Onouchi et al., Suppl. Data - 1 -

Supplemental Research Data

Influence of the GST tag on the CGS1 exon 1-mediated down-regulation

The influence of the GST tag on the CGS1 exon 1-mediated posttranscriptional regulation was

examined by a reporter assay. As shown in Supplementary Figure S1, when GST:Ex1(WT):Luc

RNA was translated in wheat germ extract, LUC activity was downregulated in response to

AdoMet, as seen when Ex1(WT):Luc RNA without GST tag was used (Chiba et al. , 2003).

Therefore, addition of the GST tag did not affect the CGS1 exon 1-mediated regulation.

Effect of AdoMet concentration on translation elongation arrest

Effect of AdoMet concentration on the 55-kDa peptidyl-RNA accumulation was tested by

translating GST:Ex1(WT) RNA at different AdoMet concentrations. As shown in Supplementary

Figure S2, the AdoMet-dependent 55-kDa band was observed even at 0.1 mM AdoMet although

the intensity of the 55-kDa band was lower than that at 1 mM AdoMet. When GST:Ex1 (mto1-1)

RNA carrying the mto1-1 mutation was translated, no such band was observed irrespective of the

AdoMet concentration.

Effect of frameshift mutations of the MTO1 region in the ice plant homolog of the CGS1 gene on

the AdoMet-induced posttranscriptional regulation

To test the importance of the amino acid sequence, the effect of frameshift mutations of the MTO1

Onouchi et al., Suppl. Data - 2 -

region was analyzed. In this experiment, the ice plant ortholog (McCGS) of Arabidopsis CGS1

(AtCGS1) (Supplementary Fig. S3A) was used, because the introduction of frameshift mutations

of the MTO1 region in AtCGS1, but not the corresponding region of McCGS , causes nonsense

mutations. As shown in Supplementary Figure S3B and C, the wild-type McCGS corresponding to

the exon 1 region of AtCGS1 responded to AdoMet in the same manner as the wild-type AtCGS1

exon 1 did in downregulating reporter activity and producing the peptidyl-RNA, whereas -1 (fs1)

or -2 (fs2) nt frameshift mutations of the McCGS MTO1 region abolished the response. Both the

AdoMet-induced down-regulation of reporter activity and production of the peptidyl-RNA were

restored by the -3 nt frameshift mutation (fs3). These results indicate the importance of the amino

acid sequence of the MTO1 region in the AdoMet-induced translation arrest.

Effects of W93A and W93G substitutions in vivo

To test the importance of Trp-93 in vivo, effects of W93A and W93G substitutions on down-

regulation of reporter activity were compared by transient expression study after transfection of

Arabidopsis protoplasts by electroporation. In our previous report in which effects of amino acid

substitutions were tested to determine the MTO1 region, W93A substitution showed only a weak

effect (Ominato et al. , 2002). In the present study, we carried out three sets of transient expression

experiments using independently prepared protoplasts, with each transient expression experiment

consisting of triplicated electroporation experiments. As shown in Supplementary Fig. S4, CGS1

exon 1 carrying W93G substitution showed stronger mutant phenotype on reporter activity (i.e. ,

reduced down-regulation) than that carrying W93A substitution, although the effect was less

Onouchi et al., Suppl. Data - 3 -

prominent than that carrying mto1-1 mutation.

Supplemental Materials and methods

Plasmid construction

The ice plant (Mesembryanthemum crystallinum) ortholog of the CGS1 exon 1 (Genbank acc.

