Polyamine-responsive ribosomal arrest at the stop codon of an upstream open reading frame of the AdoMetDC1 gene triggers nonsense-mediated mRNA decay in Arabidopsis thaliana.

Plant and Cell Physiology Advance Access published June 14, 2014

Title: Polyamine-Responsive Ribosomal Arrest at the Stop Codon of an Upstream Open Reading Frame of the AdoMetDC1 Gene Triggers Nonsense-Mediated mRNA Decay in Arabidopsis thaliana

Running title: Ribosomal arrest at a uORF triggers NMD in plants

Graduate School of Agriculture, Hokkaido University Kita-9, Nishi-9, Kita-ku, Sapporo 060-8589, Japan Phone: +81-11-706-3887, Fax: +81-11-706-4932 E-mail: [email protected]

Subject Areas: (3) regulation of gene expression, (4) proteins, enzymes and metabolism

Number of black and white figures, colour figures, tables and type and number of supplementary material:

Black and white figures: 1 Color figures: 3 Tables: 0 Supplementary materials: 5 figures, 1 table

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Corresponding author: Dr. H. Onouchi

Title: Polyamine-Responsive Ribosomal Arrest at the Stop Codon of an Upstream Open Reading Frame of the AdoMetDC1 Gene Triggers Nonsense-Mediated mRNA Decay in Arabidopsis thaliana

Running title: Ribosomal arrest at a uORF triggers NMD in plants

Koyanagi3,6, Katsunori Murota1,7, Satoshi Naito1,2 and Hitoshi Onouchi2*

Authors’ Addresses: 1

Graduate School of Life Science, Hokkaido University, Sapporo 060-0810, Japan

2

Graduate School of Agriculture, Hokkaido University, Sapporo 060-8589, Japan

3

Faculty of Agriculture, Hokkaido University, Sapporo 060-8589, Japan

*Corresponding author: Fax: +81-11-706-4932; E-mail, [email protected]

Abbreviations: ACE, Arabidopsis cell-free extract; ActD, actinomycin D; AdoMetDC, S-adenosylmethionine decarboxylase; CPuORF, conserved peptide uORF; GST, glutathione S-transferase; GUS, β-glucuronidase; LUC, firefly luciferase; NMD, nonsense-mediated mRNA decay; ORF, open reading frame; RLUC, Renilla reniformis luciferase; Spd, spermidine; Spm, spermine; S-uORF, small uORF; uORF, upstream ORF; UTR, untranslated region; WGE, wheat germ extract

Footnotes: 4

Present address: Chifure Corporation, Kawagoe 350-0833, Japan

2


Authors: Naoko Uchiyama-Kadokura1,4, Karin Murakami2, Mariko Takemoto2,5, Naoto

5

Present address: SRD Corporation, Chuo-ku, Tokyo 104-0032, Japan

6

Present address: Institute of Medical Science, The University of Tokyo, Tokyo 108-8639, Japan

7

Present address: Bioproduction Research Institute, National Institute of Advanced Industrial Science and Technology, Sapporo 062-8517, Japan


3

Abstract During mRNA translation, nascent peptides with certain specific sequences cause arrest of ribosomes that have synthesized themselves. In some cases, such ribosomal arrest is coupled with mRNA decay. In yeast, mRNA quality control systems have been shown to be involved in mRNA decay associated with ribosomal arrest. However, a link between ribosomal arrest and mRNA quality control systems has not been found in

ribosomal arrest and mRNA decay in plants. For this purpose, we used an upstream open reading frame (uORF) of the Arabidopsis thaliana AdoMetDC1 gene, in which the uORF-encoded peptide is involved in polyamine-responsive translational repression of the main coding sequence. Our in vitro analyses revealed that the AdoMetDC1 uORF encoded-peptide caused ribosomal arrest at the uORF stop codon in response to polyamine. Using transgenic calli harboring an AdoMetDC1 uORF-containing reporter gene,

we

showed

that

polyamine

promoted

mRNA

decay

in

a

uORF

sequence-dependent manner. These results suggest that the polyamine-responsive ribosomal arrest mediated by the uORF-encoded peptide is coupled with mRNA decay. Our results also showed that the polyamine-responsive acceleration of mRNA decay was compromised by defects in factors that are essential for the nonsense-mediated mRNA decay (NMD), an mRNA quality control system that degrades mRNAs with premature stop codons, suggesting that NMD is involved in AdoMetDC1 uORF peptide-mediated mRNA decay. Collectively, these findings suggest that AdoMetDC1 uORF peptide-mediated ribosomal arrest at the uORF stop codon induces NMD.

4


multicellular organisms. In this study, we aimed to explore the relationship between

Keywords: Arabidopsis thaliana, nonsense-mediated mRNA decay, polyamine, ribosome, translational regulation, upstream ORF


5

Introduction Nascent peptide-mediated ribosomal regulation has been recognized as a mechanism of gene expression control (Ito and Chiba 2013, Lovett and Rogers 1996, Morris and Geballe 2000, Tenson and Ehrenberg 2002). In eukaryotes, most known regulatory nascent peptides are encoded by upstream ORFs (uORFs), which are small ORFs located in the 5′ untranslated regions (5′ UTRs) of certain eukaryotic mRNAs. In

the cytomegalovirus gpUL4, fungal arg-2 and CPA1, and mammalian AdoMetDC uORFs, the uORF-encoded nascent peptides have been shown to cause ribosomal arrest at the stop codon of the uORFs (Cao and Geballe 1996a, Law et al. 2001, Wang et al. 1999, Wang and Sachs 1997). Among these, the uORF-encoded peptides of Neurospora crassa arg-2 and its Saccharomyces cerevisiae homologue, CPA1, respond to arginine to cause ribosomal arrest, whereas the mammalian AdoMetDC uORF-encoded peptide responds to polyamine. In these genes, a ribosome stalled at the uORF stop codon prevents other scanning ribosomes from reaching the initiation codon of the main ORF, resulting in translational repression of the main ORF (Law et al. 2001, Wang and Sachs 1997). Links between nascent peptide-mediated ribosomal arrest and mRNA decay have been demonstrated in several systems. Expression of the A. thaliana CGS1 gene, which encodes an enzyme involved in methionine biosynthesis, is controlled by the N-terminal regulatory region of the nascent polypeptide encoded by the main ORF (Chiba et al. 1999, Ominato et al. 2002). The regulatory region of the nascent CGS1 polypeptide causes translation elongation arrest in response to S-adenosylmethionine, and this translation arrest triggers mRNA decay (Onouchi et al. 2005, Onoue et al.

