCTACGGAGCAATCCTAC AAAGACGGTTTGTCTAAGAATGATACAAGACTGTTTGGTCAGAAAATTGGTAGTCTTTCTCCA AATGCTATACTTAACACGACTGTTCCAGCTCACGGTATCGCCTTCTATAGGTTGAGACCCTCTTC TTGA-3 Figure S1 Gene in BDF2 reporter plasmid from the GAL1 promoter to the end of the gene. The start codon and stop codon are highlighted. Site-directed mutagenesis to generate hadar1-d libraries and inactive hadar1-d mutant. Quick change II XL site-directed mutagenesis kit (Agilent) was used to generate mutant libraries or single mutant at desired sites in hadar-d (in YEpTOP2PGAL1) following manufacturer s protocol (1). The hadar1-d expression plasmid has a GAL1 promotor, a URA3 auxotrophic marker for selection of transformed yeast, and an ampicillin resistance marker for selection of transformed E. coli. The randomized codons are designed as NNS in the mutagenesis primers, in which N is any natural nucleotide and S is either guanosine or cytosine. This is to encompass every possible residue and exclude the three stop codons at the mutation sites. Resulting PCR products were transformed into XL 10 Gold E. coli cells using ampicillin selection following manufacturer s protocol. For making hadar1-d libraries, hundreds of bacterial colonies were pooled together and cultured in LB ampicillin media overnight at 37 C with shaking. For making single mutations, single colonies were harvested and cultured. Plasmids were extracted using QIAprep Spin Miniprep kit (Qiagen) and then sequenced to confirm the mutation or randomization at desired sites of hadar1-d or hadar2-d. S6

7 Coexpression of reporter plasmid and hadar1-d in yeast and evaluation of editing level on reporter mrna. S. cerevisiae INVSc1 strain (Invitrogen) was sequentially transformed with BDF2 reporter plasmid with TRP1 selection followed by hadar1-d expression plasmid with URA3 selection using a lithium acetate protocol. Double transformants were plated on complete media (CM) ura -trp + 2% glucose plates to let colonies develop. Yeast colonies growing on these plates were transferred either by streaking with an inoculating loop or replica plating to plates containing CM -ura -trp +2% raffinose +3% galactose that had been pretreated three times with 200 µl of a 6 mg/ml solution of X-α-Gal in DMSO and dried at 30 C overnight. The plates were then incubated in 30 C incubator for 2-4 days to allow green color to develop. To determine the editing level on the reporter mrna by hadar1-d in yeast, single colonies were harvested and transferred to 5 ml CM ura -trp + 2% glucose media and cultured for 24 h at 30 C with shaking. The culture (0.5 ml) was then pelleted and washed twice with autoclaved double distilled H 2 O and then resuspended in 15 ml CM ura trp + 2% raffinose + 3% galactose to induce expression. The galactose culture was diluted at times to maintain the OD 600 under 1.5. After 48 h of induction, yeast cells in the galactose culture were pelleted and total RNA was isolated from the cell pellets using RiboPure-yeast kit (Ambion). RT-PCR was then performed on the isolated RNA using Access RT-PCR kit (Promega) with primers specific to reporter mrna. The extent of editing was accessed by sequencing the resulting RT-PCR product. S7

8 Overexpression and purification of hadar-d and derived mutants Site-directed mutagenesis reactions were performed to produce hadar1-d mutants of interest selected in the screening. The identities of mutations in the resulting plasmids were confirmed with Sanger sequencing. Wild type hadar-d and all mutants of interest were overexpressed and purified from S. cerevisiae BCY123 strain as described previously (2). Purified hadar1-d and its mutants were stored in 50 mm Tris-HCl, ph=8.0, 10% glycerol, 0.01% NP-40, 1 mm DTT, 5 mm EDTA and 200 mm KCl at -80 C. Purified hadar2-d and E488H mutant were stored in 20mM Tris-HCl, ph 8.0, 20% glycerol, 1mM BME and 100mM NaCl at -80 C. The concentrations of wild type hadar-d and all mutants were estimated by quantifying on the same SDS polyacrylamide gel using one common BSA standard visualized by SYPRO Orange staining and Typhoon scanning. In vitro RNA transcription and purification The plasmids containing RNA sequences to be transcribed were linearized using the necessary restriction enzyme. In vitro transcription was carried out with the linearized plasmids using MEGAscript kit (Life technology) following the manufacturer s protocol. The sequence between the T7 promoter and the substrate is incorporated in each transcript. Transcribed RNA was purified using a denaturing polyacrylamide gel. Bands were visualized by UV shadowing and extracted using the crush and soak method overnight at 4 C in 0.5 M NH 4 OAc, 0.1% SDS, and 0.1 mm EDTA. Polyacrylamide fragments were removed using a 0.2 µm Centrex filter S8

9 followed by phenol-chloroform extraction and ethanol precipitation. The RNA solution was lyophilized to dryness, resuspended in nuclease-free water and quantified by absorbance at 260 nm. Refolding was carried out by diluting the RNA to the desired concentration in annealing buffer (100 mm NaCl, 10 mm Tris-HCl, ph =8, 1 mm EDTA) and heating at 95 C for 5 minutes and then slowly cooled to room temperature. Solutions of folded RNA were stored at -20 C. Figure S2 Alignment of sequences of ADARs from different organisms in the location corresponding to amino acids in human ADAR1. S9

10 Figure S3 Off target editing in BDF2 RNA in vitro. hadar1-d E1008Q at 35 nm concentration was allowed to react with 10 nm in vitro transcribed BDF2 RNA. At 15 min. editing at target site is complete and low levels (<20%) of editing are detectable at the off target sites. No deamination is detected at these sites in RT-PCR products amplified from reporter mrna isolated from yeast. Figure S4 Rate constants for deamination of the primary editing site for in vitro transcribed BDF2 RNA by hadar2-d and hadar2-d E488H. Deamination assays were performed with 1 µm enzyme and 10 nm RNA substrate under conditions of 20 mm Tris-HCl ph 7.0, 60 mm KCl, 30 mm NaCl, 1.5 mm EDTA, 8% glycerol, 0.003% Nonidet P-40, 0.5 mm DTT, 0.25 mm BME, 160 U/ml RNasin, and 1.0 µg/ml yeast trna Phe in a final volume of 10 µl. Each experiment was carried out in triplicate and rate constants are plotted as average value ± standard deviation. S10

11 References 1. Pokharel, S., and Beal, P. A. (2006) High throughput screening for functional A to I RNA editing systems, ACS Chem. Biol. 1, Macbeth, M. R., and Bass, B. L. (2007) Large-Scale Overexpression and Purification of ADARs from Saccharomyces cerevisiae for Biophysical and Biochemical Studies, Methods Enzymol. 424, S11