Regulation of CLB6 expression by the cytoplasmic deadenylase Ccr4 through its coding and 3' UTR regions

RNA stability control contributes to the proper expression of gene products. Messenger RNAs (mRNAs) in eukaryotic cells possess a 5' cap structure and the 3' poly(A) tail which are important for mRNA stability and efficient translation. The Ccr4-Not complex is a major cytoplasmic deadenylase and functions in mRNA degradation. The CLB1-6 genes in Saccharomyces cerevisiae encode B-type cyclins which are involved in the cell cycle progression together with the cyclin-dependent kinase Cdc28. The CLB genes consist of CLB1/2, CLB3/4, and CLB5/6 whose gene products accumulate at the G2-M, S-G2, and late G1 phase, respectively. These Clb protein levels are thought to be mainly regulated by the transcriptional control and the protein stability control. Here we investigated regulation of CLB1-6 expression by Ccr4. Our results show that all CLB1-6 mRNA levels were significantly increased in the ccr4 Delta mutant compared to those in wild-type cells. Clb1, Clb4, and Clb6 protein levels were slightly increased in the ccr4 Delta mutant, but the Clb2, Clb3, and Clb5 protein levels were similar to those in wild-type cells. Since both CLB6 mRNA and Clb6 protein levels were most significantly increased in the ccr4 Delta mutant, we further analyzed the cis-elements for the Ccr4-mediated regulation within CLB6 mRNA. We found that there were destabilizing sequences in both coding sequence and 3' untranslated region (3' UTR). The destabilizing sequences in the coding region were found to be both within and outside the sequences corresponding the cyclin domain. The CLB6 3' UTR was sufficient for mRNA destabilization and decrease of the reporter GFP gene and this destabilization involved Ccr4. Our results suggest that CLB6 expression is regulated by Ccr4 through the coding sequence and 3' UTR of CLB6 mRNA.

Automated summary

The stability of RNA has an important role in gene expression, and the Ccr4-Not complex is involved in mRNA degradation. This study investigated the regulation of CLB1-6 genes by Ccr4, and found that Ccr4 affects the mRNA levels of CLB1-6 and consequently influences the protein levels of Clb1, Clb4, and Clb6. Further analysis identified destabilizing sequences in both the coding sequence and 3' untranslated region (3' UTR) of CLB6 mRNA, and it was determined that Ccr4 is involved in the destabilization process.