6


well-characterized examples of peptide sequence-dependent regulatory uORFs, such as

2011). In yeast, it has been shown that mRNA quality control systems are involved in decay of mRNAs with stalled ribosomes. When the CPA1 uORF-encoded peptide causes ribosomal arrest at the uORF stop codon, the CPA1 mRNA is targeted to NMD, which is an mRNA quality control system that degrades mRNAs with premature stop codons (Gaba et al. 2005). Another mRNA quality control system, no-go decay (NGD), targets mRNAs with ribosomes stalled during translation elongation and causes

2006). Recently, ribosomal arrest caused by a nascent peptide with 12 successive positively charged amino acid residues has also been shown to trigger endonucleolytic cleavage of mRNA in a similar fashion to NGD (Kuroha et al. 2010). In multicellular organisms, other than CGS1 gene regulation, little is known about the relationship between nascent peptide-mediated ribosomal arrest and mRNA decay. In particular, no report has shown a link between ribosomal arrest and an mRNA quality control system. To gain further insight on the relationship between nascent peptide-mediated ribosomal arrest and mRNA decay in higher plants, we utilized a uORF of the A. thaliana AdoMetDC1 gene, which encodes S-adenosylmethionine decarboxylase (AdoMetDC; EC 4.1.1.50) that catalyzes a key step in the biosynthesis of spermidine (Spd) and spermine (Spm), some of the most abundant polyamines in cells. The AdoMetDC1 gene has two overlapping conserved uORFs in its 5′ UTR. The first uORF, “tiny uORF”, is 12 nucleotides (nt) long, and the second uORF, “small uORF (S-uORF)”, is 159 nt long (Franceschetti et al. 2001, Fig. 1A). The peptide sequence encoded by the S-uORF has been shown to be involved in polyamine-responsive translational repression of the downstream main ORF (Hanfrey et al. 2005). However, it is not known whether the S-uORF-encoded peptide causes ribosomal arrest.

7


endonucleolytic cleavage in the vicinity of the stalled ribosome (Doma and Parker

In this study, by using cell-free systems, we showed that the S-uORF-encoded peptide sequence causes ribosomal arrest at the S-uORF stop codon in response to polyamine. In addition, using transgenic A. thaliana calli, we demonstrated that decay of S-uORF-containing mRNA is accelerated in response to polyamine in an S-uORF peptide

sequence-dependent

manner,

and

that

NMD

is

involved

in

the

polyamine-responsive acceleration of mRNA decay. These results suggest that

triggers NMD.

Results The AdoMetDC1 S-uORF-encoded peptide causes ribosomal arrest in wheat germ extract To investigate the relationship between ribosomal arrest and mRNA decay in A. thaliana by using the AdoMetDC1 S-uORF, we first examined whether the S-uORF-encoded peptide caused ribosomal arrest by using in vitro translation systems. In the previously characterized sequence-dependent regulatory uORFs, such as the cytomegalovirus gpUL4 and mammalian AdoMetDC uORFs, it was shown that uORF peptide-mediated ribosomal arrest at the translation termination step results in inhibition of peptide release from the peptidyl-tRNA (Cao and Geballe 1996b, Raney et al. 2002). Therefore, to address whether ribosomal arrest occurs in the S-uORF, we examined accumulation of a peptidyl-tRNA after translating the S-uORF in vitro. To facilitate detection of in vitro translation products of the S-uORF, a glutathione S-transferase (GST) tag sequence was fused in-frame to the 5′ end of the S-uORF (Fig. 1A, Supplementary Fig. S1). The first five nucleotides of the AdoMetDC1

8


AdoMetDC1 S-uORF peptide-mediated ribosomal arrest at the uORF stop codon

main ORF, which is located 155 nt downstream of the S-uORF stop codon, was fused in-frame to a Renilla luciferase (RLUC) coding sequence. This dicistronic construct was designated GST:S-uORF(WT):RLUC. To test the effect of the amino acid sequence of the

S-uORF,

we

generated

a

frameshift

mutant

version

construct,

GST:S-uORF(fs):RLUC, in which the tenth nucleotide of the S-uORF was deleted and an extra nucleotide was inserted three nucleotides upstream of the stop codon according

52 amino acids encoded by the S-uORF were altered. As an in vitro translation system, we first used a commercially available wheat germ extract (WGE), which contained a final concentration of 500 µM Spd for optimal translation efficiency. Because polyamine concentration could be critical to ribosomal arrest in the S-uORF, as seen in the mammalian AdoMetDC uORF (Law et al. 2001), we tested three polyamine concentrations, as indicated in Fig. 1B. After RNAs carrying GST:S-uORF(WT):RLUC and GST:S-uORF(fs):RLUC were translated separately in WGE for 30 min, translation products were subjected to immunoblot analysis with an anti-GST antibody. At all polyamine concentrations tested, in addition to the full-length GST:S-uORF(WT) translation product (Fig. 1B, white arrowhead), a band with an apparent molecular weight of 52 kDa was observed specifically when the wild-type S-uORF was translated (Fig. 1B, red arrowheads). If the wild-type S-uORF-specific 52-kDa product accumulated due to ribosomal arrest, it is expected to be a peptidyl-tRNA. To test this possibility, the translation products were treated with RNase A. After RNase A treatment of the translation products of GST:S-uORF(WT):RLUC RNA, the 52-kDa band disappeared, whereas the amounts of full-length products increased (Fig. 1B, lanes 2, 6 and 10), suggesting that the 52-kDa bands were shifted to the position of the full-length

9


to Hanfrey et al. (2005) (Supplementary Fig. S1). By these frameshift mutations, 45 of

band by RNase A treatment. In contrast, RNase A treatment of the translation products of GST:S-uORF(fs):RLUC RNA did not increase the amounts of full-length products (Fig. 1B, lanes 4, 8 and 12). These results indicate that the wild-type S-uORF-specific 52-kDa product contains an RNA moiety, whose apparent molecular weight in SDS-PAGE is consistent with that of a tRNA, and therefore, it is most likely that the 52-kDa product is a peptidyl-tRNA.

arrest at the translation termination step, as seen in the previously characterized uORFs, the RNA moiety of the 52-kDa peptidyl-tRNA should be a tRNA decoding the Ser-52 codon, which is the last amino acid-coding codon of the S-uORF. To address this, we changed the Ser-52 codon to an alanine codon (GCC), and examined whether this substitution affected the mobility of the 52-kDa peptidyl-tRNA. The mobilities of translation products of GST:S-uORF(WT):RLUC, GST:S-uORF(S52A):RLUC, and GST:S-uORF(fs):RLUC RNAs were compared to those of corresponding non-stop RNAs, GST:S-uORF(WT)ns, GST:S-uORF(S52A)ns, and GST:S-uORF(fs)ns RNAs, which are 3′-truncated forms of GST:S-uORF RNAs without a stop codon and terminated immediately before the stop codon of the S-uORF (Fig. 1A). In vitro translation of non-stop GST:S-uORF RNAs produces peptidyl-tRNAs that contain a tRNA decoding the last codon, because they lack a stop codon and, therefore, normal peptide release does not occur (Onouchi et al. 2005, Ramu et al. 2011, Wei et al. 2012). As shown in Fig. 1C, the peptidyl-tRNA band was shifted down by the S52A substitution (lanes 3 and 6, red arrowheads). The peptidyl-tRNAs produced from GST:S-uORF(WT)ns and GST:S-uORF(S52A)ns RNAs co-migrated with the largest products

of

GST:S-uORF(WT):RLUC

and

10

GST:S-uORF(S52A):RLUC

RNAs,


If the accumulation of the 52-kDa peptidyl-tRNA resulted from ribosomal

respectively (Fig. 1C, lanes 1-6). After RNase A treatment of the translation products, the largest bands disappeared and the difference in mobility of the full-length GST:S-uORF translation products between the wild-type and S52A constructs was not discernible (Fig. 1D, lanes 1, 2, 4 and 5), indicating that the difference in mobility of the largest bands between the wild-type and S52A constructs was due to the tRNA moieties. These results indicate that the 52-kDa peptidyl-tRNA produced from the

therefore, suggest that the S-uORF-mediated ribosomal arrest occurs at the translation termination step after the Ser-52 codon is decoded. The largest in vitro translation product of GST:S-uORF(fs)ns RNA, whose last codon is a threonine codon, showed slightly faster mobility than that of GST:S-uORF(WT)ns RNA (Fig. 1C, lanes 3 and 9, red arrowheads). Although a faint band produced from GST:S-uORF(fs):RLUC RNA co-migrated with the largest product of GST:S-uORF(fs)ns RNA (Fig. 1C, lanes 7-9), the band was much fainter at 700 µM Spd than the 52-kDa band produced from GST:S-uORF(WT):RLUC RNA (Fig. 1C, lanes 2 and 8), consistent with the result shown in Fig. 1B.

