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Vgl1, a multi-KH domain protein, is a novel component of the fission yeast stress granules required for cell survival under thermal stress.

Wen WL, Stevenson AL, Wang CY, Chen HJ, Kearsey SE, Norbury CJ, Watt S, Bähler J, Wang SW - Nucleic Acids Res. (2010)

Bottom Line: Unlike its counterpart in Saccharomyces cerevisiae, we found no indication that Vgl1 is required for the maintenance of cell ploidy in Schizosaccharomyces pombe.Under thermal stress, Vgl1 is rapidly relocalized from the ER to cytoplasmic foci that are distinct from P-bodies but contain stress granule markers such as poly(A)-binding protein and components of the translation initiation factor eIF3.Together, these observations demonstrated in S. pombe the presence of RNA granules with similar composition as mammalian stress granules and identified Vgl1 as a novel component that required for cell survival under thermal stress.

View Article: PubMed Central - PubMed

Affiliation: Division of Molecular and Genomic Medicine, National Health Research Institutes, 35 Keyan Road, Zhunan Town, Miaoli County 350, Taiwan.

ABSTRACT
Multiple KH-domain proteins, collectively known as vigilins, are evolutionarily highly conserved proteins that are present in eukaryotic organisms from yeast to metazoa. Proposed roles for vigilins include chromosome segregation, messenger RNA (mRNA) metabolism, translation and tRNA transport. As a step toward understanding its biological function, we have identified the fission yeast vigilin, designated Vgl1, and have investigated its role in cellular response to environmental stress. Unlike its counterpart in Saccharomyces cerevisiae, we found no indication that Vgl1 is required for the maintenance of cell ploidy in Schizosaccharomyces pombe. Instead, Vgl1 is required for cell survival under thermal stress, and vgl1Δ mutants lose their viability more rapidly than wild-type cells when incubated at high temperature. As for Scp160 in S. cerevisiae, Vgl1 bound polysomes accumulated at endoplasmic reticulum (ER) but in a microtubule-independent manner. Under thermal stress, Vgl1 is rapidly relocalized from the ER to cytoplasmic foci that are distinct from P-bodies but contain stress granule markers such as poly(A)-binding protein and components of the translation initiation factor eIF3. Together, these observations demonstrated in S. pombe the presence of RNA granules with similar composition as mammalian stress granules and identified Vgl1 as a novel component that required for cell survival under thermal stress.

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Colocalization of Vgl1 with poly(A)-binding protein (PABP) and eIF3b, a component of the translation initiation complex, but not with the decapping enzyme Dcp2 under thermal stress. (A) Merged images of fluorescence micrographs showing Dcp2-tdTomato (red) and Vgl1-GFP (green) localization in living cells grown at 30°C and after a 15-min incubation at 42°C. Bar: 5 µm. (B) Colocalization of Pabp-tdTomato (red) and Vgl1-GFP (green) in living cells under thermal stress (15 min at 42°C) that is inhibited by the addition of 100 µg/ml cycloheximide (CHX) 1 min before temperature shift. (C) Fluorescence micrographs of the vgl1Δ mutants expressing Pabp-GFP grown at 30°C and after a 15-min incubation at 42°C. (D) Fluorescence micrographs of pabpΔ mutants expressing Vgl1-GFP grown at 30°C and after a 15-min incubation at 42°C. (E) Merged images of fluorescence micrographs showing Vgl1-GFP (green) and eIF3b-tdTomato (red) localization in living cells grown at 30°C and after a 15-min incubation at 42°C that is inhibited by the addition of 100 µg/ml cycloheximide (CHX) 1 min before temperature shift.
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Figure 6: Colocalization of Vgl1 with poly(A)-binding protein (PABP) and eIF3b, a component of the translation initiation complex, but not with the decapping enzyme Dcp2 under thermal stress. (A) Merged images of fluorescence micrographs showing Dcp2-tdTomato (red) and Vgl1-GFP (green) localization in living cells grown at 30°C and after a 15-min incubation at 42°C. Bar: 5 µm. (B) Colocalization of Pabp-tdTomato (red) and Vgl1-GFP (green) in living cells under thermal stress (15 min at 42°C) that is inhibited by the addition of 100 µg/ml cycloheximide (CHX) 1 min before temperature shift. (C) Fluorescence micrographs of the vgl1Δ mutants expressing Pabp-GFP grown at 30°C and after a 15-min incubation at 42°C. (D) Fluorescence micrographs of pabpΔ mutants expressing Vgl1-GFP grown at 30°C and after a 15-min incubation at 42°C. (E) Merged images of fluorescence micrographs showing Vgl1-GFP (green) and eIF3b-tdTomato (red) localization in living cells grown at 30°C and after a 15-min incubation at 42°C that is inhibited by the addition of 100 µg/ml cycloheximide (CHX) 1 min before temperature shift.

