ABSTRACT
Circadian clocks are an endogenous internal timekeeping mechanism that drives the rhythmic expression of genes, controlling the 24 h oscillatory pattern in behaviour and physiology. It has been recently shown that post-transcriptional mechanisms are essential for controlling rhythmic gene expression. Controlling the stability of mRNA through poly(A) tail length modulation is one such mechanism. In this study, we show that Cnot1, encoding the scaffold protein of the CCR4-NOT deadenylase complex, is highly expressed in the suprachiasmatic nucleus, the master timekeeper. CNOT1 deficiency in mice results in circadian period lengthening and alterations in the mRNA and protein expression patterns of various clock genes, mainly Per2. Per2 mRNA exhibited a longer poly(A) tail and increased mRNA stability in Cnot1+/- mice. CNOT1 is recruited to Per2 mRNA through BRF1 (ZFP36L1), which itself oscillates in antiphase with Per2 mRNA. Upon Brf1 knockdown, Per2 mRNA is stabilized leading to increased PER2 expression levels. This suggests that CNOT1 plays a role in tuning and regulating the mammalian circadian clock.
Subject(s)
Circadian Rhythm , Period Circadian Proteins , Animals , Mice , Circadian Rhythm/genetics , Mammals/genetics , Period Circadian Proteins/genetics , Period Circadian Proteins/metabolism , RNA Stability , RNA, Messenger/genetics , RNA, Messenger/metabolism , Suprachiasmatic Nucleus/metabolismABSTRACT
Control of mRNA stability and degradation is essential for appropriate gene expression, and its dysregulation causes various disorders, including cancer, neurodegenerative diseases, diabetes, and obesity. The 5'-3' exoribonuclease XRN1 executes the last step of RNA decay, but its physiological impact is not well understood. To address this, forebrain-specific Xrn1 conditional knockout mice (Xrn1-cKO) were generated, as Xrn1 null mice were embryonic lethal. Xrn1-cKO mice exhibited obesity with leptin resistance, hyperglycemia, hyperphagia, and decreased energy expenditure. Obesity resulted from dysregulated communication between the central nervous system and peripheral tissues. Moreover, expression of mRNAs encoding proteins that regulate appetite and energy expenditure was dysregulated in the hypothalamus of Xrn1-cKO mice. Therefore, we propose that XRN1 function in the hypothalamus is critical for maintenance of metabolic homeostasis.
ABSTRACT
The biological significance of deadenylation in global gene expression is not fully understood. Here, we show that the CCR4-NOT deadenylase complex maintains expression of mRNAs, such as those encoding transcription factors, cell cycle regulators, DNA damage response-related proteins, and metabolic enzymes, at appropriate levels in the liver. Liver-specific disruption of Cnot1, encoding a scaffold subunit of the CCR4-NOT complex, leads to increased levels of mRNAs for transcription factors, cell cycle regulators, and DNA damage response-related proteins because of reduced deadenylation and stabilization of these mRNAs. CNOT1 suppression also results in an increase of immature, unspliced mRNAs (pre-mRNAs) for apoptosis-related and inflammation-related genes and promotes RNA polymerase II loading on their promoter regions. In contrast, mRNAs encoding metabolic enzymes become less abundant, concomitant with decreased levels of these pre-mRNAs. Lethal hepatitis develops concomitantly with abnormal mRNA expression. Mechanistically, the CCR4-NOT complex targets and destabilizes mRNAs mainly through its association with Argonaute 2 (AGO2) and butyrate response factor 1 (BRF1) in the liver. Therefore, the CCR4-NOT complex contributes to liver homeostasis by modulating the liver transcriptome through mRNA deadenylation.
Subject(s)
Homeodomain Proteins/metabolism , Liver/metabolism , Receptors, CCR4/metabolism , Animals , Cytoplasm/metabolism , Female , Homeodomain Proteins/genetics , Homeostasis , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Poly A/genetics , RNA Stability , RNA, Messenger/genetics , Receptors, CCR4/genetics , Ribonucleases/genetics , TATA-Binding Protein Associated Factors/metabolism , Transcription Factors/geneticsABSTRACT
Somatic cells can be reprogrammed as induced pluripotent stem cells (iPSCs) by introduction of the transcription factors, OCT3/4, KLF4, SOX2, and c-MYC. The CCR4-NOT complex is the major deadenylase in eukaryotes. Its subunits Cnot1, Cnot2, and Cnot3 maintain pluripotency and self-renewal of mouse and human embryonic stem (ES) cells and contribute to the transition from partial to full iPSCs. However, little is known about how the CCR4-NOT complex post-transcriptionally regulates the reprogramming process. Here, we show that the CCR4-NOT deadenylase subunits Cnot6, Cnot6l, Cnot7, and Cnot8, participate in regulating iPSC generation. Cnot1 knockdown suppresses expression levels of Cnot6, Cnot6l, Cnot7, and Cnot8 in mouse embryonic fibroblasts (MEFs) and decreases the number of alkaline phosphatase (ALP)-positive colonies after iPSC induction. Intriguingly, Cnot1 depletion allows Eomes and p21 mRNAs to persist, increasing their expression levels. Both mRNAs have longer poly(A) tails in Cnot1-depleted cells. Conversely, forced expression of a combination of Cnot6, Cnot6l, Cnot7, and Cnot8 increases the number of ALP-positive colonies after iPSC induction and decreases expression levels of Eomes and p21 mRNAs. Based on these observations, we propose that the CCR4-NOT deadenylase activity contributes to iPSC induction.
