Reconstitution of recombinant human CCR4-NOT reveals molecular insights into regulated deadenylation

Raisch, Tobias; Chang, Chung Te; Levdansky, Yevgen; Muthukumar, Sowndarya; Raunser, Stefan; Valkov, Eugene

doi:10.1038/s41467-019-11094-z

Cited by 71 publications

(105 citation statements)

References 59 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…A similar observation was reported in a previous study [26]. It is possible that CNOT6/6L might be able to function independently of the complex to deadenylate mRNAs, because purified CNOT6/6L and CNOT7/8 exhibit deadenylase activity in vitro [17,39,40]. It would be intriguing to examine whether there are any biological contexts where CNOT6/6L function independently in vivo.…”

Section: Discussionsupporting

confidence: 85%

Essential functions of the CNOT7/8 catalytic subunits of the CCR4-NOT complex in mRNA regulation and cell viability

et al. 2020

View full text Add to dashboard Cite

Shortening of mRNA poly(A) tails (deadenylation) to trigger their decay is mediated mainly by the CCR4-NOT deadenylase complex. While four catalytic subunits (CNOT6, 6L 7, and 8) have been identified in the mammalian CCR4-NOT complex, their individual biological roles are not fully understood. In this study, we addressed the contribution of CNOT7/8 to viability of primary mouse embryonic fibroblasts (MEFs). We found that MEFs lacking CNOT7/8 expression [Cnot7/8-double knockout (dKO) MEFs] undergo cell death, whereas MEFs lacking CNOT6/6L expression (Cnot6/6l-dKO MEFs) remain viable. Co-immunoprecipitation analyses showed that CNOT6/6L are also absent from the CCR4-NOT complex in Cnot7/8-dKO MEFs. In contrast, either CNOT7 or CNOT8 still interacts with other subunits in the CCR4-NOT complex in Cnot6/6l-dKO MEFs. Exogenous expression of a CNOT7 mutant lacking catalytic activity in Cnot7/8-dKO MEFs cannot recover cell viability, even though CNOT6/6L exists to some extent in the CCR4-NOT complex, confirming that CNOT7/8 is essential for viability. Bulk poly(A) tail analysis revealed that mRNAs with longer poly(A) tails are more numerous in Cnot7/8-dKO MEFs than in Cnot6/6l-dKO MEFs. Consistent with elongated poly(A) tails, more mRNAs are upregulated and stabilized in Cnot7/8-dKO MEFs than in Cnot6/6l-dKO MEFs. Importantly, Cnot6/6l-dKO mice are viable and grow normally to adulthood. Taken together, the CNOT7/8 catalytic subunits are essential for deadenylation, which is necessary to maintain cell viability, whereas CNOT6/6L are not.

show abstract

Section: Discussionsupporting

confidence: 85%

Essential functions of the CNOT7/8 catalytic subunits of the CCR4-NOT complex in mRNA regulation and cell viability

et al. 2020

View full text Add to dashboard Cite

show abstract

“…Purified human CCR4-NOT complex (50 µg) was then added to the beads. The reconstitution of the human CCR4-NOT complex was described elsewhere (57) and includes the following eight components: CNOT1 (amino acids 1-2376), CNOT2 (1-540), CNOT3 (1-753), CNOT10 (25-707), CNOT11 (257-498), CAF1 (1-285), CCR4a (1-558), CAF40 (1-299). After 1 hour incubation, the beads were washed three times with binding buffer and the proteins were eluted with binding buffer supplemented with 2.5 mM D-desthiobiotin.…”

Section: Methodsmentioning

confidence: 99%

Unique repression domains of Pumilio utilize deadenylation and decapping factors to accelerate destruction of target mRNAs

Arvola

Chang

Buytendorp

et al. 2019

Preprint

Self Cite

View full text Add to dashboard Cite

Pumilio is an RNA-binding protein that represses a network of mRNAs to control embryogenesis, stem cell fate, fertility, and neurological functions in Drosophila. We sought to identify the mechanism of Pumilio-mediated repression and find that it accelerates degradation of target mRNAs, mediated by three N-terminal Repression Domains (RDs), which are unique to Pumilio orthologs. We show that the repressive activities of the Pumilio RDs depend on specific subunits of the Ccr4-Not (CNOT) deadenylase complex. Depletion of Pop2, Not1, Not2, or Not3 subunits alleviates Pumilio RD-mediated repression of protein expression and mRNA decay, whereas depletion of other CNOT components had little or no effect. Moreover, the catalytic activity of Pop2 deadenylase is important for Pumilio RD activity. Further, we show that the Pumilio RDs directly bind to the CNOT complex. We also report that the decapping enzyme, Dcp2, participates in repression by the N-terminus of Pumilio. These results support a model wherein Pumilio utilizes CNOT deadenylase and decapping complexes to accelerate destruction of target mRNAs. Because the N-terminal RDs are conserved in mammalian Pumilio orthologs, the results of this work broadly enhance our understanding of Pumilio function and roles in diseases including cancer, neurodegeneration, and epilepsy.

show abstract

“…CNOT9 has been proposed to play regulatory roles within the complex in a manner that assists mRNA decay by interacting with CNOT1 and stimulating catalysis by subunits CNOT6, CNOT6L, CNOT7 and CNOT8 (Pavanello et al, 2018).This idea was further perpetuated by an in-vitro study of the human CCR4-NOT complex, which suggested that stimulus-dependent CNOT9 interaction with RNA binding proteins results in targeted mRNA decay (Raisch et al, 2019). In congruity with this idea we observed a similar trend within our RNA-Seq data.…”

Section: Discussionmentioning

confidence: 99%

Loss of CNOT9 begets impairment in gastrulation leading to embryonic lethality

Sarmah

Ito

Kaneko

et al. 2020

Preprint

View full text Add to dashboard Cite

The multi-subunit eukaryotic CCR4-NOT complex imparts gene expression control primarily via messenger RNA (mRNA) decay. Here, we present the role of subunit CNOT9 in target mRNA decay during embryonic development. CNOT9 null mice appear normal by the onset of gastrulation (E7.0), however, exhibit growth and differentiation defects accompanied by extensive cell death by embryonic day 9.5 (E9.5). Sox-2 Cre conditional CNOT9 knockout mice show almost identical phenotype with brief delay in onset and progression, suggesting defects to be epiblast-dominant. Among various identified targets, we show that Lefty2 mRNA expression is post-transcriptionally regulated by CNOT9. Lefty2 3'-UTR containing mRNA has significantly higher stability in cells expressing mutant form of CNOT9, relative to cells expressing wild-type CNOT9. In addition, CNOT9 primarily localizes within the cytoplasm and bridges interactions between the CCR4-NOT complex and miRNA-RISC complex in gastrulating embryos.

show abstract

Reconstitution of recombinant human CCR4-NOT reveals molecular insights into regulated deadenylation

Cited by 71 publications

References 59 publications

Essential functions of the CNOT7/8 catalytic subunits of the CCR4-NOT complex in mRNA regulation and cell viability

Essential functions of the CNOT7/8 catalytic subunits of the CCR4-NOT complex in mRNA regulation and cell viability

Unique repression domains of Pumilio utilize deadenylation and decapping factors to accelerate destruction of target mRNAs

Loss of CNOT9 begets impairment in gastrulation leading to embryonic lethality

Contact Info

Product

Resources

About