Mentions: Several different types of RNA granules have been described (26). Cytoplasmic processing bodies, also known as P-bodies (PBs), are observed in actively growing unstressed cells, that contain untranslating mRNA in conjunction with enzymes involved in translation repression and mRNA decapping and degradation. In contrast, SGs are cytoplasmic phase-dense structures that are not seen in cells growing under favorable conditions but are rapidly induced in response to environmental stress. Although SGs and PBs share some protein and mRNA components, they also contain a number of unique markers specific to each structure. To catalog the Vgl1 granules, we used fluorescence microscopy to determine the protein composition of Vgl1 granules. The signature constituents of PBs are components of the decay machinery that removes the m7G (7-methylguanosine) cap and degrades mRNA from the 5′ ends, for example, the Dcp1–Dcp2 complex. To determine if these proteins were components of a single granule type, we examined their localization with a Dcp2-tdTomato fusion protein. As shown in Figure 6A, Dcp2 formed cytoplasmic foci in actively growing unstressed cells that we refer to as fission yeast PBs. Thermal stress has little effect on these structures that are largely distinct from the Vgl1 cytoplasmic granules. In contrast, we observed that, under thermal stress, poly(A) binding protein Pabp-tdTomato (the signature constituents of SGs) colocalized almost completely with GFP fusions of Vgl1 (Figure 6B). This colocalization indicated that these proteins are components of a single granule with similar composition to mammalian SGs. This is further supported by the fact that, as in mammalian cells, drugs such as cycloheximide (that stabilize polysomes by freezing ribosomes on translating mRNA) inhibited the assembly of these granules (Figure 6B + CHX). We refer to these granules as fission yeast SGs and note that they are almost certainly the same eIF3-positive granules identified earlier (22).Figure 6.


Vgl1, a multi-KH domain protein, is a novel component of the fission yeast stress granules required for cell survival under thermal stress.

Wen WL, Stevenson AL, Wang CY, Chen HJ, Kearsey SE, Norbury CJ, Watt S, Bähler J, Wang SW - Nucleic Acids Res. (2010)

