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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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AtRlg1 proteins can splice HAC1 mRNA, whereas AtRLG1 strains are defective in the UPR. (A) Lysates were prepared from cultures of indicated strains in the absence (−) or presence (+) of 2.0 μg/ml Tm and then analyzed by Western blotting with anti-Hac1p antibodies (top). Appearance of unglycosylated Pdi1p (◁, ug-Pdi1p) was monitored to confirm effects of Tm (middle), and Por2p (mitochondrial porin) was used as a loading control (bottom). In the left part, yeast strains harboring ScRLG1 genes with no mutation (wt), rlg1-4 mutation (defective only in tRNA ligation), or rlg1-100 mutation (defective only in HAC1 mRNA ligation) on their chromosome were analyzed as control. Right, lysates from rlg1Δ strains complemented with various RLG1 genes were analyzed. In the bottom square, twofold more of lysates and the fivefold higher concentration of the anti-Hac1p antibodies were used to detect small amounts of Hac1p produced in the AtRLG1[M74] and AtRLG1[M54] cells. (B) Total RNAs (0.10 μg) prepared from the same set of strains in A was subjected to RT-PCR of HAC1 with a set of primers whose target was represented in the schematic drawing (arrows). ERO1 RT-PCR was also carried out to monitor the UPR, and ACT1 RT-PCR was done as a loading control. The top part represents RT-PCR products from yeast cells with ScRLG1 alleles on their chromosome. In the bottom panel, RT-PCR products from rlg1Δ strains complemented with various RLG1 genes on plasmids were analyzed. RT, reverse-transcription. ▶, RT-PCR fragment derived from HAC1u mRNA; ◁, that from HAC1i mRNA; gray triangles, RT-PCR fragments of ERO1 and ACT1 mRNAs. (C) A typical electrogram of sequencing reactions of the RT-PCR fragment amplified from the AtRLG1[M74] cells treated with Tm. Corresponding sequence is shown beneath the electrogram. The exon–exon junction is indicated by a vertical arrow.
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Figure 2: AtRlg1 proteins can splice HAC1 mRNA, whereas AtRLG1 strains are defective in the UPR. (A) Lysates were prepared from cultures of indicated strains in the absence (−) or presence (+) of 2.0 μg/ml Tm and then analyzed by Western blotting with anti-Hac1p antibodies (top). Appearance of unglycosylated Pdi1p (◁, ug-Pdi1p) was monitored to confirm effects of Tm (middle), and Por2p (mitochondrial porin) was used as a loading control (bottom). In the left part, yeast strains harboring ScRLG1 genes with no mutation (wt), rlg1-4 mutation (defective only in tRNA ligation), or rlg1-100 mutation (defective only in HAC1 mRNA ligation) on their chromosome were analyzed as control. Right, lysates from rlg1Δ strains complemented with various RLG1 genes were analyzed. In the bottom square, twofold more of lysates and the fivefold higher concentration of the anti-Hac1p antibodies were used to detect small amounts of Hac1p produced in the AtRLG1[M74] and AtRLG1[M54] cells. (B) Total RNAs (0.10 μg) prepared from the same set of strains in A was subjected to RT-PCR of HAC1 with a set of primers whose target was represented in the schematic drawing (arrows). ERO1 RT-PCR was also carried out to monitor the UPR, and ACT1 RT-PCR was done as a loading control. The top part represents RT-PCR products from yeast cells with ScRLG1 alleles on their chromosome. In the bottom panel, RT-PCR products from rlg1Δ strains complemented with various RLG1 genes on plasmids were analyzed. RT, reverse-transcription. ▶, RT-PCR fragment derived from HAC1u mRNA; ◁, that from HAC1i mRNA; gray triangles, RT-PCR fragments of ERO1 and ACT1 mRNAs. (C) A typical electrogram of sequencing reactions of the RT-PCR fragment amplified from the AtRLG1[M74] cells treated with Tm. Corresponding sequence is shown beneath the electrogram. The exon–exon junction is indicated by a vertical arrow.

Mentions: Next, we asked whether the RLG1 homologues can substitute for ScRLG1 in the UPR. The yeast strains with KlRLG1 and SpRLG as the sole RLG1 gene, as well as the ScRLG1 strain, grew in the presence of 0.25 μg/ml Tm, which caused accumulation of unfolded proteins in the ER through inhibition of N-glycosylation of newly synthesized secretory proteins (Figure 1B; see also Figure 2A). On the other hand, the AtRLG1[M54] and AtRLG1[M74] strains showed growth defects on the Tm plate, whereas the former grew better than the latter. Tm sensitivity of AtRLG1-expressing cells was quantified with the disk assay. An average diameter of the inhibition zone caused by a 6-mm filter disk absorbing 0.25 μg of Tm was 14.3 ± 0.3 mm for the wild-type yeast, 17.5 ± 0.3 mm for the HA-AtRLG1[M54] strain, and 18.5 ± 0.4 mm for the HA-AtRLG1[M74] strain, which confirms the higher sensitivity of HA-AtRLG1[M74] to Tm. These results suggest that the fungal homologues but not the plant homologue of Rlg1p can replace ScRLG1 in the UPR in vivo.


