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Dual functions of yeast tRNA ligase in the unfolded protein response: unconventional cytoplasmic splicing of HAC1 pre-mRNA is not sufficient to release translational attenuation.

Mori T, Ogasawara C, Inada T, Englert M, Beier H, Takezawa M, Endo T, Yoshihisa T - Mol. Biol. Cell (2010)

Bottom Line: In the AtRLG1 cells, the HAC1 intron is circularized after splicing and remains associated on polysomes, impairing relief of the translational repression of HAC1(i) mRNA.RNA IP revealed that yeast Rlg1p is integrated in HAC1 mRNP, before Ire1p cleaves HAC1(u) mRNA.These results indicate that the splicing and the release of translational attenuation of HAC1 mRNA are separable steps and that Rlg1p has pivotal roles in both of these steps.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry and Division of Biological Science, Graduate School of Science, and Research Center for Materials Science, Nagoya University, Nagoya 464-8602, Japan.

ABSTRACT
The unfolded protein response (UPR) is an essential signal transduction to cope with protein-folding stress in the endoplasmic reticulum. In the yeast UPR, the unconventional splicing of HAC1 mRNA is a key step. Translation of HAC1 pre-mRNA (HAC1(u) mRNA) is attenuated on polysomes and restarted only after splicing upon the UPR. However, the precise mechanism of this restart remained unclear. Here we show that yeast tRNA ligase (Rlg1p/Trl1p) acting on HAC1 ligation has an unexpected role in HAC1 translation. An RLG1 homologue from Arabidopsis thaliana (AtRLG1) substitutes for yeast RLG1 in tRNA splicing but not in the UPR. Surprisingly, AtRlg1p ligates HAC1 exons, but the spliced mRNA (HAC1(i) mRNA) is not translated efficiently. In the AtRLG1 cells, the HAC1 intron is circularized after splicing and remains associated on polysomes, impairing relief of the translational repression of HAC1(i) mRNA. Furthermore, the HAC1 5' UTR itself enables yeast Rlg1p to regulate translation of the following ORF. RNA IP revealed that yeast Rlg1p is integrated in HAC1 mRNP, before Ire1p cleaves HAC1(u) mRNA. These results indicate that the splicing and the release of translational attenuation of HAC1 mRNA are separable steps and that Rlg1p has pivotal roles in both of these steps.

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HAC1 intron is circularized and retained in the AtRLG1 strain. Wild-type (RLG1), rlg1-4, rlg1-100, ire1Δ, RLG1-HA, and rlg1Δ/HA-AtRLG1[M74] strains were cultured in the presence (+ lanes) or absence (− lanes) of 2.0 μg/ml Tm for 60 min. Total RNAs were prepared and electrophoresed with 1.5% agarose/2.2 M formaldehyde gel. (A) HAC1 RNA species were detected by Northern blotting with an anti-HAC1u probe (top). ACT1 mRNA was also detected as a loading control (bottom). (B) The total RNAs from the AtRLG1[M74] cells were subjected to Northern blotting with an anti-HAC1 intron probe. Signal enhancement enables to observe the minor HAC1u mRNA in the presence of Tm. (C) Total RNAs prepared from HA-AtRLG1 cells were hybridized with probes a or b and treated with RNase H. In the lane labeled −, no oligonucleotide was added. RNAs recovered from the digestion were analyzed on a polyacrylamide gel. The HAC1 intron and its fragments were visualized by Northern blotting with either of the probe a (left) or b (right). Hybridization positions of these probes on putative linear or circular HAC1 introns are schematically described in the right.
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Figure 3: HAC1 intron is circularized and retained in the AtRLG1 strain. Wild-type (RLG1), rlg1-4, rlg1-100, ire1Δ, RLG1-HA, and rlg1Δ/HA-AtRLG1[M74] strains were cultured in the presence (+ lanes) or absence (− lanes) of 2.0 μg/ml Tm for 60 min. Total RNAs were prepared and electrophoresed with 1.5% agarose/2.2 M formaldehyde gel. (A) HAC1 RNA species were detected by Northern blotting with an anti-HAC1u probe (top). ACT1 mRNA was also detected as a loading control (bottom). (B) The total RNAs from the AtRLG1[M74] cells were subjected to Northern blotting with an anti-HAC1 intron probe. Signal enhancement enables to observe the minor HAC1u mRNA in the presence of Tm. (C) Total RNAs prepared from HA-AtRLG1 cells were hybridized with probes a or b and treated with RNase H. In the lane labeled −, no oligonucleotide was added. RNAs recovered from the digestion were analyzed on a polyacrylamide gel. The HAC1 intron and its fragments were visualized by Northern blotting with either of the probe a (left) or b (right). Hybridization positions of these probes on putative linear or circular HAC1 introns are schematically described in the right.

Mentions: Oligonucleotide-directed RNase H cleavage was performed as described as in Murray and Schoenberg (2008). Briefly, 10 μg of total RNA prepared from the HA-AtRLG1 cells was hybridized with 200 pmol of anti-sense probes a (HAC1_1310-291c) or b (HAC1_1360-41c) shown in Figure 3C, and treated with 20 U of RNase H at 37°C for 30 min. RNAs were extracted by phenol/chloroform and ethanol-precipitated, and then separated with 7 M urea/5% polyacrylamide gel. The HAC1 intron and its fragments were detected by Northern blotting with anti-sense probes a or b. Complete digestion of the HAC1 intron was confirmed by the absence of the Northern signal when the same probe was used for the digestion and detection.


