RNA binding proteins Smaug and Cup induce CCR4–NOT-dependent deadenylation of the <i>nanos</i> mRNA in a reconstituted system

Pekovic, Filip; Rammelt, Christiane; Kubíková, Jana; Metz, J; Jeske, Mandy; Wahle, Elmar

doi:10.1093/nar/gkad159

Cited by 16 publications

(12 citation statements)

References 131 publications

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“…Whether MARF1 or Smaug directly associates with the CCR4-NOT complex was unknown. Using ReLo, we found that Smaug interacts with the NOT3 subunit 63 and that MARF1 binds to the NOT1 subunit (Fig. 4e and Supplementary Fig.…”

Section: Resultsmentioning

confidence: 98%

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ReLo is a simple and rapid colocalization assay to identify and characterize direct protein–protein interactions

Salgania,

Metz,

Jeske

2024

Nat Commun

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The characterization of protein-protein interactions (PPIs) is fundamental to the understanding of biochemical processes. Many methods have been established to identify and study direct PPIs; however, screening and investigating PPIs involving large or poorly soluble proteins remains challenging. Here, we introduce ReLo, a simple, rapid, and versatile cell culture-based method for detecting and investigating interactions in a cellular context. Our experiments demonstrate that ReLo specifically detects direct binary PPIs. Furthermore, we show that ReLo bridging experiments can also be used to determine the binding topology of subunits within multiprotein complexes. In addition, ReLo facilitates the identification of protein domains that mediate complex formation, allows screening for interfering point mutations, and it is sensitive to drugs that mediate or disrupt an interaction. In summary, ReLo is a simple and rapid alternative for the study of PPIs, especially when studying structurally complex proteins or when established methods fail.

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Section: Resultsmentioning

confidence: 98%

“…Interaction data for Bruno and Cup are shown in Supplementary Fig. 7 and data for Smaug are published in 63 . b Bam interacted with CAF40.…”

Section: Resultsmentioning

confidence: 99%

ReLo is a simple and rapid colocalization assay to identify and characterize direct protein–protein interactions

Salgania,

Metz,

Jeske

2024

Nat Commun

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“…Given the large number of regulated mRNAs, it is not surprising that expression of Nos protein is tightly controlled. During oogenesis and embryogenesis, much of this control is exerted via the 3’-UTR of its mRNA to control the timing and location of protein accumulation (Forrest et al, 2004; Pekovic et al, 2023; Peng & Gavis, 2022); we therefore intentionally designed all of the transgenes used in this work to express mRNAs bearing the native nos 3’-UTR. Our results suggest an additional level of control–– negative autoregulation.…”

Section: Discussionmentioning

confidence: 99%

Widespread regulation of the maternal transcriptome by Nanos in Drosophila

Marhabaie,

Wharton,

Kim

et al. 2023

Preprint

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The translational repressor Nanos (Nos) regulates a single target, maternalhunchback(hb)mRNA, to govern abdominal segmentation in the early Drosophila embryo. Nos is recruited specifically to sites in the 3’-UTR ofhbmRNA in collaboration with the sequence-specific RNA-binding protein Pumilio (Pum); on its own, Nos has no binding specificity. Nos is expressed at other stages of development, but very few mRNA targets that might mediate its action at these stages have been described. Nor has it been clear whether Nos is targeted to other mRNAs in concert with Pum or via other mechanisms. In this report, we identify mRNAs targeted by Nos via two approaches. In the first method, we identify mRNAs depleted upon expression of a chimera bearing Nos fused to the nonsense mediated decay (NMD) factor Upf1. We find that, in addition tohb, Upf1-Nos depletes ∼2600 mRNAs from the maternal transcriptome in early embryos. Virtually all of these appear to be targeted in a canonical,hb-like manner in concert with Pum. In a second, more conventional approach, we identify mRNAs that are stabilized during the maternal zygotic transition (MZT) in embryos fromnos-females. Most (86%) of the 1185 mRNAs regulated by Nos are also targeted by Upf1-Nos, validating use of the chimera. Approximately 60% of mRNAs targeted by Upf1-Nos are not stabilized in the absence of Nos. However, Upf1-Nos mRNA targets are hypo-adenylated and inefficiently translated at the ovary-embryo transition, whether or not they suffer Nos-dependent degradation in the embryo. We suggest that the late ovarian burst of Nos represses a large fraction of the maternal transcriptome, priming it for later degradation by other factors during the MZT in the embryo.

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“…The trans-acting factors recruited by these sequence elements typically induce mRNA degradation via deadenylation coupled to the 5′-3′ degradation pathway ( Muhlrad et al, 1994 ; Stoecklin et al, 2006 ; Bonisch et al, 2007 ). Well-characterised examples include the A/U-rich element (ARE) ( Stoecklin et al, 2006 ), microRNA binding sites ( Behm-Ansmant et al, 2006 ; Piao et al, 2010 ), and sequence elements recognised by developmental regulators such as Smaug and Bicaudal-C ( Zaessinger et al, 2006 ; Chicoine et al, 2007 ; Pekovic et al, 2023 ). In addition to recruitment by mRNA-specific factors, the 5′-3′ degradation pathway can be induced by general mechanisms involving the interaction between the cytoplasmic poly(A)-binding protein (PABPC) 1 and members of the BTG/Tob family of proteins, which include TOB1 and its paralogue TOB2, and the BTG1 and BTG2 paralogues that are frequently mutated in non-Hodgkin lymphoma ( Mauxion et al, 2009 ; Winkler, 2010 ; Yuniati et al, 2018 ; Almasmoum et al, 2021 ).…”

Section: Introductionmentioning

confidence: 99%

Structure and function of molecular machines involved in deadenylation-dependent 5′-3′ mRNA degradation

Zhao,

Pavanello,

Bartlam

et al. 2023

Front. Genet.

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In eukaryotic cells, the synthesis, processing, and degradation of mRNA are important processes required for the accurate execution of gene expression programmes. Fully processed cytoplasmic mRNA is characterised by the presence of a 5′cap structure and 3′poly(A) tail. These elements promote translation and prevent non-specific degradation. Degradation via the deadenylation-dependent 5′-3′ degradation pathway can be induced by trans-acting factors binding the mRNA, such as RNA-binding proteins recognising sequence elements and the miRNA-induced repression complex. These factors recruit the core mRNA degradation machinery that carries out the following steps: i) shortening of the poly(A) tail by the Ccr4-Not and Pan2-Pan3 poly (A)-specific nucleases (deadenylases); ii) removal of the 5′cap structure by the Dcp1-Dcp2 decapping complex that is recruited by the Lsm1-7-Pat1 complex; and iii) degradation of the mRNA body by the 5′-3′ exoribonuclease Xrn1. In this review, the biochemical function of the nucleases and accessory proteins involved in deadenylation-dependent mRNA degradation will be reviewed with a particular focus on structural aspects of the proteins and enzymes involved.

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RNA binding proteins Smaug and Cup induce CCR4–NOT-dependent deadenylation of the nanos mRNA in a reconstituted system

Cited by 16 publications

References 131 publications

ReLo is a simple and rapid colocalization assay to identify and characterize direct protein–protein interactions

ReLo is a simple and rapid colocalization assay to identify and characterize direct protein–protein interactions

Widespread regulation of the maternal transcriptome by Nanos in Drosophila

Structure and function of molecular machines involved in deadenylation-dependent 5′-3′ mRNA degradation

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