Cytoplasmic deadenylase Ccr4 is required for translational repression of LRG1 mRNA in the stationary phase

Ccr4 is a major cytoplasmic deadenylase involved in mRNA poly(A) tail shortening in Saccharomyces cerevisiae. We have previously shown that Ccr4 negatively regulates expression of LRG1 mRNA encoding a GTPase-activating protein for the small GTPase Rho1, a component of cell wall integrity pathway, and deletion of LRG1 suppresses the temperature-sensitive growth defect of the ccr4Δ mutant. We have also shown that the slow growth of the ccr4Δ mutant is suppressed by deletion of another gene, PBP1, encoding a poly(A)-binding protein (Pab1)-binding protein 1; however, the underlying mechanism still remains unknown. In this study, we investigated how ccr4Δ and pbp1Δ mutations influence on the length of poly(A) tail and LRG1 mRNA and protein levels during long-term cultivation. In the log-phase ccr4Δ mutant cells, LRG1 poly(A) tail was longer and LRG1 mRNA level was higher than those in the log-phase wild-type (WT) cells. Unexpectedly, Lrg1 protein level in the ccr4Δ mutant cells was comparable with that in WT. In the stationary-phase ccr4Δ mutant cells, LRG1 poly(A) tail length was still longer and LRG1 mRNA level was still higher than those in WT cells. In contrast to the log phase, Lrg1 protein level in the stationary-phase ccr4Δ mutant cells was maintained much higher than that in the stationary-phase WT cells. Consistently, active translating ribosomes still remained abundant in the stationary-phase ccr4Δ mutant cells, whereas they were strongly decreased in the stationary-phase WT cells. Loss of PBP1 reduced the LRG1 poly(A) tail length as well as LRG1 mRNA and protein levels in the stationary-phase ccr4Δ mutant cells. Our results suggest that Ccr4 regulates not only LRG1 mRNA level through poly(A) shortening but also the translation of LRG1 mRNA, and that Pbp1 is involved in the Ccr4-mediated regulation of mRNA stability and translation.

Ccr4是酿酒酵母（Saccharomyces cerevisiae）中参与mRNA poly(A)尾（poly(A) tail）缩短的主要胞质脱腺苷酸酶。我们此前已证实，Ccr4负调控LRG1 mRNA的表达，该mRNA编码小GTP酶Rho1的GTP酶激活蛋白（GTPase-activating protein），而Rho1是细胞壁完整性通路的组成组分；LRG1基因缺失可挽救ccr4Δ突变体的温度敏感生长缺陷。我们还证实，另一基因PBP1的缺失可挽救ccr4Δ突变体的生长迟缓表型，该基因编码poly(A)结合蛋白（poly(A)-binding protein）结合蛋白1；但其背后的分子机制仍未阐明。本研究旨在探究ccr4Δ与pbp1Δ突变在长期培养过程中，对poly(A)尾长度、LRG1 mRNA及蛋白水平的影响。对数生长期的ccr4Δ突变体细胞中，LRG1的poly(A)尾长度更长，LRG1 mRNA水平也较野生型（WT）对数生长期细胞更高。出乎意料的是，ccr4Δ突变体细胞中的Lrg1蛋白水平与野生型细胞相当。稳定期的ccr4Δ突变体细胞中，LRG1的poly(A)尾长度与mRNA水平仍较野生型细胞更高。与对数生长期不同的是，稳定期ccr4Δ突变体细胞的Lrg1蛋白水平显著高于稳定期野生型细胞。与之相符的是，稳定期ccr4Δ突变体细胞中仍存在大量活跃翻译的核糖体，而野生型稳定期细胞中的活跃翻译的核糖体则显著减少。在稳定期ccr4Δ突变体细胞中，PBP1基因缺失会降低LRG1的poly(A)尾长度，同时下调LRG1 mRNA与蛋白水平。本研究结果表明，Ccr4不仅可通过缩短poly(A)尾调控LRG1 mRNA的稳定性，还可调控LRG1 mRNA的翻译过程；同时证实Pbp1参与了Ccr4介导的mRNA稳定性与翻译调控。