Colocalization of Vgl1 with poly(A)-binding protein (PABP) and eIF3b, a component of the translation initiation complex, but not with the decapping enzyme Dcp2 under thermal stress. (A) Merged images of fluorescence micrographs showing Dcp2-tdTomato (red) and Vgl1-GFP (green) localization in living cells grown at 30°C and after a 15-min incubation at 42°C. Bar: 5 µm. (B) Colocalization of Pabp-tdTomato (red) and Vgl1-GFP (green) in living cells under thermal stress (15 min at 42°C) that is inhibited by the addition of 100 µg/ml cycloheximide (CHX) 1 min before temperature shift. (C) Fluorescence micrographs of the vgl1Δ mutants expressing Pabp-GFP grown at 30°C and after a 15-min incubation at 42°C. (D) Fluorescence micrographs of pabpΔ mutants expressing Vgl1-GFP grown at 30°C and after a 15-min incubation at 42°C. (E) Merged images of fluorescence micrographs showing Vgl1-GFP (green) and eIF3b-tdTomato (red) localization in living cells grown at 30°C and after a 15-min incubation at 42°C that is inhibited by the addition of 100 µg/ml cycloheximide (CHX) 1 min before temperature shift.
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Figure 6: Colocalization of Vgl1 with poly(A)-binding protein (PABP) and eIF3b, a component of the translation initiation complex, but not with the decapping enzyme Dcp2 under thermal stress. (A) Merged images of fluorescence micrographs showing Dcp2-tdTomato (red) and Vgl1-GFP (green) localization in living cells grown at 30°C and after a 15-min incubation at 42°C. Bar: 5 µm. (B) Colocalization of Pabp-tdTomato (red) and Vgl1-GFP (green) in living cells under thermal stress (15 min at 42°C) that is inhibited by the addition of 100 µg/ml cycloheximide (CHX) 1 min before temperature shift. (C) Fluorescence micrographs of the vgl1Δ mutants expressing Pabp-GFP grown at 30°C and after a 15-min incubation at 42°C. (D) Fluorescence micrographs of pabpΔ mutants expressing Vgl1-GFP grown at 30°C and after a 15-min incubation at 42°C. (E) Merged images of fluorescence micrographs showing Vgl1-GFP (green) and eIF3b-tdTomato (red) localization in living cells grown at 30°C and after a 15-min incubation at 42°C that is inhibited by the addition of 100 µg/ml cycloheximide (CHX) 1 min before temperature shift.
Mentions: Several different types of RNA granules have been described (26). Cytoplasmic processing bodies, also known as P-bodies (PBs), are observed in actively growing unstressed cells, that contain untranslating mRNA in conjunction with enzymes involved in translation repression and mRNA decapping and degradation. In contrast, SGs are cytoplasmic phase-dense structures that are not seen in cells growing under favorable conditions but are rapidly induced in response to environmental stress. Although SGs and PBs share some protein and mRNA components, they also contain a number of unique markers specific to each structure. To catalog the Vgl1 granules, we used fluorescence microscopy to determine the protein composition of Vgl1 granules. The signature constituents of PBs are components of the decay machinery that removes the m7G (7-methylguanosine) cap and degrades mRNA from the 5′ ends, for example, the Dcp1–Dcp2 complex. To determine if these proteins were components of a single granule type, we examined their localization with a Dcp2-tdTomato fusion protein. As shown in Figure 6A, Dcp2 formed cytoplasmic foci in actively growing unstressed cells that we refer to as fission yeast PBs. Thermal stress has little effect on these structures that are largely distinct from the Vgl1 cytoplasmic granules. In contrast, we observed that, under thermal stress, poly(A) binding protein Pabp-tdTomato (the signature constituents of SGs) colocalized almost completely with GFP fusions of Vgl1 (Figure 6B). This colocalization indicated that these proteins are components of a single granule with similar composition to mammalian SGs. This is further supported by the fact that, as in mammalian cells, drugs such as cycloheximide (that stabilize polysomes by freezing ribosomes on translating mRNA) inhibited the assembly of these granules (Figure 6B + CHX). We refer to these granules as fission yeast SGs and note that they are almost certainly the same eIF3-positive granules identified earlier (22).Figure 6.

Bottom Line: Unlike its counterpart in Saccharomyces cerevisiae, we found no indication that Vgl1 is required for the maintenance of cell ploidy in Schizosaccharomyces pombe.Under thermal stress, Vgl1 is rapidly relocalized from the ER to cytoplasmic foci that are distinct from P-bodies but contain stress granule markers such as poly(A)-binding protein and components of the translation initiation factor eIF3.Together, these observations demonstrated in S. pombe the presence of RNA granules with similar composition as mammalian stress granules and identified Vgl1 as a novel component that required for cell survival under thermal stress.

View Article: PubMed Central - PubMed

Affiliation: Division of Molecular and Genomic Medicine, National Health Research Institutes, 35 Keyan Road, Zhunan Town, Miaoli County 350, Taiwan.

ABSTRACT
Multiple KH-domain proteins, collectively known as vigilins, are evolutionarily highly conserved proteins that are present in eukaryotic organisms from yeast to metazoa. Proposed roles for vigilins include chromosome segregation, messenger RNA (mRNA) metabolism, translation and tRNA transport. As a step toward understanding its biological function, we have identified the fission yeast vigilin, designated Vgl1, and have investigated its role in cellular response to environmental stress. Unlike its counterpart in Saccharomyces cerevisiae, we found no indication that Vgl1 is required for the maintenance of cell ploidy in Schizosaccharomyces pombe. Instead, Vgl1 is required for cell survival under thermal stress, and vgl1Δ mutants lose their viability more rapidly than wild-type cells when incubated at high temperature. As for Scp160 in S. cerevisiae, Vgl1 bound polysomes accumulated at endoplasmic reticulum (ER) but in a microtubule-independent manner. Under thermal stress, Vgl1 is rapidly relocalized from the ER to cytoplasmic foci that are distinct from P-bodies but contain stress granule markers such as poly(A)-binding protein and components of the translation initiation factor eIF3. Together, these observations demonstrated in S. pombe the presence of RNA granules with similar composition as mammalian stress granules and identified Vgl1 as a novel component that required for cell survival under thermal stress.

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