Structural and biochemical analysis of a NOT1 MIF4G-like domain of the CCR4-NOT complex

Raisch, Tobias; Sandmeir, Felix; Weichenrieder, Oliver; Valkov, Eugene; Izaurralde, Elisa

doi:10.1016/j.jsb.2018.10.009

Cited by 18 publications

(13 citation statements)

References 39 publications

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“…The protein was then flash-frozen in liquid nitrogen for storage. Recombinant CCR4–NOT complex components were purified as previously described (17,43,44). The purification of the recombinant EVH1 domain of Dcp1 from fission yeast, as well as the recombinant PatL1-C, were also described previously (39,45).…”

Section: Methodsmentioning

confidence: 99%

A low-complexity region in human XRN1 directly recruits deadenylation and decapping factors in 5′–3′ messenger RNA decay

Chang

Muthukumar

Weber

et al. 2019

Nucleic Acids Research

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XRN1 is the major cytoplasmic exoribonuclease in eukaryotes, which degrades deadenylated and decapped mRNAs in the last step of the 5′–3′ mRNA decay pathway. Metazoan XRN1 interacts with decapping factors coupling the final stages of decay. Here, we reveal a direct interaction between XRN1 and the CCR4–NOT deadenylase complex mediated by a low-complexity region in XRN1, which we term the ‘C-terminal interacting region’ or CIR. The CIR represses reporter mRNA deadenylation in human cells when overexpressed and inhibits CCR4–NOT and isolated CAF1 deadenylase activity in vitro. Through complementation studies in an XRN1-null cell line, we dissect the specific contributions of XRN1 domains and regions toward decay of an mRNA reporter. We observe that XRN1 binding to the decapping activator EDC4 counteracts the dominant negative effect of CIR overexpression on decay. Another decapping activator PatL1 directly interacts with CIR and alleviates the CIR-mediated inhibition of CCR4–NOT activity in vitro. Ribosome profiling revealed that XRN1 loss impacts not only on mRNA levels but also on the translational efficiency of many cellular transcripts likely as a consequence of incomplete decay. Our findings reveal an additional layer of direct interactions in a tightly integrated network of factors mediating deadenylation, decapping and 5′–3′ exonucleolytic decay.

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Section: Methodsmentioning

confidence: 99%

A low-complexity region in human XRN1 directly recruits deadenylation and decapping factors in 5′–3′ messenger RNA decay

Chang

Muthukumar

Weber

et al. 2019

Nucleic Acids Research

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“…CNOT1 serves as a scaffold for additional CNOT subunits (i.e. CNOT2, 3, 9, 10, and 11) that mediate protein interactions with RBPs, microRNA-induced silencing complex, mRNA decay factors, and translational regulators (Fabian et al 2013;Chen et al 2014;Bhandari et al 2014;Sgromo et al 2017;Mathys et al 2014;Raisch et al 2018Raisch et al , 2019. Multiple CNOT components were reported to copurify with human PUMs, including the deadenylases (Goldstrohm et al 2006;Van Etten et al 2012), but precise contacts were not mapped and functional roles of the CNOT components in PUM-mediated repression remained unknown.…”

Section: Introductionmentioning

confidence: 99%

Human Pumilio proteins directly bind the CCR4-NOT deadenylase complex to regulate the transcriptome

Enwerem

Elrod

Chang

et al. 2020

Preprint

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Pumilio paralogs, PUM1 and PUM2, are sequence-specific RNA-binding proteins that are essential for vertebrate development and neurological functions. PUM1&2 negatively regulate gene expression by accelerating degradation of specific mRNAs. Here, we determined the repression mechanism and impact of human PUM1&2 on the transcriptome. We identified subunits of the CCR4-NOT (CNOT) deadenylase complex required for stable interaction with PUM1&2 and to elicit CNOT-dependent repression. Isoform-level RNA sequencing revealed broad co-regulation of target mRNAs through the PUM-CNOT repression mechanism.Functional dissection of the domains of PUM1&2 identified a conserved N-terminal region that confers the predominant repressive activity via direct interaction with CNOT. In addition, we show that the mRNA decapping enzyme, DCP2, has an important role in repression by PUM1&2 N-terminal regions. Our results support a molecular model of repression by human PUM1&2 via direct recruitment of CNOT deadenylation machinery in a decapping-dependent mRNA decay pathway.

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“…The catalytically active nucleases CAF1 (or its paralog POP2) and CCR4a (or its paralog CCR4b) bind to a central MIF4G (middle-domain of eIF4G)-like domain of NOT1 (Lau et al, 2009; Basquin et al, 2012; Petit et al, 2012) adjacent to the CAF40-binding domain (CC) of NOT1 (Chen et al, 2014; Mathys et al, 2014). The CC domain is followed by a short connector domain in NOT1, recently identified to be an additional MIF4G-like domain, termed MIF4G-C (Raisch et al, 2018). NOT2 and NOT3 assemble on the C-terminal part of NOT1 (Bhaskar et al, 2013; Boland et al, 2013).…”

Section: Resultsmentioning

confidence: 99%

HELZ directly interacts with CCR4–NOT and causes decay of bound mRNAs

et al. 2019

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Eukaryotic superfamily (SF) 1 helicases have been implicated in various aspects of RNA metabolism, including transcription, processing, translation, and degradation. Nevertheless, until now, most human SF1 helicases remain poorly understood. Here, we have functionally and biochemically characterized the role of a putative SF1 helicase termed “helicase with zinc-finger,” or HELZ. We discovered that HELZ associates with various mRNA decay factors, including components of the carbon catabolite repressor 4-negative on TATA box (CCR4–NOT) deadenylase complex in human and Drosophila melanogaster cells. The interaction between HELZ and the CCR4–NOT complex is direct and mediated by extended low-complexity regions in the C-terminal part of the protein. We further reveal that HELZ requires the deadenylase complex to mediate translational repression and decapping-dependent mRNA decay. Finally, transcriptome-wide analysis of Helz-null cells suggests that HELZ has a role in the regulation of the expression of genes associated with the development of the nervous system.

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Structural and biochemical analysis of a NOT1 MIF4G-like domain of the CCR4-NOT complex

Cited by 18 publications

References 39 publications

A low-complexity region in human XRN1 directly recruits deadenylation and decapping factors in 5′–3′ messenger RNA decay

A low-complexity region in human XRN1 directly recruits deadenylation and decapping factors in 5′–3′ messenger RNA decay

Human Pumilio proteins directly bind the CCR4-NOT deadenylase complex to regulate the transcriptome

HELZ directly interacts with CCR4–NOT and causes decay of bound mRNAs

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