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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 associated with polysomes in the HA-AtRLG1 strain. Distribution of HAC1 RNA species was examined by polysome analysis. Extracts from HA-ScRLG1 cells (A and C) and HA-AtRLG1[M74] cells (B and D) cultured in the absence (A and B, −Tm) or the presence (C and D, +Tm) of 2.0 μg/ml Tm were subjected to 10–50% sucrose density gradient ultracentrifugation. Fractions were collected from the top (left) to the bottom (right). In each set of three panels, the top, middle, and bottom panels represent A260 trace, HAC1 RNAs, and ACT1 RNA, respectively. In the top panel of A, peak assignments are shown. In the middle panel, positions of HAC1 RNA species were indicated in the right. Amounts of HAC1-related RNAs in the sucrose gradient fractions of HA-AtRLG1 extracts from control (E) or Tm-treated (F) cells were compared with standard RNAs transcribed in vitro. In the bottom panel, Northern signals of HAC1u mRNA (□), HCA1i mRNA (▩) and HAC1 intron (■) were quantified and expressed in arbitrary units. L, cell extract; Std, 0.22 fmol each of HAC1u mRNA, HAC1i mRNA, and HAC1 intron.
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Figure 4: HAC1 intron is associated with polysomes in the HA-AtRLG1 strain. Distribution of HAC1 RNA species was examined by polysome analysis. Extracts from HA-ScRLG1 cells (A and C) and HA-AtRLG1[M74] cells (B and D) cultured in the absence (A and B, −Tm) or the presence (C and D, +Tm) of 2.0 μg/ml Tm were subjected to 10–50% sucrose density gradient ultracentrifugation. Fractions were collected from the top (left) to the bottom (right). In each set of three panels, the top, middle, and bottom panels represent A260 trace, HAC1 RNAs, and ACT1 RNA, respectively. In the top panel of A, peak assignments are shown. In the middle panel, positions of HAC1 RNA species were indicated in the right. Amounts of HAC1-related RNAs in the sucrose gradient fractions of HA-AtRLG1 extracts from control (E) or Tm-treated (F) cells were compared with standard RNAs transcribed in vitro. In the bottom panel, Northern signals of HAC1u mRNA (□), HCA1i mRNA (▩) and HAC1 intron (■) were quantified and expressed in arbitrary units. L, cell extract; Std, 0.22 fmol each of HAC1u mRNA, HAC1i mRNA, and HAC1 intron.

Mentions: The existence of the stable intron in the AtRLG1 strain suggests that base-pairing between the 5′ UTR of the HAC1i mRNA and the cleaved intron is still maintained in this strain to attenuate Hac1p translation. To examine this possibility, we performed sucrose density gradient centrifugation to compare distribution of HAC1 RNA species and polysomes. HAC1u and HAC1i mRNAs both in ScRLG1 and AtRLG1 extracts appeared in two peaks in the sucrose density gradient as reported (Rüegsegger et al., 2001; Kuhn et al., 2001): one peak sedimented near 40S and 60S ribosomal subunits, and the other cosedimented with polysomes (Figure 4). More HAC1i was associated with polysomes than HAC1u. In the AtRLG1 extracts, some HAC1 intron was detected in the lighter (nonpolysomal) fraction, but its significant portion was cosedimented with polysomes (Figure 4, B and D). No intron was detected in the fractions of ScRLG1 extracts (Figure 4, A and C).


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 associated with polysomes in the HA-AtRLG1 strain. Distribution of HAC1 RNA species was examined by polysome analysis. Extracts from HA-ScRLG1 cells (A and C) and HA-AtRLG1[M74] cells (B and D) cultured in the absence (A and B, −Tm) or the presence (C and D, +Tm) of 2.0 μg/ml Tm were subjected to 10–50% sucrose density gradient ultracentrifugation. Fractions were collected from the top (left) to the bottom (right). In each set of three panels, the top, middle, and bottom panels represent A260 trace, HAC1 RNAs, and ACT1 RNA, respectively. In the top panel of A, peak assignments are shown. In the middle panel, positions of HAC1 RNA species were indicated in the right. Amounts of HAC1-related RNAs in the sucrose gradient fractions of HA-AtRLG1 extracts from control (E) or Tm-treated (F) cells were compared with standard RNAs transcribed in vitro. In the bottom panel, Northern signals of HAC1u mRNA (□), HCA1i mRNA (▩) and HAC1 intron (■) were quantified and expressed in arbitrary units. L, cell extract; Std, 0.22 fmol each of HAC1u mRNA, HAC1i mRNA, and HAC1 intron.
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Figure 4: HAC1 intron is associated with polysomes in the HA-AtRLG1 strain. Distribution of HAC1 RNA species was examined by polysome analysis. Extracts from HA-ScRLG1 cells (A and C) and HA-AtRLG1[M74] cells (B and D) cultured in the absence (A and B, −Tm) or the presence (C and D, +Tm) of 2.0 μg/ml Tm were subjected to 10–50% sucrose density gradient ultracentrifugation. Fractions were collected from the top (left) to the bottom (right). In each set of three panels, the top, middle, and bottom panels represent A260 trace, HAC1 RNAs, and ACT1 RNA, respectively. In the top panel of A, peak assignments are shown. In the middle panel, positions of HAC1 RNA species were indicated in the right. Amounts of HAC1-related RNAs in the sucrose gradient fractions of HA-AtRLG1 extracts from control (E) or Tm-treated (F) cells were compared with standard RNAs transcribed in vitro. In the bottom panel, Northern signals of HAC1u mRNA (□), HCA1i mRNA (▩) and HAC1 intron (■) were quantified and expressed in arbitrary units. L, cell extract; Std, 0.22 fmol each of HAC1u mRNA, HAC1i mRNA, and HAC1 intron.
Mentions: The existence of the stable intron in the AtRLG1 strain suggests that base-pairing between the 5′ UTR of the HAC1i mRNA and the cleaved intron is still maintained in this strain to attenuate Hac1p translation. To examine this possibility, we performed sucrose density gradient centrifugation to compare distribution of HAC1 RNA species and polysomes. HAC1u and HAC1i mRNAs both in ScRLG1 and AtRLG1 extracts appeared in two peaks in the sucrose density gradient as reported (Rüegsegger et al., 2001; Kuhn et al., 2001): one peak sedimented near 40S and 60S ribosomal subunits, and the other cosedimented with polysomes (Figure 4). More HAC1i was associated with polysomes than HAC1u. In the AtRLG1 extracts, some HAC1 intron was detected in the lighter (nonpolysomal) fraction, but its significant portion was cosedimented with polysomes (Figure 4, B and D). No intron was detected in the fractions of ScRLG1 extracts (Figure 4, A and C).

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