The S-uORF sequence-dependent ribosomal arrest occurs in a polyamine concentration-dependent manner Because the commercially available WGE contains 500 µM Spd, only a limited range of concentrations can be used for determination of the polyamine-concentration dependency of the S-uORF-mediated ribosomal arrest. To test the effect of a wider range of polyamine concentrations on the S-uORF-mediated ribosomal arrest, we next used Arabidopsis cell-free extract (ACE), which is an in vitro translation system 11


GST:S-uORF(WT):RLUC RNA contains a tRNA decoding the Ser-52 codon and,

recently developed by Murota et al. (2011). GST:S-uORF(WT):RLUC RNA was translated in ACE supplemented with 200, 300, 400, 500, 600, or 700 µM Spd for 30 min. As shown in Fig. 2B, immunoblot analysis with an anti-GST antibody indicated that the intensity of the 52-kDa band increased as the concentration of Spd increased in the range of 200-400 µM. While the 52-kDa band intensity did not markedly change in the range of 400-700 µM Spd, the level of full-length product dramatically decreased as

level is probably due to general influence of Spd on mRNA translation, because polyamine is known to affect translation (Igarashi and Kashiwagi 2000). Consistent with this, when an RNA encoding a firefly luciferase (LUC) reporter without GST:S-uORF was translated in ACE supplemented with a various concentration of Spd, LUC activity decreased as the Spd concentration increased in the range of 300-700 µM (Supplementary Fig. S2). Because the chance of ribosomal arrest depends on translation efficiency of GST:S-uORF, we therefore evaluated the efficiency of ribosomal arrest by normalizing the amount of the 52-kDa product by that of the full-length product. As shown in Fig. 2C, the ratios of the amount of the 52-kDa product to the full-length translation product increased as the concentration of Spd increased. This result suggests that the efficiency of the S-uORF-mediated ribosomal arrest depends on Spm concentration. Next, using ACE supplemented with 200 and 600 µM Spd, we compared in vitro

translation

products

between

GST:S-uORF(WT):RLUC

and

GST:S-uORF(fs):RLUC RNAs. When GST:S-uORF(WT):RLUC RNA was translated in ACE supplemented with 600 µM Spd, a higher amount of the 52-kDa peptidyl-tRNA

12


the Spd concentration increased in this range. This decrease in the full-length product

band was observed compared to that at 200 µM Spd (Fig. 2D, lanes 1 and 2, red arrowhead). In contrast, when GST:S-uORF(fs):RLUC RNA was translated in ACE, the largest peptidyl-tRNA band, which should contain a tRNA decoding the last amino acid-coding codon, Thr-52, did not increase at 600 µM Spd compared to at 200 µM Spd (Fig. 2D, lanes 3 and 4, green arrowhead). In addition, the intensities of the largest bands produced from GST:S-uORF(fs):RLUC RNA were much lower than those from

polyamine-responsive ribosomal arrest occurs in an S-uORF sequence-dependent manner in ACE.

The S-uORF-mediated ribosomal arrest occurs at the S-uORF stop codon We next performed toeprint (primer-extension inhibition) analysis to determine the exact

position

of

the

S-uORF-mediated

ribosomal

arrest.

In

this

assay,

ribosome-associated mRNAs are subjected to primer extension with a radiolabeled primer. The reverse transcription reaction is blocked by a ribosome, and therefore positions of stalled ribosomes can be determined by analyzing the sizes of primer extension products (Hartz et al. 1988).

For this analysis, we used the full-length

AdoMetDC1 5′ UTR without the GST tag sequence, but the tiny uORF was removed by substituting the initiation AUG codon of the tiny uORF to AUA (Supplementary Fig. S3), because our immunoblot analysis showed that the S-uORF can cause ribosomal arrest in the absence of the tiny uORF (Fig. 1, 2). Also, the tiny uORF has been suggested to have a negative effect on translation efficiency of the S-uORF (Hanfrey et al. 2005), which may result in a reduction in efficiency of ribosomal arrest. The mutated AdoMetDC1 5′ UTR was placed upstream of the RLUC coding sequence to generate the 13


GST:S-uORF(WT):RLUC RNA at both concentrations. These results suggest that the

S-uORF:RLUC constructs (Fig. 3A). After translating S-uORF(WT):RLUC and S-uORF(fs):RLUC RNAs separately in WGE supplemented with a final concentration of 500 or 700 µM Spd for 30 min, ribosomes were fixed on mRNAs by treating with a translation elongation inhibitor, hygromycin B, which stabilizes the S-uORF-mediated ribosomal arrest (Supplementary Fig. S4). This fixation is necessary for detection of the ribosomal arrest by toeprint analysis, because a pulse-chase analysis using edeine, an

is temporal (Supplementary Fig. S4) and, therefore, stalled ribosomes may be released during primer extension reaction. As shown in Fig. 3B and C, in the wild-type S-uORF, the strongest toeprint signal was detected 11 nt downstream of the S-uORF stop codon at 700 µM Spd (lanes 3 and 5, red arrowheads). The intensity of this toeprint signal was higher in the wild-type S-uORF than in the frameshift mutant S-uORF (Fig. 3B, C, lanes 3-6, Fig. 3D). When the in vitro translation reaction mixture was treated with hygromycin B prior to the translation reaction or treated with EDTA after the 30-min translation reaction, the intensity of the toeprint signal decreased to the background level (Fig. 3C, lanes 7-10), suggesting that ribosomes were responsible for the signals. Based on the relationship between the positions of the toeprint signal and the P and A sites of eukaryotic ribosomes (Anthony and Merrick 1992, Sachs et al. 2002), this toeprint signal represents a ribosome that was stalled with its A site at the stop codon of the S-uORF (Fig. 3E). These results suggest that the S-uORF-encoded peptide promotes ribosome stalling at the S-uORF stop codon, consistent with the results of the immunoblot analysis with the S52A mutant (Fig. 1C).

