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. 2005 Aug 1;19(15):1799-810.
doi: 10.1101/gad.1317105. Epub 2005 Jul 18.

Nascent peptide-mediated translation elongation arrest coupled with mRNA degradation in the CGS1 gene of Arabidopsis

Hitoshi Onouchi  1 , Yoko Nagami, Yuhi Haraguchi, Mari Nakamoto, Yoshiko Nishimura, Ryoko Sakurai, Nobuhiro Nagao, Daisuke Kawasaki, Yoshitomo Kadokura, Satoshi Naito
Affiliations

Nascent peptide-mediated translation elongation arrest coupled with mRNA degradation in the CGS1 gene of Arabidopsis

Hitoshi Onouchi et al. Genes Dev. .

Abstract

Expression of the Arabidopsis CGS1 gene that codes for cystathionine gamma-synthase is feedback regulated at the step of mRNA stability in response to S-adenosyl-L-methionine (AdoMet). A short stretch of amino acid sequence, called the MTO1 region, encoded by the first exon of CGS1 itself is involved in this regulation. Here, we demonstrate, using a cell-free system, that AdoMet induces temporal translation elongation arrest at the Ser-94 codon located immediately downstream of the MTO1 region, by analyzing a translation intermediate and performing primer extension inhibition (toeprint) analysis. This translation arrest precedes the formation of a degradation intermediate of CGS1 mRNA, which has its 5' end points near the 5' edge of the stalled ribosome. The position of ribosome stalling also suggests that the MTO1 region in nascent peptide resides in the ribosomal exit tunnel when translation elongation is temporarily arrested. In addition to the MTO1 region amino acid sequence, downstream Trp-93 is also important for the AdoMet-induced translation arrest. This is the first example of nascent peptide-mediated translation elongation arrest coupled with mRNA degradation in eukaryotes. Furthermore, our data suggest that the ribosome stalls at the step of translocation rather than at the step of peptidyl transfer.

