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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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ScRlg1p also interacts with the HAC1 intron spliced by AtRlg1p. (A) HA-AtRlg1[M74]p and FLAG-ScRlg1p were coexpressed under the rlg1Δ background, and their ability to compliment UPR defects was tested on a 0.25 μg/ml Tm-containing plate (right). (B) Cell extracts were prepared from the HA-AtRLG1[M74] strain (left set) and the HA-AtRLG1[M74]/FLAG-ScRLG1 strain (right set), and tagged proteins were immunoprecipitated with immobilized antibody-resins indicated in the row IP. Top, Northern blotting with the anti-HAC1u probe. T, 1/10 of total sample; U, 1/10 of unbound fraction; B, 3/4 of bound fraction. Bottom, protein staining of the SDS-gel analyzing the same samples.
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Figure 8: ScRlg1p also interacts with the HAC1 intron spliced by AtRlg1p. (A) HA-AtRlg1[M74]p and FLAG-ScRlg1p were coexpressed under the rlg1Δ background, and their ability to compliment UPR defects was tested on a 0.25 μg/ml Tm-containing plate (right). (B) Cell extracts were prepared from the HA-AtRLG1[M74] strain (left set) and the HA-AtRLG1[M74]/FLAG-ScRLG1 strain (right set), and tagged proteins were immunoprecipitated with immobilized antibody-resins indicated in the row IP. Top, Northern blotting with the anti-HAC1u probe. T, 1/10 of total sample; U, 1/10 of unbound fraction; B, 3/4 of bound fraction. Bottom, protein staining of the SDS-gel analyzing the same samples.

Mentions: Then we devised a setup to examine whether ScRlg1p binds the HAC1 intron because the intron accumulates only in the AtRLG1 strains. When we constructed an rlg1Δ strain harboring both HA-AtRLG1[M74] and FLAG-ScRLG1 on plasmids, it showed intermediate Tm sensitivity (Figure 8A). Then, we immunoprecipitated FLAG-ScRlg1p from Tm-treated cell extracts of this strain and found that HAC1u mRNA, HAC1i mRNA, and HAC1 intron were coprecipitated as in the case of AtRlg1p, whereas no such RNA was detected in the immunoprecipitate from the control strain that did not express FLAG-ScRlg1p (Figure 8B, top). When we analyzed protein compositions of the immunoprecipitates, only negligible amounts of HA-AtRlg1p were found in anti-FLAG immunoprecipitates and vice versa (Figure 8B, bottom). These results indicate that ScRlg1p can bind the HAC1 intron produced by splicing reaction with HA-AtRlg1p, as well.


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)

ScRlg1p also interacts with the HAC1 intron spliced by AtRlg1p. (A) HA-AtRlg1[M74]p and FLAG-ScRlg1p were coexpressed under the rlg1Δ background, and their ability to compliment UPR defects was tested on a 0.25 μg/ml Tm-containing plate (right). (B) Cell extracts were prepared from the HA-AtRLG1[M74] strain (left set) and the HA-AtRLG1[M74]/FLAG-ScRLG1 strain (right set), and tagged proteins were immunoprecipitated with immobilized antibody-resins indicated in the row IP. Top, Northern blotting with the anti-HAC1u probe. T, 1/10 of total sample; U, 1/10 of unbound fraction; B, 3/4 of bound fraction. Bottom, protein staining of the SDS-gel analyzing the same samples.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC2965688&req=5

Figure 8: ScRlg1p also interacts with the HAC1 intron spliced by AtRlg1p. (A) HA-AtRlg1[M74]p and FLAG-ScRlg1p were coexpressed under the rlg1Δ background, and their ability to compliment UPR defects was tested on a 0.25 μg/ml Tm-containing plate (right). (B) Cell extracts were prepared from the HA-AtRLG1[M74] strain (left set) and the HA-AtRLG1[M74]/FLAG-ScRLG1 strain (right set), and tagged proteins were immunoprecipitated with immobilized antibody-resins indicated in the row IP. Top, Northern blotting with the anti-HAC1u probe. T, 1/10 of total sample; U, 1/10 of unbound fraction; B, 3/4 of bound fraction. Bottom, protein staining of the SDS-gel analyzing the same samples.
Mentions: Then we devised a setup to examine whether ScRlg1p binds the HAC1 intron because the intron accumulates only in the AtRLG1 strains. When we constructed an rlg1Δ strain harboring both HA-AtRLG1[M74] and FLAG-ScRLG1 on plasmids, it showed intermediate Tm sensitivity (Figure 8A). Then, we immunoprecipitated FLAG-ScRlg1p from Tm-treated cell extracts of this strain and found that HAC1u mRNA, HAC1i mRNA, and HAC1 intron were coprecipitated as in the case of AtRlg1p, whereas no such RNA was detected in the immunoprecipitate from the control strain that did not express FLAG-ScRlg1p (Figure 8B, top). When we analyzed protein compositions of the immunoprecipitates, only negligible amounts of HA-AtRlg1p were found in anti-FLAG immunoprecipitates and vice versa (Figure 8B, bottom). These results indicate that ScRlg1p can bind the HAC1 intron produced by splicing reaction with HA-AtRlg1p, as well.

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