CNOT1 regulates circadian behaviour through Per2 mRNA decay in a deadenylation-dependent manner

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.journal articl

ARE-binding protein ZFP36L1 interacts with CNOT1 to directly repress translation via a deadenylation-independent mechanism

Eukaryotic gene expression can be spatiotemporally tuned at the post-transcriptional level by cis-regulatory elements in mRNA sequences. An important example is the AU-rich element (ARE), which induces mRNA destabilization in a variety of biological contexts in mammals and can also mediate translational control. Regulation is mediated by trans-acting factors that recognize the ARE, such as Tristetraprolin (TTP) and BRF1/ZFP36L1. Although both proteins can destabilize their target mRNAs through the recruitment of the CCR4-NOT deadenylation complex, TTP also directly regulates translation. Whether ZFP36L1 can directly repress translation remains unknown. Here, we used an in vitro translation system derived from mammalian cell lines to address this key mechanistic issue in ARE regulation by ZFP36L1. Functional assays with mutant proteins reveal that ZFP36L1 can repress translation via AU-Rich elements independent of deadenylation. ZFP36L1-mediated translation repression requires interaction between ZFP36L1 and CNOT1, suggesting that it might use a repression mechanism similar to either TPP or miRISC. However, several lines of evidence suggest that the similarity ends there. Unlike, TTP, it does not efficiently interact with either 4E-HP or GIGYF2, suggesting it does not repress translation by recruiting these proteins to the mRNA cap. Moreover, ZFP36L1 could not repress ECMV-IRES driven translation and was resistant to pharmacological eIF4A inhibitor silvestrol, suggesting fundamental differences with miRISC repression via eIF4A. Collectively, our results reveal that ZFP36L1 represses translation directly and suggest that it does so via a novel mechanism distinct from other translational regulators that interact with the CCR4-NOT deadenylase complex

The CCR4-NOT deadenylase complex controls Atg7-dependent cell death and heart function

Shortening and removal of the polyadenylate [poly(A)] tail of mRNA, a process called deadenylation, is a key step in mRNA decay that is mediated through the CCR4-NOT (carbon catabolite repression 4-negative on TATA-less) complex. In our investigation of the regulation of mRNA deadenylation in the heart, we found that this complex was required to prevent cell death. Conditional deletion of the CCR4-NOT complex components Cnot1 or Cnot3 resulted in the formation of autophagic vacuoles and cardiomyocyte death, leading to lethal heart failure accompanied by long QT intervals. Cnot3 bound to and shortened the poly(A) tail of the mRNA encoding the key autophagy regulator Atg7. In Cnot3-depleted hearts, Atg7 expression was posttranscriptionally increased. Genetic ablation of Atg7, but not Atg5, increased survival and partially restored cardiac function of Cnot1 or Cnot3 knockout mice. We further showed that in Cnot3-depleted hearts, Atg7 interacted with p53 and modulated p53 activity to induce the expression of genes encoding cell death-promoting factors in cardiomyocytes, indicating that defects in deadenylation in the heart aberrantly activated Atg7 and p53 to promote cell death. Thus, mRNA deadenylation mediated by the CCR4-NOT complex is crucial to prevent Atg7-induced cell death and heart failure, suggesting a role for mRNA deadenylation in targeting autophagy genes to maintain normal cardiac homeostasis

Two distinct nuclear stress bodies containing different sets of RNA-binding proteins are formed with HSATIII architectural noncoding RNAs upon thermal stress exposure

Nuclear stress bodies (nSBs) are thermal stress-inducible membrane-less nuclear bodies that are formed on highly repetitive satellite Ill architectural noncoding RNAs (HSATIII arcRNAs). Upon thermal stress exposure, HSATIII expression is induced to sequestrate specific sets of RNA-binding proteins and form nSBs. The major population of nSBs contain SAFB as a marker, whereas the minor population are SAFB-negative. Here, we found that HNRNPM, which was previously reported to localize in nuclear foci adjacent to SAFB-positive foci upon thermal stress, localizes in a minor population of HSATIII-dependent nSBs. Hence, we used the terms nSB-S and nSB-M to distinguish the SAFB foci and HNRNPM foci, respectively. Analysis of the components of the nSBs revealed that each set contains distinct RNA-binding proteins, including SLTM and NCO5A in nSB-Ss and HNRNPA1 and HNRNPH1 in nSB-Ms. Overall, our findings indicate that two sets of nSBs containing HSATIII arcRNAs and distinct sets of RNA-binding proteins are formed upon thermal stress exposure

LncRNA-dependent nuclear stress bodies promote intron retention through SR protein phosphorylation