AF069317) was amplified by PCR from ice plant genomic DNA using primers Mc1 and Mc2. The

amplified fragment was digested with X baI and BamHI, and inserted between the X baI and BamHI

sites of pYN10∆Xba(WT) to produce GST:McEx1(WT):Luc DNA. In pYN10∆Xba(WT), the

X baI site (5' -TCTAGA-3' ) immediately 5' of the GST coding region of pYN10(WT) was mutated

to 5' -ACTAGA-3' to facilitate subcloning. Frameshift mutagenesis was carried out by the overlap

extension PCR method. In the GST:McEx1(fs1):Luc construct, the 63rd nucleotide from the first

AUG of McCGS was deleted using complementary oligonucleotides FS1-1f and FS1-1r, and a

guanine residue was inserted immediately 3' of the 379th nucleotide using complementary

oligonucleotides FS2-1f and FS2-1r. In the GST:McEx1(fs2):Luc construct, the 62nd and 63rd

nucleotide were deleted using complementary oligonucleotides FS1-2f and FS1-2r, and guanine

and thymine residues were inserted immediately 3' of the 379th nucleotide using complementary

oligonucleotides and FS2-2r. In the GST:McEx1(fs3):Luc construct, the 61st-63rd nucleotides

were deleted using complementary oligonucleotides FS1-3f and FS1-3r. The primers used are

listed in Supplementary Table S1).

pMI4(WT), pMI4(mto1-1), and pMI4(W93A) have been described in Ominato et al.

(2002). pMI4(W93G) was constructed by replacing the X baI-BamHI fragment of pMN1(W93G)

Onouchi et al., Suppl. Data - 4 -

with the corresponding fragment of pMI4(WT).

In all of the constructs, the integrity of the PCR-amplified regions was confirmed by

sequence analysis.

Transfection experiments and reporter assay

Transfection of Arabidopsis protoplasts by electroporation, and β-glucuronidase (GUS), LUC, and

Renilla luciferase (RLUC) assays were performed as described (Ominato et al. , 2002; Chiba et al. ,

2003).

Onouchi et al., Suppl. Data - 5 -

Supplementary Table S1. Primers and oligonucleotides used.

Name Sequence

CD3Xr 5'-CACACCATGGAAGGTAACGCGGAGGAT-3'

Ex1P1 5'-CGAATCTAGAATGGCCGTCTC-3'

Ex1stop1 5'-GATCCTAGTCTTGAGCGGAGCT-3'

Ex1stop2 5'-CCGCTCAAGACTAG-3'

FS1-1f 5'-AATGCCGCCCTAGCCAGCAGACTT-3'

FS1-1r 5'-AAGTCTGCTGGCTAGGGCGGCATT-3'

FS1-2f 5'-AATGCCGCCCTGCCAGCAGACTT-3'

FS1-2r 5'-AAGTCTGCTGGCAGGGCGGCATT-3'

FS1-3f 5'-AATGCCGCCCTCCAGCAGACTT-3'

FS1-3r 5'-AAGTCTGCTGGAGGGCGGCATT-3'

FS2-1f 5'-ATGGTGGTGCTGGTAGTTGAGGAT-3'

FS2-1r 5'-ATCCTCAACTACCAGCACCACCAT-3'

FS2-2f 5'-ATGGTGGTGCTGTGTAGTTGAGGAT-3'

FS2-2r 5'-ATCCTCAACTACACAGCACCACCAT-3'

GSTf 5'-ACATCTAGAATGTCCCCTATACTAGG-3'

GSTr 5'-GGATCTAGAGGGCCCCTGGAAC-3'

K92r 5'-CTTAGCCGCCACGAT-3'

LucTP3 5'-TCCATCTTCCAGCGGATAGA-3'

Lys(CUU)f 5'-CGGAATTCTAATACGACTCACTATAGCCCGTCTAGCTCAGTTGGT-3'

Lys(CUU)r 5'-CGGAATTCGGGGTACCTGGCGCCCACCGTGGGGCTCGAA-3'

Mc1 5'-GCTCTAGAATGGCCGCCATCACGA-3'

Mc2 5'-CGGGATCCACCGGCATGAATTGCAAC-3'

N95Ar 5'-GGCGGACCACTTAGC-3'

N95r 5'-GTTGGACCACTTAGCCG-3'

S94Ar 5'-GGCCCACTTAGCCGC-3'

S94r 5'-GGACCACTTAGCCGC-3'

SP65'fP 5'-CATCAGAGCAGATTGTACTG-3'

