Limits...
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.

Show MeSH
Assembly of SGs in S. pombe is independent of eIF2α phosphorylation. (A) Whole-cell protein extracts from wild-type cells and hri2Δ gcn2Δ mutants grown at 30°C and after a 15-min incubation at 42°C were prepared by alkaline extraction followed by trichloroacetic acid precipitation. The extracts were separated by SDS–PAGE and subjected to immunoblotting using antibodies that specifically recognize eIF2α phosphorylated at serine 51 and total eIF2α· (B) Fluorescence micrographs of the hri2Δgcn2Δ mutants expressing Vgl1-GFP (top), Pabp-GFP (middle) and eIF3b-tdTomato (lower) grown at 30°C and after a 15-min incubation at 42°C. One-hundred micrograms per milliliter of cycloheximide (CHX) were added 1 min before temperature shift. Bar: 5 µm.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2965253&req=5

Figure 7: Assembly of SGs in S. pombe is independent of eIF2α phosphorylation. (A) Whole-cell protein extracts from wild-type cells and hri2Δ gcn2Δ mutants grown at 30°C and after a 15-min incubation at 42°C were prepared by alkaline extraction followed by trichloroacetic acid precipitation. The extracts were separated by SDS–PAGE and subjected to immunoblotting using antibodies that specifically recognize eIF2α phosphorylated at serine 51 and total eIF2α· (B) Fluorescence micrographs of the hri2Δgcn2Δ mutants expressing Vgl1-GFP (top), Pabp-GFP (middle) and eIF3b-tdTomato (lower) grown at 30°C and after a 15-min incubation at 42°C. One-hundred micrograms per milliliter of cycloheximide (CHX) were added 1 min before temperature shift. Bar: 5 µm.

Mentions: Although SG formation can occur as a consequence of impaired translation initiation independently of eIF2α phosphorylation (27), SG assembly usually requires the stress-induced phosphorylation of eIF2α (28). To determine how fission yeast SGs assemble, we examine how the absence of eIF2α phosphorylation affected the assembly of SGs under thermal stress. It has been previously demonstrated that Hri2 and Gcn2 are the primary and secondary eIF2 kinases in response to heat shock in S. pombe (29), and the double-knockout strain contained no phosphorylated eIF2α (Figure 7A). We found that the relocalization of Vgl1, Pabp and eIF3b still occurred in the absence of Hri2 and Gcn2, which can be inhibited by the addition of cycloheximide (Figure 7B). We conclude that assembly of SGs in S. pombe is independent of eIF2α phosphorylation.Figure 7.


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)

Assembly of SGs in S. pombe is independent of eIF2α phosphorylation. (A) Whole-cell protein extracts from wild-type cells and hri2Δ gcn2Δ mutants grown at 30°C and after a 15-min incubation at 42°C were prepared by alkaline extraction followed by trichloroacetic acid precipitation. The extracts were separated by SDS–PAGE and subjected to immunoblotting using antibodies that specifically recognize eIF2α phosphorylated at serine 51 and total eIF2α· (B) Fluorescence micrographs of the hri2Δgcn2Δ mutants expressing Vgl1-GFP (top), Pabp-GFP (middle) and eIF3b-tdTomato (lower) grown at 30°C and after a 15-min incubation at 42°C. One-hundred micrograms per milliliter of cycloheximide (CHX) were added 1 min before temperature shift. Bar: 5 µm.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2965253&req=5

Figure 7: Assembly of SGs in S. pombe is independent of eIF2α phosphorylation. (A) Whole-cell protein extracts from wild-type cells and hri2Δ gcn2Δ mutants grown at 30°C and after a 15-min incubation at 42°C were prepared by alkaline extraction followed by trichloroacetic acid precipitation. The extracts were separated by SDS–PAGE and subjected to immunoblotting using antibodies that specifically recognize eIF2α phosphorylated at serine 51 and total eIF2α· (B) Fluorescence micrographs of the hri2Δgcn2Δ mutants expressing Vgl1-GFP (top), Pabp-GFP (middle) and eIF3b-tdTomato (lower) grown at 30°C and after a 15-min incubation at 42°C. One-hundred micrograms per milliliter of cycloheximide (CHX) were added 1 min before temperature shift. Bar: 5 µm.
Mentions: Although SG formation can occur as a consequence of impaired translation initiation independently of eIF2α phosphorylation (27), SG assembly usually requires the stress-induced phosphorylation of eIF2α (28). To determine how fission yeast SGs assemble, we examine how the absence of eIF2α phosphorylation affected the assembly of SGs under thermal stress. It has been previously demonstrated that Hri2 and Gcn2 are the primary and secondary eIF2 kinases in response to heat shock in S. pombe (29), and the double-knockout strain contained no phosphorylated eIF2α (Figure 7A). We found that the relocalization of Vgl1, Pabp and eIF3b still occurred in the absence of Hri2 and Gcn2, which can be inhibited by the addition of cycloheximide (Figure 7B). We conclude that assembly of SGs in S. pombe is independent of eIF2α phosphorylation.Figure 7.

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.

Show MeSH