A number of long noncoding RNAs (lncRNAs) are induced in response to specific stresses to construct membrane-less nuclear bodies; however, their function remains poorly understood. Here, we report the role of nuclear stress bodies (nSBs) formed on highly repetitive satellite III (HSATIII) lncRNAs derived from primate-specific satellite III repeats upon thermal stress exposure. A transcriptomic analysis revealed that depletion of HSATIII lncRNAs, resulting in elimination of nSBs, promoted splicing of 533 retained introns during thermal stress recovery. A HSATIII-Comprehensive identification of RNA-binding proteins by mass spectrometry (ChIRP-MS) analysis identified multiple splicing factors in nSBs, including serine and arginine-rich pre-mRNA splicing factors (SRSFs), the phosphorylation states of which affect splicing patterns. SRSFs are rapidly de-phosphorylated upon thermal stress exposure. During stress recovery, CDC like kinase 1 (CLK1) was recruited to nSBs and accelerated the re-phosphorylation of SRSF9, thereby promoting target intron retention. Our findings suggest that HSATIII-dependent nSBs serve as a conditional platform for phosphorylation of SRSFs by CLK1 to promote the rapid adaptation of gene expression through intron retention following thermal stress exposure

ZFP36L2 is a cell cycle-regulated CCCH protein necessary for DNA lesion-induced S-phase arrest

ZFP36L2 promotes the destruction of AU-rich element-containing transcripts, while its regulation and functional significance in cell cycle control are scarcely identified. We show that ZFP36L2 is a cell cycle-regulated CCCH protein, the abundance of which is regulated post-translationally at the respective stages of the cell cycle. Indeed, ZFP36L2 protein was eliminated after release from M phase, and ZYG11B-based E3 ligase plays a role in its polyubiquitination in interphase. Although ZFP36L2 is dispensable for normal cell cycle progression, we found that endogenous ZFP36L2 played a key role in cisplatin-induced S-phase arrest, a process in which the suppression of G1/S cyclins is necessary. The accumulation of ZFP36L2 was stimulated under DNA replication stresses and altered interactions with a subset of RNA-binding proteins. Notably, silencing endogenous ZFP36L2 led to impaired cell viability in the presence of cisplatin-induced DNA lesions. Thus, we propose that ZFP36L2 is a key protein that controls S-phase progression in the case of genome instability

Post-transcriptional Stabilization of Ucp1 mRNA Protects Mice from Diet-Induced Obesity

Uncoupling protein 1 (Ucp1) contributes to thermogenesis, and its expression is regulated at the transcriptional level. Here, we show that Ucp1 expression is also regulated post-transcriptionally. In inguinal white adipose tissue (iWAT) of mice fed a high-fat diet (HFD), Ucp1 level decreases concomitantly with increases in Cnot7 and its interacting partner Tob. HFD-fed mice lacking Cnot7 and Tob express elevated levels of Ucp1 mRNA in iWAT and are resistant to diet-induced obesity. Ucp1 mRNA has an elongated poly(A) tail and persists in iWAT of Cnot7−/− and/or Tob−/− mice on a HFD. Ucp1 3′-UTR-containing mRNA is more stable in cells expressing mutant Tob that is unable to bind Cnot7 than in WT Tob-expressing cells. Tob interacts with BRF1, which binds to an AU-rich element in the Ucp1 3′-UTR. BRF1 knockdown partially restores the stability of Ucp1 3′-UTR-containing mRNA. Thus, the Cnot7-Tob-BRF1 axis inhibits Ucp1 expression and contributes to obesity

Codon bias confers stability to human mRNAs

ヒト細胞のコドン(遺伝暗号)に隠された暗号を解明 --ヒトコドン最適化制御による治療戦略の開発へ--. 京都大学プレスリリース. 2019-09-18.Codon bias has been implicated as one of the major factors contributing to mRNA stability in several model organisms. However, the molecular mechanisms of codon bias on mRNA stability remain unclear in humans. Here, we show that human cells possess a mechanism to modulate RNA stability through a unique codon bias. Bioinformatics analysis showed that codons could be clustered into two distinct groups--codons with G or C at the third base position (GC3) and codons with either A or T at the third base position (AT3): the former stabilizing while the latter destabilizing mRNA. Quantification of codon bias showed that increased GC3‐content entails proportionately higher GC‐content. Through bioinformatics, ribosome profiling, and in vitro analysis, we show that decoupling the effects of codon bias reveals two modes of mRNA regulation, one GC3‐ and one GC‐content dependent. Employing an immunoprecipitation‐based strategy, we identify ILF2 and ILF3 as RNA‐binding proteins that differentially regulate global mRNA abundances based on codon bias. Our results demonstrate that codon bias is a two‐pronged system that governs mRNA abundance