Polyamine induces mRNA decay in an S-uORF sequence-dependent manner 14


inhibitor of translation initiation, suggested that the S-uORF-mediated ribosomal arrest

To determine whether the S-uORF peptide-mediated ribosomal arrest is coupled with mRNA decay,

we

generated

transgenic

A.

thaliana

plants

harboring

the

35S::S-uORF(WT):GUS or 35S::S-uORF(fs):GUS constructs, in which the A. thaliana AdoMetDC1 5′ UTR without the tiny uORF and with the wild-type or frameshift mutant S-uORF was fused to an Escherichia coli β-glucuronidase (GUS) coding sequence and placed under control of the cauliflower mosaic virus 35S RNA promoter (Fig. 4A,

plants, we first confirmed that the S-uORF can mediate polyamine-responsive translational repression even in the absence of the tiny uORF. As shown in Supplementary Fig. S5, translation of the GUS coding sequence was strongly repressed when the transgenic calli carrying 35S::S-uORF(WT):GUS was cultured in the presence of 800 µM Spd. In addition, the effect of the wild-type S-uORF was much stronger than that of the frame-shift mutant S-uORF, indicating that the S-uORF has a sequence-dependent inhibitory effect on main ORF translation under this condition. We next examined the effect of polyamine on decay of the S-uORF-containing mRNAs. After liquid callus cultures prepared from the transgenic seedlings were incubated in the presence or absence of 800 µM Spd for 2 h, the cultures were treated with actinomycin D (ActD), an inhibitor of mRNA synthesis, and the time course of changes in S-uORF:GUS mRNA levels were monitored by Northern blot analysis to determine the half-lives of S-uORF:GUS mRNAs under each condition. As shown in Fig. 4B and F, the half-life of S-uORF(WT):GUS mRNA was approximately fourfold shorter in the presence of added Spd than in its absence. In contrast, as shown in Fig. 4C and F, the half-lives of S-uORF(fs):GUS mRNA were not significantly different under both conditions. These results suggest that polyamine promotes degradation of 15


Supplementary Fig. S3). Using liquid callus cultures prepared from these transgenic

S-uORF-containing mRNA in an S-uORF sequence-dependent manner. In yeast, it has been shown that ribosomal arrest at the stop codon of the CPA1 uORF promotes NMD of CPA1 mRNA (Gaba et al. 2005). Therefore, our finding from in vitro studies that ribosomal arrest occurs at the S-uORF stop codon raises the possibility that NMD is involved in the polyamine-responsive acceleration of S-uORF(WT):GUS mRNA decay. To address this possibility, we tested the influence of

S-uORF(WT):GUS mRNA decay. For this purpose, we used the atupf1-1 and atupf3-1 mutants. The atupf1-1 mutant carries a missense mutation in the AtUPF1 gene (Yoine et al. 2006a, Yoine et al. 2006b), and the atupf3-1 mutant harbors a T-DNA insertion at the 5′ splice site of the fifth intron in the AtUPF3 gene (Hori and Watanabe 2005). Both mutants show a severe defect in NMD (Hori and Watanabe 2005, Yoine et al. 2006a, Yoine et al. 2006b). We introduced the 35S::S-uORF(WT):GUS transgene into the atupf1-1 and atupf3-1 mutant backgrounds separately, and measured the half-life of S-uORF(WT):GUS mRNA in the absence and presence of 800 µM Spd. As shown in Fig. 4B, D, E, and F, the atupf1-1 and atupf3-1 mutations significantly increased the half-life of S-uORF(WT):GUS mRNA in the presence of added Spd, whereas no significant effect was observed in the absence of added Spd. These results suggest that NMD is involved in polyamine-responsive degradation of the S-uORF-containing mRNA.

Discussion In this study, to address the relationship between ribosomal arrest and mRNA decay in plants, we used the S-uORF of the A. thaliana AdoMetDC1 gene. We showed that the

16


defects in essential NMD factors, AtUPF1 and AtUPF3, on polyamine-induced

S-uORF causes ribosomal arrest at the S-uORF stop codon in response to polyamine and, using this system, we demonstrated that ribosomal arrest at a uORF stop codon induces NMD in A. thaliana.

The S-uORF-encoded peptide mediates polyamine-responsive ribosomal arrest at the uORF stop codon

the S-uORF, mediate polyamine-responsive translational repression of AdoMetDC1 mRNA, and that the S-uORF-encoded peptide has a role in it. The present study revealed that the underlying mechanism of the AdoMetDC1 translational regulation involves ribosomal arrest mediated by the S-uORF-encoded peptide. Immunoblot analyses with WGE and ACE showed that S-uORF sequence-dependent ribosomal arrest occurs in a polyamine concentration-dependent manner (Fig. 1, 2). We examined the effect of Spd concentration in a range of 200-700 µM, whereas levels of free Spd contents in plants are 101-102 nmol g-1 fresh weight order of magnitude (Imai et al. 2004, Paschalidis and Roubelakis-Angelakis 2005, Serafini-Fracassini et al. 2010). Therefore, the Spd concentrations used in this study do not seem much different from the physiological concentration, although cytosolic Spd concentration in plant cells has not been investigated to our best knowledge. Our in vitro analysis also showed that the S-uORF-mediated ribosomal arrest resulted in accumulation of the peptidyl-tRNA with a tRNA decoding the last amino acid-coding codon of the S-uORF (Fig. 1C). This observation suggests that the S-uORF-mediated ribosomal arrest occurs at the translation termination step. Consistent with this, toeprint analysis revealed that ribosome stalling occurs at the S-uORF stop codon in an S-uORF sequence-dependent

17


Hanfrey et al. (2005) have reported that the two overlapping uORFs, the tiny uORF and

manner (Fig. 3). Collectively, these results suggest that the S-uORF-encoded peptide mediates polyamine-responsive ribosomal arrest at the S-uORF stop codon. In plants, besides the S-uORF of AdoMetDC1, a uORF of the A. thaliana bZIP11 gene has been shown to mediate translational regulation in a sequence-dependent manner (Rahmani et al. 2009). However, no example of uORF peptide-mediated ribosomal arrest has been reported. Therefore, this study is the first report of uORF peptide-mediated ribosomal

To date, uORF peptide-mediated ribosomal arrest has been reported in the cytomegalovirus gpUL4, fungal arg-2 and CPA1, and mammalian AdoMetDC genes. In all of these, the uORF-encoded peptide causes ribosomal arrest at the uORF stop codon (Cao and Geballe 1996, Wang and Sachs 1997, Wang et al. 1999, Law et al. 2001), as seen in the S-uORF of the A. thaliana AdoMetDC1 gene. In addition, we showed that the S-uORF-mediated ribosomal arrest is temporal (Supplementary Fig. S4). This feature is also consistent with previously characterized nascent peptide-mediated ribosomal arrests in the arg-2 gene and the CGS1 gene (Fang et al. 2004, Onouchi et al. 2005). Among the previously identified genes regulated by a nascent peptide, the mammalian AdoMetDC gene is homologous to the A. thaliana AdoMetDC1 gene. The mammalian AdoMetDC uORF-encoded peptide causes ribosomal arrest in response to polyamine, as seen in A. thaliana AdoMetDC1 (Raney et al. 2002). However, the mammalian AdoMetDC uORF encodes only six amino acid residues, MAGDIS, whereas the A. thaliana AdoMetDC1 uORF encodes 52 amino acid residues. In addition, a sequence similar to the ‘MAGDIS’ sequence was not found in the A. thaliana AdoMetDC1 uORF sequence. Thus, uORF-encoded peptide sequences of the

18


arrest in plants.

mammalian AdoMetDC and A. thaliana AdoMetDC1 genes are not similar, despite the similarity in the regulatory mechanisms. It is, therefore, unlikely that these uORFs are evolutionarily derived from a common ancestor sequence. Rather, it is more likely that they arose independently by parallel evolution.

