The Arabidopsis homologs of CCR4-associated factor 1 show mRNA deadenylation activity and play a role in plant defence responses

Messenger RNA （mRNA） turnover in eukaryotic cells begins with shortening of the poly （A） tail at the 3＇ end, a process called deadenylation. In yeast, the deadenylation reaction is predominantly mediated by CCR4 and CCR4- associated factor 1 （CAF1）, two components of the well-characterised protein complex named CCR4-NOT. We report here that AtCAF1a and AtCAF1b, putative Arabidopsis homologs of the yeast CAF1 gene, partially complement the growth defect of the yeast call mutant in the presence of caffeine or at high temperatures. The expression of At-CAF1a and AtCAFlb is induced by multiple stress-related hormones and stimuli. Both AtCAF1a and AtCAFlb show deadenylation activity in vitro and point mutations in the predicted active sites disrupt this activity. T-DNA insertion mutants disrupting the expression of AtCAF1a and/or AtCAF1b are defective in deadenylation of stress-related mRNAs, indicating that the two AtCAF1 proteins are involved in regulated mRNA deadenylation in vivo. Interestingly, the single and double mutants of AtCAF1a and AtCAFlb show reduced expression of pathogenesis-related （PR） genes PR1 and PR2 and are more susceptible to Pseudomonas syringae pv tomato DC3000 （Pst DC3000） infection, whereas transgenic plants over-expressing AtCAFla show elevated expression of PR1 and PR2 and increased resis-tance to the same pathogen. Our results suggest roles of the AtCAF1 proteins in regulated mRNA deadenylation and defence responses to pathogen infections.

SPT5 affects the rate of mRNA degradation and physically interacts with CCR4 but does not control mRNA deadenylation

The CCR4-NOT complex has been shown to have multiple roles in mRNA metabolism, including that of transcriptional elongation, mRNA transport, and nuclear exosome function, but the primary function of CCR4 and CAF1 is in the deadenylation and degradation of cytoplasmic mRNA. As previous genetic analysis supported an interaction between SPT5, known to be involved in transcriptional elongation, and that of CCR4, the physical association of SPT5 with CCR4 was examined. A two-hybrid screen utilizing the deadenylase domain of CCR4 as a bait identified SPT5 as a potential interacting protein. SPT5 at its physiological concentration was shown to immunoprecipitate CCR4 and CAF1, and in vitro purified SPT5 specifically could bind to CAF1 and the deadenylase domain of CCR4. We additionally demonstrated that mutations in SPT5 or an spt4 deletion slowed the rate of mRNA degradation, a phenotype associated with defects in the CCR4 mRNA deadenylase complex. Yet, unlike ccr4 and caf1 deletions, spt5 and spt4 defects displayed little effect on the rate of deadenylation. They also did not affect decapping or 5' - 3' degradation of mRNA. These results suggest that the interactions between SPT5/SPT4 and the CCR4-NOT complex are probably the consequences of effects involving nuclear events and do not involve the primary role of CCR4 in mRNA deadenylation and turnover.

The role of the CNOT1 subunit of the CCR4-NOT complex in mRNA deadenylation and cell viability

The human CCR4-NOT deadenylase complex consists of at least nine enzymatic and non-enzymatic subunits.Accumulating evidence suggests that the non-enzymatic subunits are involved in the regulation of mRNA deadenylation,although their precise roles remain to be established.In this study,we addressed the function of the CNOT1 subunit by depleting its expression in HeLa cells.Flow cytometric analysis revealed that the sub G1 fraction was increased in CNOT1-depleted cells.Virtually,the same level of the sub G1 fraction was seen when cells were treated with a mixture of siRNAs targeted against all enzymatic subunits,suggesting that CNOT1 depletion induces apoptosis by destroying the CCR4-NOT-associated deadenylase activity.Further analysis revealed that CNOT1 depletion leads to a reduction in the amount of other CCR4-NOT subunits.Importantly,the specific activity of the CNOT6L immunoprecipitates-associated deadenylase from CNOT1-depleted cells was less than that from control cells.The formation of P-bodies,where mRNA decay is reported to take place,was largely suppressed in CNOT1-depleted cells.Therefore,CNOT1 has an important role in exhibiting enzymatic activity of the CCR4-NOT complex,and thus is critical in control of mRNA deadenylation and mRNA decay.We further showed that CNOT1 depletion enhanced CHOP mRNA levels and activated caspase-4,which is associated with endoplasmic reticulum ER stress-induced apoptosis.Taken together,CNOT1 depletion structurally and functionally deteriorates the CCR4-NOTcomplex and induces stabilization of mRNAs,which results in the increment of translation causing ER stress-mediated apoptosis.We conclude that CNOT1 contributes to cell viability by securing the activity of the CCR4-NOT deadenylase.

RNF219 interacts with CCR4-NOT in regulating stem cell differentiation

Regulation of RNA stability plays a crucial role in gene expression control.Deadenylation is the initial rate-limiting step for the majority of RNA decay events.Here,we show that RING finger protein 219(RNF219)interacts with the CCR4-NOT deadenylase complex.RNF219-CCR4-NOT exhibits deadenylation activity in vitro.RNA-seq analyses identify some of the 2-cell-specific genes and the neuronal genes significantly downregulated upon RNF219 knockdown,while upregulated after depletion of the CCR4-NOT subunit CNOTIO in mouse embryonic stem(ES)cells.RNF219 depletion leads to impaired neuronal lineage commitment during ES cell differentiation.Our study suggests that RNF219 is a novel interacting partner of CCR4-NOT and required for maintenance of ES cell pluripotency.