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Figures

Figure 1.
Figure 1.
Translation elongation arrest and RNA degradation. (A) Schematic representation of GST:Ex1:Luc RNA. (B,C) Immunoblot analysis. GST:Ex1(WT):Luc RNA or GST:Ex1(mto1-1): Luc RNA was translated for 30 min in the presence (+) or absence (-) of 1 mM AdoMet. The translation products were separated by SDS-PAGE and subjected to immunoblot analysis with anti-GST antibody. In C, the WT samples were treated with RNase A as indicated before separation by SDS-PAGE. The 106-kDa full-length translation product (full-length), the 55-kDa AdoMet-dependent peptidyl-RNA (arrest-PR), and the AdoMet-dependent band shifted by the RNase A treatment (arrest-P) are marked. (D,E) Time course analyses. GST:Ex1(WT):Luc RNA was translated in the presence (right) or absence (left) of AdoMet. Aliquots were withdrawn at the indicated time points and subjected to immunoblot analysis with anti-GST antibody (D) or Northern blot analysis with LUC 3′ probe (E). The full-length RNA (FL RNA) and the 5′-truncated RNA (5′-trunc) are marked in E. Representative results of duplicate to triplicate experiments are shown.
Figure 2.
Figure 2.
Pulse-chase analysis of the translation arrest products. (A) Schematic representation of GST:Ex1(WT) RNA. Positions of methionine codons are marked with vertical bars. (B) GST:Ex1(WT) RNA was translated in the presence of [35S]methionine with (right) or without (left) AdoMet. Edeine was added 5 min after the start of translation. Aliquots were withdrawn at the indicated time points, and the radioactive signals were detected after separation by SDS-PAGE. The 45-kDa full-length translation product (full-length) and the 55-kDa AdoMet-dependent peptidyl RNA band (arrest-PR) are marked. The asterisk indicates a 45-kDa background signal detected at 15 min. (C) The radioactive signals in B were quantified by NIH Image software. Open circle indicates 45-kDa full-length product; solid circle indicates 55-kDa AdoMet-dependent peptidyl RNA. (Inset) The 45-kDa background signal in B was ignored as this signal was no longer detectable after RNase A treatment. Representative results of triplicate experiments are shown.
Figure 3.
Figure 3.
Association of the peptidyl-RNA with ribosomes. (A) GST:Ex1(WT) RNA was translated for 60 min in the presence (right panels) or absence (left panels) of AdoMet. The samples were fractionated by 10%-30% (w/v) sucrose density gradient centrifugation. UV absorbance profile at 254 nm (upper panels) and immunoblot analysis of each fraction with anti-GST antibody (lower panels) are shown. (B) The same as in A except that the samples were incubated for 30 min with 1 mM puromycin before centrifugation. The 45-kDa full-length translation product (full-length), the 55-kDa AdoMet-dependent peptidyl-RNA band (arrest-PR), and a 35-kDa band, possibly a peptidyl-puromycin (arrest-P★), are marked. Lanes BC represent samples before centrifugation. Representative results of quadruplicate (A) or duplicate (B) experiments are shown.
Figure 4.
Figure 4.
Determination of the position of the translation elongation arrest. (A,B) Effects of stop codon substitutions. GST:Ex1:Luc RNAs carrying the stop codon substitution constructs were translated for 30 min (A) or 90 min (B) in the presence (right) or absence (left) of AdoMet. Aliquots were subjected to immunoblot analysis with anti-GST antibody (A) or Northern blot analysis with LUC 3′ probe (B). The full-length polypeptide (106 kDa) is not seen in A. Brackets in A mark the complete translation products terminated at the introduced stop codon. (C) Toeprint analysis. GST:Ex1Δsl(WT):Luc RNA (lanes 1,2,5-10) or GST:Ex1Δsl(mto1-1):Luc RNA (lanes 3,4,11,12) was translated for 30 min in the presence (+) or absence (-) of AdoMet. In lanes 5 and 6, EDTA was added to a final concentration of 5 mM at 30 min, and the samples were incubated for another 5 min. MgCl2 was added to 10 mM prior to the primer extension reaction. In lanes 7 and 8, puromycin (Puro) was added to 1 mM at 30 min, and the reaction mixtures were incubated for an additional 10 min. In lanes 9-12, 0.5 mg/mL CHX was added at 30 min. The red arrowheads mark the most 5′-proximal AdoMet-dependent toeprint signals at the 293rd nucleotide position. The sequence ladder (shown in the sense strand sequence) was synthesized using the same primer as used for toeprinting. The MTO1 region and the Δsl:Luc junction are indicated. (D) Schematic representation of the translation elongation arrest position. The red arrowhead indicates the position of the most 5′-proximal AdoMet-dependent toeprint signal. The positions of the P and A sites of the stalled ribosome deduced from the toeprint signal are shown. Open squares indicate codons whose substitution to a stop codon abolished production of the AdoMet-dependent peptidyl-tRNA and 5′-truncated RNA, while solid squares indicate codons without any effect. Amino acid residues that are conserved among plant CGSs are reversed in purple and those with conservative changes are shaded in light blue (Ominato et al. 2002). The pink diamonds indicate the positions of the 5′ ends of the 5′-truncated RNA species with the large one pointing to the most prominent one (Chiba et al. 2003). Representative results of triplicate to quadruplicate experiments are shown.
Figure 5.
Figure 5.
Amino acid changes affecting translation elongation arrest and determination of tRNA species in the arrest product. (A) Schematic representation of GST:Ex1 RNAs carrying mutations and GST:Ex1′ nonstop RNAs. (B) GST:Ex1 RNAs carrying different alleles of the mto1 mutation were translated for 30 min in the presence or absence of AdoMet, and the translation products were analyzed by immunoblot analysis with anti-GST antibody. (C) GST:Ex1 RNAs carrying amino acid substitutions or insertion were translated and analyzed as in B. The open arrow marks the AdoMet-dependent band shifted by substitution of Ser-94 to alanine (S94A). (D) Comparison of the mobility in SDS-PAGE. GST:Ex1 RNAs carrying alanine substitutions and GST:Ex1′ nonstop RNAs were translated and analyzed as in B. The samples were separated by SDS-PAGE before (upper) or after (lower) RNase A treatment. The solid and open arrows mark the peptidyl-tRNASer and peptidyl-tRNAAla, respectively. (E) In vitro translation in the presence of labeled tRNAs. 32P-labeled tRNASer(AGA) (left) or tRNALys(CUU) (right) (2 × 105 cpm) was preincubated for 10 min at 25°C in wheat germ extract without an RNA template. Then, GST:Ex1(WT) RNA was translated in a 20-μL reaction mixture for 30 min with (+) or without (-) AdoMet, and 5 μL of each sample was separated by SDS-PAGE. The radioactive signals were visualized by an image analyzer. Nonstop RNAs GST:Ex1′(S94-ns) (left) or GST:Ex1′(K92-ns) (right) was used as a positive control. Representative results of duplicate to triplicate experiments are shown.
Figure 6.
Figure 6.
Determination of the arrested step in translation elongation. (A,B) Schematic representations of the ribosome stalled at the translocation step at Ser-94 (A) or at one of the steps after the translocation (B), and the reactions that follow the resumption of translation. The steps inhibited by CHX, Hyg, and Fus are indicated. The solid and open arrowheads represent the toeprint signals at the 293rd and 296th nucleotides, respectively. (C) Effects of translation elongation inhibitors on the first round of peptidyl transfer after the resumption of translation. GST:Ex1(S94A) RNA was translated in the presence of AdoMet. Edeine was added at 5 min. Hyg (1 mM) or CHX (1 mg/mL) was added at 30 min, and the reaction mixtures were further incubated as indicated. Positions of the peptidyl-tRNAAla (pink arrow) and peptidyl-tRNAAsn (blue arrow) are marked. (D) Effects of translation elongation inhibitors on toeprinting. GST:Ex1Δsl(WT):Luc RNA was translated in the presence (+) or absence (-) of AdoMet. Hyg (1 mM), CHX (1 mg/mL), or Fus (1 mM) was added at 30 min, and the reaction mixtures were incubated for additional 20 min prior to the primer extension reaction. The solid and open arrowheads mark the AdoMet-dependent toeprint signals at the 293rd and 296th nucleotides, respectively. Representative results of triplicate (C) or duplicate (D) experiments are shown.
Figure 7.
Figure 7.
Model for CGS1 exon 1-mediated post-transcriptional regulation. When the cellular concentration of AdoMet is high, translation of CGS1 mRNA is temporarily arrested when the A site of the ribosome resides at the Ser-94 codon of CGS1. The MTO1 region in the nascent polypeptide acts within the ribosomal exit tunnel to cause translation elongation arrest. Following the translation arrest, mRNA degradation occurs upstream of the stalled ribosome, resulting in production of the 5′-truncated RNA species. The ribonuclease (Pacman) responsible for the mRNA degradation remains to be identified.
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