NMD is induced by polyamine-responsive ribosomal arrest at the S-uORF stop

Our in vitro analyses revealed that S-uORF sequence-dependent ribosomal arrest occurs in response to polyamine. In addition, using transgenic calli, we showed that polyamine specifically promoted decay of wild-type S-uORF-containing mRNA. These results indicate that polyamine induces two events, ribosomal arrest and mRNA decay, in an S-uORF sequence-dependent manner. This suggests that ribosomal arrest mediated by the S-uORF-encoded peptide is coupled with mRNA decay. Although we do not have in vivo evidence for the S-uORF-mediated ribosomal arrest, using transgenic calli we showed that the S-uORF mediates polyamine-responsive translational repression of the downstream coding sequence in a sequence-dependent manner (Supplementary Fig. S5). This implies that the S-uORF-mediated ribosomal regulation seen in vitro also occurs in vivo. Furthermore, we showed that NMD is involved in the polyamine-responsive acceleration of mRNA decay, suggesting that S-uORF-mediated ribosomal arrest at the S-uORF stop codon promotes NMD. Although it has been shown that NMD is promoted by ribosomal arrest at the stop codon of a uORF in yeast (Gaba et al. 2005), a link between ribosomal arrest and NMD, or any other mRNA quality control system, has not been reported in multicellular organisms. Therefore, this study is the first report showing a link between ribosomal arrest and an mRNA quality control system in a

19


codon

multicellular organism. The atupf1-1 and atupf3-1 mutations compromised the polyamine-responsive acceleration of S-uORF(WT):GUS mRNA decay, but showed no significant effect on S-uORF(WT):GUS mRNA decay in the absence of exogenously applied polyamine, suggesting that the S-uORF-containing mRNA is not subject to NMD when ribosomal arrest does not occur. Nyikó et al. (2009) have shown that a 50-amino-acid-long uORF

uORFs trigger NMD in a uORF size-dependent manner in plants. Inconsistent with this observation, despite the S-uORF being 52 amino acids long, our above result indicates that the S-uORF-containing mRNA is hardly targeted to NMD in the absence of polyamine. This suggests that the NMD sensitivities of uORF-containing mRNAs are not determined by uORF length alone in plants. In yeast, it has been suggested that inefficient translation termination at a premature stop codon is a critical determinant of NMD targeting (Amrani et al. 2004). Taking this into account, translation termination of the S-uORF might be efficient in the absence of polyamine and, therefore, NMD may be strongly induced only when ribosomal arrest at the S-uORF stop codon results in inefficient translation termination. In a previous study, it has been shown that endogenous AdoMetDC1 mRNA levels did not decrease after A. thaliana cell suspensions were treated with 0.5 mM each of Spd and Spm for 16 h (Hanfrey et al. 2002). This implies that polyamine does not induce NMD of endogenous AdoMetDC1 mRNA. Because the principal purpose of the present study was to address the relationship between ribosomal arrest and mRNA decay in plants, the tiny uORF, which has been suggested to reduce translation efficiency of the S-uORF (Hanfrey et al. 2005), was eliminated from the AdoMetDC1 5′

20


triggered NMD efficiently but a 35-amino-acid-long uORF did not, and suggested that

UTR of our reporter constructs to efficiently induce ribosomal arrest. It is, therefore, possible that the NMD efficiency of the endogenous AdoMetDC1 mRNA may be too low to cause a detectable reduction in mRNA level, because of the low translation efficiency of the S-uORF due to the presence of the tiny uORF. This study revealed that the mechanism of NMD induction by ribosomal arrest at a uORF stop codon is widely conserved in eukaryotes, from yeast to plants. Recent

that eight genes containing uORFs with conserved peptide sequences (CPuORFs) are natural NMD targets (Rayson et al. 2012). Although it is not yet known whether these CPuORFs cause ribosomal arrest, uORF peptide-mediated ribosomal arrest at the uORF stop codon is a likely mechanism that explains why mRNAs harboring these CPuORFs are targeted to NMD. Therefore, it is likely that the molecular mechanism revealed in this study is not specific to the S-uORF of AdoMetDC1 but may also be involved in expression of other CPuORF-containing genes in A. thaliana. Although approximately 30% of A. thaliana genes contain one or more uORFs in their 5′ UTRs (Kawaguchi and Bailey-Serres 2005, Takahashi et al. 2012), how each uORF affects gene expression is largely unknown. This study provides a better understanding of uORF-mediated gene expression regulatory mechanisms and an insight on determinants of NMD targeting for uORF-containing mRNAs in plants.

Materials and Methods Plant materials Arabidopsis thaliana (L.) Heynh. Col-0 ecotype was used for plant transformations. The atupf1-1 mutant seeds were kindly provided by Dr. Kenzo Nakamura (Chubu

21


microarray analyses with A. thaliana mutants deficient in NMD factors have suggested

University) and the atupf3-1 mutant seeds by Dr. Yuichiro Watanabe (the University of Tokyo).

Chemicals Spd, Spm, and actinomycin D (ActD) were purchased from Sigma-Aldrich (www.sigmaaldrich.com).

Sequences of primers used for plasmid construction are shown in Supplementary Table S1.

Plasmids

pSY209

S-uORF(fs):RLUC

and

constructs

pSY214

carry

in

pSP64

the

the

S-uORF(WT):RLUC

Poly(A)

vector

and

(Promega,

www.promega.com), respectively. To construct these plasmids, AdoMetDC1 5′ UTR was amplified from poly(A)+ RNA prepared from A. thaliana (Col-0) calli by using the OneStep RT-PCR Kit (Qiagen, www.qiagen.com) with primers SAMDCF1 and SAMDCR2, digested with HindIII and SalI, and cloned into pMI27 (Chiba et al. 2003) between the HindIII and SalI sites. Using the overlap extension PCR method (Ho et al. 1989), the initiation ATG codon of the tiny uORF was changed to ATA to create pSY209, and the frameshift mutations were introduced into the S-uORF of pSY209 to generate pSY214. Plasmids pNU14, pNU15, and pHS1 harbor the GST:S-uORF(WT):RLUC, GST:S-uORF(fs):RLUC, and GST:S-uORF(S52A):RLUC constructs in the pSP64 Poly(A) vector, respectively. To generate pNU14 and pNU15, the GST coding region was amplified from pYN10 (Onouchi et al. 2005) with primer sets, sp64seqF/GSTwt1R and sp64seqF/GSTfs1R, respectively. The 5′-truncated AdoMetDC1 5′ UTR containing

22


Plasmid construction

from the first codon of S-uORF to the first five nucleotides of the main ORF was amplified from pSY209 or pSY214 with primer sets, SAMDCwtG1F/RLUCr or SAMDCfsG1F/RLUCr, respectively. The PCR fragments containing GST and S-uORF(WT or fs) were fused by the overlap extension PCR method, and digested with HindIII and BamHI. The 471 bp HindIII-BamHI fragment of pSY209 were replaced with the HindIII- and BamHI-digested PCR fragments to make pNU14 and pNU15. To

site-directed mutagenesis (Weiner et al. 1994) with primers S52AsdmF and S52AsdmR. Plasmids pNU10 and pNU11 carry the 35S::S-uORF(WT):GUS and 35S::S-uORF(fs):GUS constructs, respectively, in a binary vector plasmid, pBI121 (Jefferson, 1987). To construct these plasmids, the AdoMetDC1 5′ UTRs containing S-uORF(WT or fs) and the first seven nucleotides of the main ORF were amplified from pSY209 or pSY214, respectively, using primers SAMDCtg1F and SAMDCtg2R. The amplified AdoMetDC1 5′ UTR fragments were digested with XbaI and SmaI, and cloned between the XbaI and SmaI sites of pBI121 to generate pNU10 and pNU11. In all of the constructs, the integrity of the PCR-amplified regions was confirmed by sequence analysis.

