Abstract
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.
Highlights
In the nucleus of eukaryotic cells, mRNAs are transcribed and undergo modifications including addition of the cap 7-methylguanosine (m7G) to the 5’ end, addition of poly(A) tailPLOS ONE | DOI:10.1371/journal.pone.0172476 February 23, 2017Ccr4 is required for translational repression of LRG1 mRNA to the 3’ end, and splicing to remove introns [1]
In ccr4Δ mutant, the LRG1 mRNA level initially dropped during 4 h to 24 h time points, but it minimally changed and remained relatively high level after the 24 h time point (Fig 1C, ccr4Δ). These results suggest that Ccr4 negatively regulates the poly(A) tail length and the LRG1 mRNA level, and that the longer poly(A) tail seems to be more important for the mRNA level at the later time points of cell growth
In a genome-wide analysis, Subtelny et al have shown that the poly(A) tail length positively correlates to translation efficiency only in early zebrafish and frog embryo, and deadenylation primarily enhances mRNA decay [31]
Summary
In the nucleus of eukaryotic cells, mRNAs are transcribed and undergo modifications including addition of the cap 7-methylguanosine (m7G) to the 5’ end, addition of poly(A) tailPLOS ONE | DOI:10.1371/journal.pone.0172476 February 23, 2017Ccr is required for translational repression of LRG1 mRNA to the 3’ end, and splicing to remove introns [1]. The Pab (Poly[A] binding protein 1) binds to poly(A) tail of mRNAs and physically interacts with the translational initiation factor eIF4G, a component of the translational initiation complex. Another component of this complex, eIF4E, directly binds to the 5’ cap structure of mRNA to form mRNP (messenger ribonucleoprotein) loop, which is dependent on 5’ cap and 3’ poly(A) tail. MRNA degradation firstly initiates with shortening poly(A) tail by the cytoplasmic deadenylase [5, 6]. Regulation of mRNA poly(A) tail length is an important step that determines the mRNA behavior in the cell. RNA-binding proteins such as PUF (Pumilio and FBF) proteins or miRNAs, which bind to the specific sites in the 3’-untranslated region (UTR) of mRNAs, regulate mRNA degradation and/or translation through recruiting the mRNA decay machinery to the target mRNAs [7,8,9]
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