Ser(AGA)f 5'-CGGAATTCTAATACGACTCACTATAGTGGACGTGCCGGAGTGGTT-3'

Ser(AGA)r 5'-CGGAATTCGGGGTACCTGGCGTGAACGGCAGGATTCGAA-3'

Onouchi et al., Suppl. Data - 6 -

Figure Legends for Supplementary Figures

Supplementary Figure S1. Influence of the GST tag on the CGS1 exon 1-mediated down-

regulation of reporter activity. (A) Schematic representation of Ex1:Luc and GST:Ex1:Luc RNAs.

(B) Ex1:Luc RNA or GST:Ex1:Luc RNA, carrying WT (open bars) or mto1-1 (filled bars) exon 1

was translated in wheat germ extract for 120 min in the presence or absence of 1 mM AdoMet.

LUC activity was normalized with the control RLUC activity from the cotranslated Rluc RNA

(Chiba et al. , 2003), and reporter activity relative to that without AdoMet was calculated. Averages

± SD of triplicate experiments are shown.

Supplementary Figure S2. Influence of AdoMet concentration on accumulation of translation

arrest product. GST:Ex1(WT) RNA or GST:Ex1(mto1-1) RNA was translated for 30 min in the

presence of indicated concentrations of AdoMet. The translation products were separated by SDS-

PAGE and subjected to immunoblot analysis with anti-GST antibody. The 45-kDa full-length

translation product (full-length) and the 55-kDa AdoMet-dependent peptidyl-RNA band (arrest-PR)

are marked.

Onouchi et al., Suppl. Data - 7 -

Supplementary Figure S3. Effect of frameshift mutations of the MTO1 region in the ice plant

homolog of the CGS1 gene on the AdoMet-induced posttranscriptional regulation. (A) Alignment

of the deduced amino acid sequence of AtCGS1 exon 1 and the corresponding region of McCGS

(Genbank acc. AF069317). Asterisks indicate identical amino acids. Dots and colons mark similar

amino acids. The underlined amino acid sequence of McCGS is the frameshifted region. Amino

acid sequences of McEx1(fs1), McEx1(fs2), and McEx1(fs3) are shown below the alignment. The

MTO1 region is marked. (B) The effect of frameshift mutations on AdoMet-induced down-

regulation of reporter activity. GST:McEx1(WT):Luc (WT), GST:McEx1(fs1):Luc (fs1),

GST:McEx1(fs2):Luc (fs2), or GST:McEx1(fs3):Luc (fs3) RNA was translated in wheat germ

extract for 120 min in the presence or absence of 1 mM AdoMet. LUC activity was normalized with

the control RLUC activity from the co-translated Rluc RNA, and the reporter activity relative to that

without AdoMet was calculated. Averages ± SD of triplicate experiments are shown. (C) The effect

of frameshift mutations on translation arrest. GST:McEx1(WT):Luc, GST:McEx1(fs1):Luc,

GST:McEx1(fs2):Luc, or GST:McEx1(fs3):Luc RNA was translated in wheat germ extract for 60

min in the presence (+) or absence (-) of 1 mM AdoMet. Translation products were analyzed by

immunoblot analysis with anti-GST antibody. The full-length translation product (full-length) and

the AdoMet-dependent 55-kDa peptidyl-RNA (arrest-PR) are marked. The asterisks indicate the

cross-reactive bands.

Onouchi et al., Suppl. Data - 8 -

Supplementary Figure S4. Effect of W93A and W93G substitutions on down-regulation of

reporter activity in transfection experiments. pMI4 plasmid carries CGS1 exon 1 fused in-frame

with a GUS reporter gene and placed under the control of a cauliflower mosaic virus 35S RNA

promoter (Ominato et al. , 2002). Arabidopsis protoplasts were electroporated with pMI4 plasmid

DNAs carrying wild-type (WT) or mutant versions of CGS1 exon 1, and were incubated for 48 h

in the presence (+Met) or absence (-Met) of 0.1 mM methionine. GUS activity was normalized

with LUC activity from the co-transfected control plasmid, and the values for +Met were divided

with those for -Met. Averages ± SD of nine transfection experiments are shown.