Plant transformation The 35S::S-uORF(WT):GUS and 35S::S-uORF(fs):GUS constructs were introduced into the A. thaliana Col-0 ecotype using the floral dip method (Clough and Bent 1998) with Agrobacterium tumefaciens strain C58C1RifR(pGV2260) (Deblaere et al. 1985). Transgenic plants harboring a single copy of the transgene were selected by Southern blot analysis. The 35S::S-uORF(WT):GUS construct was introduced into atupf1-1 and

23


construct pHS1, the S52A substitution was introduced into the S-uORF of pNU14 by

atupf3-1 mutants by cross-pollination. Homozygous lines were established using GUS staining with 5-bromo-4-chloro-3-indolyl glucuronide (Jefferson et al. 1987) and dCAPS and PCR analyses with primers to detect the atupf1-1 (Yoine et al. 2006b) and atupf3-1 mutations, respectively (Supplementary Table S1).

In vitro transcription

described (Chiba et al. 2003). Templates for non-stop RNAs were prepared by amplifying the corresponding region from pNU14, pNU15 and pHS1 by PCR with KOD-Plus-Neo DNA polymerase (Toyobo, www.toyobo-global.com) and the primers listed in Supplementary Table S1. In vitro transcription in the presence of a cap analog, m7G[5′]ppp[5′]GTP, was accomplished as described (Chiba et al. 2003).

In vitro translation In vitro translation reactions using WGE (Promega) were carried out as described (Chiba et al. 2003). For RNase A treatment, RNase A was added at a final concentration of 0.5 mg ml–1, and the reaction mixtures were incubated for 15 min at 37°C. Preparation of ACE and in vitro translation reactions using ACE were performed as described (Murota et al. 2011).

Immunoblot analysis GST:S-uORF:RLUC RNAs (1 pmol) or GST:S-uORFns RNAs (2 pmol) were translated in a 20-µl WGE or ACE reaction mixture. After 30 min translation reaction, 2-µl aliquots were mixed with 48-µl SDS-PAGE sample buffer containing 62.5 mM

24


DNA templates in the pSP64 poly(A) vector were linearized with EcoRI and purified as

Tris-HCI, pH 6.8, 2% SDS, 100 mM DTT, 5% glycerol, and 0.002% bromophenol blue, and boiled for 3 min. The samples were separated on a NuPAGE 4%–12% Bis-Tris Gel with MOPS-SDS running buffer (Life Technologies, www.lifetechnologies.com). In this neutral pH SDS–PAGE system, peptidyl-tRNA ester bonds are retained during electrophoresis (Onouchi et al. 2005). Translation products were then transferred to an Immobilon-P membrane (Millipore, www.millipore.com), probed with a polyclonal

the Immobilon Western Chemiluminescent HRP Substrate (Millipore).

RNA analysis A. thaliana liquid callus cultures were prepared as described previously (Murota et al. 2011;

note

a

typographical

error

in

kinetin

concentration,

http://pcp.oxfordjournals.org/content/53/3/602.full). Approximately 100 one-week-old transgenic

A.

thaliana

seedlings

carrying

35S::S-uORF(WT):GUS

or

35S::S-uORF(fs):GUS were minced with a razor blade, transferred to a 200-ml Erlenmeyer flask containing 50-ml RM28 medium (Guzman and Ecker, 1988), and incubated at 23°C in the dark with shaking at 100 r.p.m. The medium was exchanged once every 6 days. On the 21st day after starting callus culture, Spd was added to a final concentration of 0 or 800 µM. At 2 h after the addition of Spd, ActD was added to a final concentration of 100 µg ml–1. Approximately 100 mg of calli were harvested at 0, 30, 60, 90, and 120 min after the addition of ActD and frozen in liquid nitrogen. Total RNAs were extracted from frozen calli using the RNeasy Plant Mini kit (Qiagen). Northern blot analysis was performed as described (Suzuki et al. 2001, Chiba et al. 2003). The GUS probe was described previously (Suzuki et al. 2001). The 25S

25


anti-GST antibody (Santa Cruz Biotechnology, www.scbt.com), and visualized using

rRNA probe was prepared by PCR amplification of A. thaliana Col-0 genomic DNA with the primers 25Sfor (5′-GGTAAAGCCAGAGGAAACTCT-3′) and 25Srev (5′-CACAAAGTCGGACTAGAGTCA-3′). The PCR product was purified with the GenElute PCR Clean-Up Kit (Sigma-Aldrich) and labeled with [α-32P]dCTP using the Megaprime

DNA

Labeling

System

(GE

Healthcare

Life

Sciences,

www.gelifesciences.com).

After 200 fmol S-uORF(WT):RLUC RNA or S-uORF(fs):RLUC RNA was translated in a 20-µl WGE reaction mixture, hygromycin B was added to a final concentration of 2 mM to fix ribosomes on RNAs unless otherwise stated in the legend for Fig. 3. Primer extension of the ribosome-bound RNAs and analysis of the reaction products with a sequencing gel were performed as described by Wang and Sachs (1997) with the following modifications. One-µl aliquots of the translation reaction mixtures were used for reverse transcription reaction in a final volume of 20 µl. The reverse transcription reaction mixtures contained hygromycin B at a final concentration of 2 mM in addition to the components described in Wang and Sachs (1997). The oligonucleotide SAMDCTP2 (5′-AGGAGCTTTGAAAGAAGAAA-3′) was labeled at its 5′ terminus with T4 polynucleotide kinase (Takara Bio, www.takara-bio.com) and [γ-32P]ATP (110 TBq/mmol; GE Healthcare Life Sciences, www.gelifesciences.com) and was purified using a QIAquick Nucleotide Removal kit (Qiagen). The reverse transcription samples were separated on an 8% polyacrylamide/7 M urea gel. DNA sequence ladders were prepared using the 5′-32P-labeled SAMDCTP2 primer, the pSY209 plasmid as a

26


Toeprint analysis

template and a Takara Taq Cycle Sequencing kit (Takara Bio). The gel image was analyzed using a Fuji BAS 1000 Bio Image Analyzer.

Supplementary data Supplementary data are available at PCP online.

This work was supported by the Japan Science and Technology Agency [Core Research for Evolutional Science and Technology (PJ34085001) to H.O.]; the Japan Society for the Promotion of Science [Grant-in-aid for Scientific Research (C) (21570032) to H.O.]; the Ministry of Education, Culture, Sports, Science and Technology of Japan [Grant-in-aid for Scientific Research on Innovative Areas (22119006) to S.N.]; and Suhara Memorial Foundation.

Disclosures Conflicts of interest: No conflicts of interest declared.

Acknowledgments We thank Ms. Hitomi Sekihara, Ms. Saeko Yasokawa, and Ms. Eriko Tanaka for skillful technical assistance, and Ms. Kumi Fujiwara, Ms. Naoe Konno, and Ms. Maki Mori for general assistance. We used the Radioisotope Laboratory and the DNA Sequencing Facility of the Graduate School of Agriculture, Hokkaido University.

27


Funding

References

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Legends to figures

Fig. 1. Analyses of in vitro translation products of the AdoMetDC1 S-uORF in WGE. (A) Schematic representation of AdoMetDC1 mRNA, GST:S-uORF:RLUC RNAs, and GST:S-uORFns RNAs. GST:S-uORF:RLUC RNAs contain the intact S-uORF, whereas GST:S-uORFns RNAs are 3′-truncated forms that lack the stop codon. The

analysis

of

translation

products

at

various

polyamine

concentrations.