Dual functions of yeast tRNA ligase in the unfolded protein response: unconventional cytoplasmic splicing of HAC1 pre-mRNA is not sufficient to release translational attenuation.

Mori T, Ogasawara C, Inada T, Englert M, Beier H, Takezawa M, Endo T, Yoshihisa T - Mol. Biol. Cell (2010)

HAC1 intron is circularized and retained in the AtRLG1 strain. Wild-type (RLG1), rlg1-4, rlg1-100, ire1Δ, RLG1-HA, and rlg1Δ/HA-AtRLG1[M74] strains were cultured in the presence (+ lanes) or absence (− lanes) of 2.0 μg/ml Tm for 60 min. Total RNAs were prepared and electrophoresed with 1.5% agarose/2.2 M formaldehyde gel. (A) HAC1 RNA species were detected by Northern blotting with an anti-HAC1u probe (top). ACT1 mRNA was also detected as a loading control (bottom). (B) The total RNAs from the AtRLG1[M74] cells were subjected to Northern blotting with an anti-HAC1 intron probe. Signal enhancement enables to observe the minor HAC1u mRNA in the presence of Tm. (C) Total RNAs prepared from HA-AtRLG1 cells were hybridized with probes a or b and treated with RNase H. In the lane labeled −, no oligonucleotide was added. RNAs recovered from the digestion were analyzed on a polyacrylamide gel. The HAC1 intron and its fragments were visualized by Northern blotting with either of the probe a (left) or b (right). Hybridization positions of these probes on putative linear or circular HAC1 introns are schematically described in the right.
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Figure 3: HAC1 intron is circularized and retained in the AtRLG1 strain. Wild-type (RLG1), rlg1-4, rlg1-100, ire1Δ, RLG1-HA, and rlg1Δ/HA-AtRLG1[M74] strains were cultured in the presence (+ lanes) or absence (− lanes) of 2.0 μg/ml Tm for 60 min. Total RNAs were prepared and electrophoresed with 1.5% agarose/2.2 M formaldehyde gel. (A) HAC1 RNA species were detected by Northern blotting with an anti-HAC1u probe (top). ACT1 mRNA was also detected as a loading control (bottom). (B) The total RNAs from the AtRLG1[M74] cells were subjected to Northern blotting with an anti-HAC1 intron probe. Signal enhancement enables to observe the minor HAC1u mRNA in the presence of Tm. (C) Total RNAs prepared from HA-AtRLG1 cells were hybridized with probes a or b and treated with RNase H. In the lane labeled −, no oligonucleotide was added. RNAs recovered from the digestion were analyzed on a polyacrylamide gel. The HAC1 intron and its fragments were visualized by Northern blotting with either of the probe a (left) or b (right). Hybridization positions of these probes on putative linear or circular HAC1 introns are schematically described in the right.
Mentions: Oligonucleotide-directed RNase H cleavage was performed as described as in Murray and Schoenberg (2008). Briefly, 10 μg of total RNA prepared from the HA-AtRLG1 cells was hybridized with 200 pmol of anti-sense probes a (HAC1_1310-291c) or b (HAC1_1360-41c) shown in Figure 3C, and treated with 20 U of RNase H at 37°C for 30 min. RNAs were extracted by phenol/chloroform and ethanol-precipitated, and then separated with 7 M urea/5% polyacrylamide gel. The HAC1 intron and its fragments were detected by Northern blotting with anti-sense probes a or b. Complete digestion of the HAC1 intron was confirmed by the absence of the Northern signal when the same probe was used for the digestion and detection.

Bottom Line: In the AtRLG1 cells, the HAC1 intron is circularized after splicing and remains associated on polysomes, impairing relief of the translational repression of HAC1(i) mRNA.RNA IP revealed that yeast Rlg1p is integrated in HAC1 mRNP, before Ire1p cleaves HAC1(u) mRNA.These results indicate that the splicing and the release of translational attenuation of HAC1 mRNA are separable steps and that Rlg1p has pivotal roles in both of these steps.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry and Division of Biological Science, Graduate School of Science, and Research Center for Materials Science, Nagoya University, Nagoya 464-8602, Japan.

ABSTRACT
The unfolded protein response (UPR) is an essential signal transduction to cope with protein-folding stress in the endoplasmic reticulum. In the yeast UPR, the unconventional splicing of HAC1 mRNA is a key step. Translation of HAC1 pre-mRNA (HAC1(u) mRNA) is attenuated on polysomes and restarted only after splicing upon the UPR. However, the precise mechanism of this restart remained unclear. Here we show that yeast tRNA ligase (Rlg1p/Trl1p) acting on HAC1 ligation has an unexpected role in HAC1 translation. An RLG1 homologue from Arabidopsis thaliana (AtRLG1) substitutes for yeast RLG1 in tRNA splicing but not in the UPR. Surprisingly, AtRlg1p ligates HAC1 exons, but the spliced mRNA (HAC1(i) mRNA) is not translated efficiently. In the AtRLG1 cells, the HAC1 intron is circularized after splicing and remains associated on polysomes, impairing relief of the translational repression of HAC1(i) mRNA. Furthermore, the HAC1 5' UTR itself enables yeast Rlg1p to regulate translation of the following ORF. RNA IP revealed that yeast Rlg1p is integrated in HAC1 mRNP, before Ire1p cleaves HAC1(u) mRNA. These results indicate that the splicing and the release of translational attenuation of HAC1 mRNA are separable steps and that Rlg1p has pivotal roles in both of these steps.

Show MeSH
Related in: MedlinePlus