Duy Duong Long, Suda Yasuyuki, Irie Kenji
Department of Molecular Cell Biology, Graduate School of Comprehensive Human Sciences and Faculty of Medicine, University of Tsukuba, Tsukuba, Japan.
Live Cell Super-resolution Imaging Research Team, RIKEN Center for Advanced Photonics, Wako, Saitama, Japan.
PLoS One. 2017 Feb 23;12(2):e0172476. doi: 10.1371/journal.pone.0172476. eCollection 2017.
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是酿酒酵母中参与mRNA聚腺苷酸（poly(A)）尾巴缩短的主要细胞质去腺苷酸化酶。我们之前已经表明，Ccr4负向调节LRG1 mRNA的表达，LRG1编码一种小GTP酶Rho1的GTP酶激活蛋白，Rho1是细胞壁完整性途径的一个组成部分，并且LRG1的缺失抑制了ccr4Δ突变体的温度敏感生长缺陷。我们还表明，另一个基因PBP1（编码一种聚腺苷酸结合蛋白（Pab1）结合蛋白1）的缺失抑制了ccr4Δ突变体的缓慢生长；然而，其潜在机制仍然未知。在本研究中，我们调查了在长期培养过程中ccr4Δ和pbp1Δ突变如何影响聚腺苷酸尾巴的长度以及LRG1 mRNA和蛋白质水平。在对数期ccr4Δ突变体细胞中，LRG1聚腺苷酸尾巴比对数期野生型（WT）细胞中的更长，并且LRG1 mRNA水平更高。出乎意料的是，ccr4Δ突变体细胞中的Lrg1蛋白水平与WT细胞中的相当。在稳定期ccr4Δ突变体细胞中，LRG1聚腺苷酸尾巴长度仍然更长，并且LRG1 mRNA水平仍然高于WT细胞中的。与对数期相反，稳定期ccr4Δ突变体细胞中的Lrg1蛋白水平维持在远高于稳定期WT细胞中的水平。一致地，在稳定期ccr4Δ突变体细胞中，活跃的翻译核糖体仍然大量存在，而在稳定期WT细胞中它们显著减少。PBP1的缺失降低了稳定期ccr4Δ突变体细胞中LRG1聚腺苷酸尾巴的长度以及LRG1 mRNA和蛋白质水平。我们的结果表明，Ccr4不仅通过聚腺苷酸缩短调节LRG1 mRNA水平，还调节LRG1 mRNA的翻译，并且Pbp1参与Ccr4介导的mRNA稳定性和翻译调节。