The

GST:S-uORF(WT):RLUC and GST:S-uORF(fs):RLUC RNAs were translated separately for 30 min in WGE supplemented with the indicated final concentrations of Spd and Spm. Aliquots of translation reaction mixtures were treated with RNase A as indicated. Translation products were separated by SDS-PAGE and immunoblotted with an anti-GST antibody. The positions of protein size markers are shown on the left. Red and white arrowheads indicate the positions of the wild-type S-uORF-specific 52-kDa peptidyl-tRNA bands and the full-length GST:S-uORF translation products, respectively. (C, D) Comparison of the translation products of GST:S-uORF:RLUC RNAs (intact) and GST:S-uORFns RNAs (ns). The wild-type (WT), S52A mutant (S52A), or frameshift mutant (fs) GST:S-uORF:RLUC and GST:S-uORFns RNAs were translated separately for 30 min in WGE supplemented with a final concentration of 500 or 700 µM Spd. In D, aliquots of translation reaction mixtures were treated with RNase A before separation by SDS-PAGE. Translation products were immunoblotted with an anti-GST antibody. Red and white arrowheads denote the positions of the peptidyl-tRNA band produced from each GST:S-uORFns RNA and the full-length

34


frame-shifted region in the frameshift mutant S-uORF is shown in red. (B) Immunoblot

GST:S-uORF translation products, respectively. For each panel, a representative result of three independent experiments is shown.

Fig. 2. Analyses of in vitro translation products of the AdoMetDC1 S-uORF in ACE. (A) Schematic representation of GST:S-uORF(WT):RLUC and GST:S-uORF(fs):RLUC RNAs. The frame-shifted region in the frameshift mutant S-uORF is shown in red. (B)

GST:S-uORF(WT):RLUC RNA was translated for 30 min in ACE supplemented with the indicated final concentration of Spd. Translation products were separated by SDS-PAGE and immunoblotted with an anti-GST antibody. Red and white arrowheads indicate the positions of the 52-kDa peptidyl-tRNA bands and the full-length GST:S-uORF translation products, respectively. (C) Efficiency of the S-uORF-mediated ribosomal arrest in ACE at various Spd concentrations. The intensities of the 52-kDa peptidyl-tRNA bands and the full-length product bands in B were quantified using MultiGauge (Fuji Photo Film), and the ratios of the amount of the 52-kDa peptidyl-tRNA to the full-length product were calculated. The ratios relative to that at 200 µM Spd were calculated, and means ± S.D. of three independent experiments is shown. (D) Comparison of translation products of the wild-type and frameshift mutant GST:S-uORF. The GST:S-uORF(WT):RLUC and GST:S-uORF(fs):RLUC RNAs were translated separately for 30 min in ACE supplemented with a final concentration of 200 or 600 µM Spd. Translation products were immunoblotted with an anti-GST antibody. The white arrowhead indicate the positions of the full-length GST:S-uORF translation products. Red and green arrowheads mark the positions of the largest peptidyl-tRNA bands produced from the GST:S-uORF(WT):RLUC and GST:S-uORF(fs):RLUC RNAs, 35


Immunoblot analysis of translation products at various Spd concentrations. The

respectively. For B and D, a representative result of three independent experiments is presented.

Fig. 3. Determination of the position of the S-uORF-mediated ribosome stalling by toeprint

analysis.

(A)

Schematic

representation

of

S-uORF(WT):RLUC

and

S-uORF(fs):RLUC RNAs. The frame-shifted region in the frameshift mutant S-uORF is

(fs) RNAs were translated separately for 30 min in WGE supplemented with a final concentration of 500 µM (lane 1, 2) or 700 µM (lane 3-10) Spd. In lanes 1-6, 2 mM hygromycin B (Hyg) was added after the translation reaction. In lanes 7 and 8, 2 mM hygromycin B was added prior to the translation reaction. In lanes 9 and 10, EDTA and hygromycin B were added to a final concentration of 5 and 2 mM, respectively, after the translation reaction, and the samples were incubated for another 5 min. MgCl2 was added to 10 mM prior to the primer extension reaction. Primer extension reactions were performed using the 32P-labeled primer SAMDCTP2. Red arrowheads mark the position of the toeprint signals detected 11 nt downstream of the S-uORF stop codon. The green arrowhead indicates the location of the toeprint signal thought to be derived from a ribosome stalled at the S-uORF initiation codon by hygromycin B. White arrowheads denotes the position of the full-length primer extension products. The positions of the initiation (AUG) and stop (UGA) codons of the S-uORF are indicated on the left. The sequence ladder (shown in the sense strand sequence) was synthesized using the same primer as for toeprinting. For each panel, a representative result of at least two independent experiments is shown. (D) Quantification of the toeprint signal. The intensities of the strongest toeprint signal detected at 700µM Spd (red arrowhead in B

36


shown in red. (B, C) Toeprint analysis. S-uORF(WT):RLUC (W) and S-uORF(fs):RLUC

and C) and the full-length primer extension signal (white arrowhead in B and C) were quantified using MultiGauge (Fuji Photo Film). The intensity of the toeprint signal was normalized to that of the full-length primer extension signal. Means ± S.D. of four independent experiments are shown. Asterisk indicates a significant difference between the wild-type (WT) and frame-shift mutant (fs) S-uORFs (p < 0.01 by t-test). (E) Schematic representation of the position of ribosome stalling. The red arrowhead

The C-terminal amino acid sequence of the S-uORF and nucleotide sequence of the corresponding region are shown. The positions of the P and A sites of the stalled ribosome deduced from the toeprint signal are indicated.

Fig. 4. Effects of polyamine and NMD mutations on decay of S-uORF-containing mRNAs. (A) Schematic representation of the reporter constructs. The frame-shifted region in the frameshift mutant S-uORF is shown in red. (B, C, D, E) Decay kinetics of S-uORF-containing mRNAs. The transgenic calli harboring 35S::S-uORF(WT or fs):GUS in Col-0 (B, C), atupf1-1 (D), or atupf3-1 (E) background were cultured in liquid RM28 medium supplemented with 0 µM (blue circle) or 800 µM (red triangle) Spd for 2 h and then treated with 100 µg ml–1 ActD. After the start of ActD treatment (0 min), samples were withdrawn from the cultures at the time points indicated, and the levels of transgene mRNA and 25S rRNA were determined by Northern blot analysis using GUS and 25S rRNA probes, respectively. Northern hybridization data were quantified using the NIH Image or MultiGauge (Fuji Photo Film) software, and the intensities of the GUS signals were normalized to those of the 25S rRNA signals. The amounts of RNAs relative to the time zero value are shown in semi-log plots. Means ±

37


indicates the position of the toeprint signals marked by the red arrowheads in B and C.

S.D. of three independent experiments are shown. (F) Half-lives of S-uORF:GUS mRNA. Half-lives of S-uORF(WT/fs):GUS mRNA in Col-0, atupf1-1, or atupf3-1 background were calculated based on data presented in B to E by non-linear least square

method.