Supplementary Figure S1 R

ela

tive r

eport

er

activity

(+A

doM

et / -A

doM

et)

mto1-1

Ex1:Luc

WT mto1-1

GST:Ex1:Luc

WT

Onouchi et al.

0

0.5

1.0

A

B

GST:Ex1:Luc A30 GST Ex1 LUC

Ex1:Luc A30 Ex1 LUC

Supplementary Figure S2

Onouchi et al.

37

50

full-length

arrest-PR

kDa

WT

AdoMet (mM) 0 0.1 0.3 1 0 0.1 1

mto1-1 no

RN

A

A

CB

Supplementary Figure S3

37

50

75

100

kDa

- + AdoMet- + - + - +

WT

no

RN

A

fs1 fs2 fs3

.: .: :. *:**.:* ..:... ..:. :: * :.. .:. ** *AtCGS1 MAVSSFQCPTIFSSSSISGFQCRSDPDLVGSPVGGSSRRRVHASAGISSSFTGDAGLSSRIMcCGS ----MAAITSSCCARVLPPFECRPEPADFSTAAAKQEKNIPKSFFGGANGRLFSSSSSSLI

McEx1(fs1) ----MAAITSSCCARVLPPFECRPSQQTSPLPPPSKRRTSPNPSSAAPMAASSPPLLPLLSMcEx1(fs2) ----MAAITSSCCARVLPPFECRPASRLLHCRRQAREEHPQILLRRRQWPPLLLLFFLSYPMcEx1(fs3) ----MAAITSSCCARVLPPFECRP-PADFSTAAAKQEKNIPKSFFGGANGRLFSSSSSSLI

************ *************:***.****..:. **.....*:::..*.: ** AtCGS1 LRFPPNFVRQLSIKARRNCSNIGVAQIVAAKWSNNPSSALTSAAAAAATSSASAVSSAASAMcCGS LRFPPNFVRQLSTKARRNCSNIGVAQVVAASWSNNSDAGATSCGGVPAATAVDAAAIAAVD

McEx1(fs1) FAFLPTLSVSSAPRLAATAATLVSHRSSLPPGPTTPMPAPPLAVAFRLLLPSTLPLSPPSIMcEx1(fs2) SLSSQLCPSAQHQGSPQLQQHWCRTGRRCLLVQQLRCRRHLLRWRSGCYCRRRCRYRRRRSMcEx1(fs3) LRFPPNFVRQLSTKARRNCSNIGVAQVVAASWSNNSDAGATSCGGVPAATAVDAAAIAAVD

. ...*. : .:* :: .:: * * : .******.:::***AtCGS1 AAASSAAAAPVAAAPPVVLKSVDEEVVVAEEGIREKIGSVQLTDSKHSFLSSDGQLTVHAGMcCGS HVEIDGGAVVEDSND-----------GGVKELSSNNSNTLGLGDFRPPFLSSDGSVAIHAG

McEx1(fs1) MWRSMVVLVVEDSND-----------GGVKELSSNNSNTLGLGDFRPPFLSSDGSVAIHAGMcEx1(fs2) CGDRWWCCVVEDSND-----------GGVKELSSNNSNTLGLGDFRPPFLSSDGSVAIHAGMcEx1(fs3) HVEIDGGAVVEDSND-----------GGVKELSSNNSNTLGLGDFRPPFLSSDGSVAIHAG

0

0.5

1.0

WT fs1 fs2 fs3

Re

lative

re

po

rte

r a

ctivity

(+A

doM

et / -A

doM

et)

*

*

Onouchi et al.

MTO1 region

full-length

arrest-PR

Rela

tive G

US

activity

(+M

et/-M

et)

0.8

0.6

0.4

0.2

0WT W93A W93G mto1-1

Supplementary Figure S4

Onouchi et al.