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)

AtRlg1 proteins can splice HAC1 mRNA, whereas AtRLG1 strains are defective in the UPR. (A) Lysates were prepared from cultures of indicated strains in the absence (−) or presence (+) of 2.0 μg/ml Tm and then analyzed by Western blotting with anti-Hac1p antibodies (top). Appearance of unglycosylated Pdi1p (◁, ug-Pdi1p) was monitored to confirm effects of Tm (middle), and Por2p (mitochondrial porin) was used as a loading control (bottom). In the left part, yeast strains harboring ScRLG1 genes with no mutation (wt), rlg1-4 mutation (defective only in tRNA ligation), or rlg1-100 mutation (defective only in HAC1 mRNA ligation) on their chromosome were analyzed as control. Right, lysates from rlg1Δ strains complemented with various RLG1 genes were analyzed. In the bottom square, twofold more of lysates and the fivefold higher concentration of the anti-Hac1p antibodies were used to detect small amounts of Hac1p produced in the AtRLG1[M74] and AtRLG1[M54] cells. (B) Total RNAs (0.10 μg) prepared from the same set of strains in A was subjected to RT-PCR of HAC1 with a set of primers whose target was represented in the schematic drawing (arrows). ERO1 RT-PCR was also carried out to monitor the UPR, and ACT1 RT-PCR was done as a loading control. The top part represents RT-PCR products from yeast cells with ScRLG1 alleles on their chromosome. In the bottom panel, RT-PCR products from rlg1Δ strains complemented with various RLG1 genes on plasmids were analyzed. RT, reverse-transcription. ▶, RT-PCR fragment derived from HAC1u mRNA; ◁, that from HAC1i mRNA; gray triangles, RT-PCR fragments of ERO1 and ACT1 mRNAs. (C) A typical electrogram of sequencing reactions of the RT-PCR fragment amplified from the AtRLG1[M74] cells treated with Tm. Corresponding sequence is shown beneath the electrogram. The exon–exon junction is indicated by a vertical arrow.
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Figure 2: AtRlg1 proteins can splice HAC1 mRNA, whereas AtRLG1 strains are defective in the UPR. (A) Lysates were prepared from cultures of indicated strains in the absence (−) or presence (+) of 2.0 μg/ml Tm and then analyzed by Western blotting with anti-Hac1p antibodies (top). Appearance of unglycosylated Pdi1p (◁, ug-Pdi1p) was monitored to confirm effects of Tm (middle), and Por2p (mitochondrial porin) was used as a loading control (bottom). In the left part, yeast strains harboring ScRLG1 genes with no mutation (wt), rlg1-4 mutation (defective only in tRNA ligation), or rlg1-100 mutation (defective only in HAC1 mRNA ligation) on their chromosome were analyzed as control. Right, lysates from rlg1Δ strains complemented with various RLG1 genes were analyzed. In the bottom square, twofold more of lysates and the fivefold higher concentration of the anti-Hac1p antibodies were used to detect small amounts of Hac1p produced in the AtRLG1[M74] and AtRLG1[M54] cells. (B) Total RNAs (0.10 μg) prepared from the same set of strains in A was subjected to RT-PCR of HAC1 with a set of primers whose target was represented in the schematic drawing (arrows). ERO1 RT-PCR was also carried out to monitor the UPR, and ACT1 RT-PCR was done as a loading control. The top part represents RT-PCR products from yeast cells with ScRLG1 alleles on their chromosome. In the bottom panel, RT-PCR products from rlg1Δ strains complemented with various RLG1 genes on plasmids were analyzed. RT, reverse-transcription. ▶, RT-PCR fragment derived from HAC1u mRNA; ◁, that from HAC1i mRNA; gray triangles, RT-PCR fragments of ERO1 and ACT1 mRNAs. (C) A typical electrogram of sequencing reactions of the RT-PCR fragment amplified from the AtRLG1[M74] cells treated with Tm. Corresponding sequence is shown beneath the electrogram. The exon–exon junction is indicated by a vertical arrow.
Mentions: Next, we asked whether the RLG1 homologues can substitute for ScRLG1 in the UPR. The yeast strains with KlRLG1 and SpRLG as the sole RLG1 gene, as well as the ScRLG1 strain, grew in the presence of 0.25 μg/ml Tm, which caused accumulation of unfolded proteins in the ER through inhibition of N-glycosylation of newly synthesized secretory proteins (Figure 1B; see also Figure 2A). On the other hand, the AtRLG1[M54] and AtRLG1[M74] strains showed growth defects on the Tm plate, whereas the former grew better than the latter. Tm sensitivity of AtRLG1-expressing cells was quantified with the disk assay. An average diameter of the inhibition zone caused by a 6-mm filter disk absorbing 0.25 μg of Tm was 14.3 ± 0.3 mm for the wild-type yeast, 17.5 ± 0.3 mm for the HA-AtRLG1[M54] strain, and 18.5 ± 0.4 mm for the HA-AtRLG1[M74] strain, which confirms the higher sensitivity of HA-AtRLG1[M74] to Tm. These results suggest that the fungal homologues but not the plant homologue of Rlg1p can replace ScRLG1 in the UPR in vivo.

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