Asterisks

indicate

significant

differences

from

the

35S::S-uORF(WT):GUS transgenic calli in Col-0 background under the same Spd condition (p < 0.01 by Welch's t-test). Downloaded from http://pcp.oxfordjournals.org/ at Maastricht University on June 26, 2014

38


Fig. 1. Analyses of in vitro translation products of the AdoMetDC1 S-uORF in WGE. (A) Schematic representation of AdoMetDC1 mRNA, GST:S-uORF:RLUC RNAs, and GST:S-uORFns RNAs. GST:SuORF:RLUC RNAs contain the intact S-uORF, whereas GST:S-uORFns RNAs are 3′-truncated forms that lack the stop codon. The frame-shifted region in the frameshift mutant S-uORF is shown in red. (B) Immunoblot analysis of translation products at various polyamine concentrations. The GST:S-uORF(WT):RLUC and GST:S-uORF(fs):RLUC RNAs were translated separately for 30 min in WGE supplemented with the indicated final concentrations of Spd and Spm. Aliquots of translation reaction mixtures were treated with RNase A as indicated. Translation products were separated by SDS-PAGE and immunoblotted with an anti-GST antibody. The positions of protein size markers are shown on the left. Red and white arrowheads indicate the positions of the wild-type S-uORF-specific 52-kDa peptidyl-tRNA bands and the full-length GST:S-uORF translation products, respectively. (C, D) Comparison of the translation products of GST:S-uORF:RLUC RNAs (intact) and GST:S-uORFns RNAs (ns). The wild-type (WT), S52A mutant (S52A), or frameshift mutant (fs) GST:SuORF:RLUC and GST:S-uORFns RNAs were translated separately for 30 min in WGE supplemented with a

final concentration of 500 or 700 µM Spd. In D, aliquots of translation reaction mixtures were treated with RNase A before separation by SDS-PAGE. Translation products were immunoblotted with an anti-GST antibody. Red and white arrowheads denote the positions of the peptidyl-tRNA band produced from each GST:S-uORFns RNA and the full-length GST:S-uORF translation products, respectively. For each panel, a representative result of three independent experiments is shown. 181x225mm (300 x 300 DPI)



Fig. 2. Analyses of in vitro translation products of the AdoMetDC1 S-uORF in ACE. (A) Schematic representation of GST:S-uORF(WT):RLUC and GST:S-uORF(fs):RLUC RNAs. The frame-shifted region in the frameshift mutant S-uORF is shown in red. (B) Immunoblot analysis of translation products at various Spd concentrations. The GST:S-uORF(WT):RLUC RNA was translated for 30 min in ACE supplemented with the indicated final concentration of Spd. Translation products were separated by SDS-PAGE and immunoblotted with an anti-GST antibody. Red and white arrowheads indicate the positions of the 52-kDa peptidyl-tRNA bands and the full-length GST:S-uORF translation products, respectively. (C) Efficiency of the S-uORFmediated ribosomal arrest in ACE at various Spd concentrations. The intensities of the 52-kDa peptidyl-tRNA bands and the full-length product bands in B were quantified using MultiGauge (Fuji Photo Film), and the ratios of the amount of the 52-kDa peptidyl-tRNA to the full-length product were calculated. The ratios relative to that at 200 µM Spd were calculated, and means ± S.D. of three independent experiments is shown. (D) Comparison of translation products of the wild-type and frameshift mutant GST:S-uORF. The GST:S-uORF(WT):RLUC and GST:S-uORF(fs):RLUC RNAs were translated separately for 30 min in ACE supplemented with a final concentration of 200 or 600 µM Spd. Translation products were immunoblotted with an anti-GST antibody. The white arrowhead indicate the positions of the full-length GST:S-uORF translation products. Red and green arrowheads mark the positions of the largest peptidyl-tRNA bands produced from the GST:S-uORF(WT):RLUC and GST:S-uORF(fs):RLUC RNAs, respectively. For B and D, a representative result of three independent experiments is presented. 127x108mm (300 x 300 DPI)


Fig. 3. Determination of the position of the S-uORF-mediated ribosome stalling by toeprint analysis. (A) Schematic representation of S-uORF(WT):RLUC and S-uORF(fs):RLUC RNAs. The frame-shifted region in the frameshift mutant S-uORF is shown in red. (B, C) Toeprint analysis. S-uORF(WT):RLUC (W) and SuORF(fs):RLUC (fs) RNAs were translated separately for 30 min in WGE supplemented with a final concentration of 500 µM (lane 1, 2) or 700 µM (lane 3-10) Spd. In lanes 1-6, 2 mM hygromycin B (Hyg) was added after the translation reaction. In lanes 7 and 8, 2 mM hygromycin B was added prior to the translation reaction. In lanes 9 and 10, EDTA and hygromycin B were added to a final concentration of 5 and 2 mM, respectively, after the translation reaction, and the samples were incubated for another 5 min. MgCl2 was added to 10 mM prior to the primer extension reaction. Primer extension reactions were performed using the 32P-labeled primer SAMDCTP2. Red arrowheads mark the position of the toeprint signals detected 11 nt downstream of the S-uORF stop codon. The green arrowhead indicates the location of the toeprint signal thought to be derived from a ribosome stalled at the S-uORF initiation codon by hygromycin B. White arrowheads denotes the position of the full-length primer extension products. The positions of the initiation

(AUG) and stop (UGA) codons of the S-uORF are indicated on the left. The sequence ladder (shown in the sense strand sequence) was synthesized using the same primer as for toeprinting. For each panel, a representative result of at least two independent experiments is shown. (D) Quantification of the toeprint signal. The intensities of the strongest toeprint signal detected at 700µM Spd (red arrowhead in B and C) and the full-length primer extension signal (white arrowhead in B and C) were quantified using MultiGauge (Fuji Photo Film). The intensity of the toeprint signal was normalized to that of the full-length primer extension signal. Means ± S.D. of four independent experiments are shown. Asterisk indicates a significant difference between the wild-type (WT) and frame-shift mutant (fs) S-uORFs (p < 0.01 by t-test). (E) Schematic representation of the position of ribosome stalling. The red arrowhead indicates the position of the toeprint signals marked by the red arrowheads in B and C. The C-terminal amino acid sequence of the S-uORF and nucleotide sequence of the corresponding region are shown. The positions of the P and A sites of the stalled ribosome deduced from the toeprint signal are indicated. 203x334mm (300 x 300 DPI)



Fig. 4. Effects of polyamine and NMD mutations on decay of S-uORF-containing mRNAs. (A) Schematic representation of the reporter constructs. The frame-shifted region in the frameshift mutant S-uORF is shown in red. (B, C, D, E) Decay kinetics of S-uORF-containing mRNAs. The transgenic calli harboring 35S::S-uORF(WT or fs):GUS in Col-0 (B, C), atupf1-1 (D), or atupf3-1 (E) background were cultured in liquid RM28 medium supplemented with 0 µM (blue circle) or 800 µM (red triangle) Spd for 2 h and then treated with 100 µg ml–1 ActD. After the start of ActD treatment (0 min), samples were withdrawn from the cultures at the time points indicated, and the levels of transgene mRNA and 25S rRNA were determined by Northern blot analysis using GUS and 25S rRNA probes, respectively. Northern hybridization data were quantified using the NIH Image or MultiGauge (Fuji Photo Film) software, and the intensities of the GUS signals were normalized to those of the 25S rRNA signals. The amounts of RNAs relative to the time zero value are shown in semi-log plots. Means ± S.D. of three independent experiments are shown. (F) Half-lives of S-uORF:GUS mRNA. Half-lives of S-uORF(WT/fs):GUS mRNA in Col-0, atupf1-1, or atupf3-1 background were calculated based on data presented in B to E by non-linear least square method. Asterisks indicate significant differences from the 35S::S-uORF(WT):GUS transgenic calli in Col-0 background under the same Spd condition (p < 0.01 by Welch's t-test).

172x200mm (300 x 300 DPI)


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