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:10.1101/2022.05.11.491288

Cited by 5 publications

(10 citation statements)

References 137 publications

(242 reference statements)

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“…An intriguing speculation is that the IDRs of Cup may actually facilitate direct binding to osk mRNA, as IDR domains are known to bind mRNAs in other species (Smith et al 2016). This speculation was supported by results of a recent study that suggest that Cup is able to directly bind to mRNAs without any other factors (Pekovic et al 2022). Surprisingly, we also found that Cup-YFP expression was decreased in bru1 RNAi egg chambers and the YFP signal was no longer concentrated in the oocyte (Fig.…”

Section: Bru1 and Cup Maintain A Stable Association With Osk Mrna In ...supporting

confidence: 75%

Cup is essential foroskarmRNA translational repression during earlyDrosophilaoogenesis

Bayer

Milano

Formel

et al. 2023

Preprint

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The proper timing of mRNA translation is crucial across many biological systems for processes such as intercellular communication, body pattern formation, and morphogenesis. The main D. melanogaster posterior determinant, oskar, is maternally transcribed, but only translated when properly localized at the oocyte's posterior cortex. Bruno 1 and Cup are two effector proteins known to participate in multiple aspects of oskar mRNA regulation. Current model describes a mechanism in which Bruno 1 is necessary for Cup's recruitment to oskar mRNA, and Bruno 1 is indispensable for its translational repression. Here, we reveal that the Bruno 1-Cup interaction, as well as their interdependent influence on each other's mRNA and protein expression, lead to precise oskar mRNA regulation during early oogenesis. We show that these factors stably associate with the oskar mRNA in vivo, but surprisingly, Bruno 1's stable association with oskar mRNA depends on Cup, while Bruno 1 is not necessary for Cup association to oskar mRNA. During early oogenesis, Cup, not Bruno 1, is the essential factor for oskar mRNA repression. Cup is a crucial P-body member that maintains proper P-body morphology during oogenesis, as well as it is necessary for oskar mRNA's association with P-bodies, thus driving the translational repression and stability of oskar mRNA. Our experimental results collectively suggest a regulatory mechanism where a feedback loop between Bruno 1 and Cup coordinates oskar mRNA regulation in the egg chamber allowing for proper development to occur.

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Section: Bru1 and Cup Maintain A Stable Association With Osk Mrna In ...supporting

confidence: 75%

Cup is essential foroskarmRNA translational repression during earlyDrosophilaoogenesis

Bayer

Milano

Formel

et al. 2023

Preprint

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“…To assess protein interactions with Smaug we used an EGFP-Smaug construct that carried an N-terminal fusion to a PH domain (Salgania et al , 2022), which directed the localization of Smaug to the plasma membrane. With the ReLo assay, we have previously evaluated the interaction between Smaug and the six core subunits of the CCR4-NOT deadenylase complex, which is responsible for the deadenylation of nanos mRNA, and have identified the NOT3 subunit as Smaug-binding protein (Pekovic et al , 2022). Here, we tested interactions between Smaug and additional proteins involved in nanos mRNA repression, including Ago1 and Ago2 (Pinder and Smibert, 2013), Aubergine and Ago3 (Barckmann et al , 2015; Rouget et al , 2010), Cup (Nelson et al , 2004) as well as Me31B, Tral, and Belle (Götze et al , 2017; Jeske et al , 2011).…”

Section: Resultsmentioning

confidence: 99%

“…All other ReLo cloning vectors were described previously (Salgania et al , 2022; Jeske et al , 2017; Behm-Ansmant et al , 2006). The Y2H cloning vectors were described previously (Pekovic et al , 2022; Jeske et al , 2015).…”

Section: Methodsmentioning

confidence: 99%

“…Drosophila Smaug mediates translation inhibition and degradation of bulk nanos mRNA in the early Drosophila embryo, a critical step during embryonic patterning (Dahanukar & Wharton, 1996; Dahanukar et al , 1999; Smibert et al , 1996). Smaug mediates its effects by recruiting the eIF4E-binding translation inhibitor Cup and the CCR4-NOT deadenylase complex (Götze et al , 2017; Jeske et al , 2011; Nelson et al , 2004; Semotok et al , 2005; Zaessinger et al , 2006; Pekovic et al , 2022). Several other RNA-binding proteins have been implicated in Smaug-mediated repression of nanos mRNA, including Argonaute 1 (Ago1), Trailer Hitch (Tral), and the DEAD-box RNA helicases ‘Maternal expression at 31B’ (Me31B; ortholog of human DDX6) and Belle (ortholog of human DDX3X/DDX3Y) (Götze et al , 2017; Pinder & Smibert, 2013).…”

Section: Introductionmentioning

confidence: 99%

“…Smaug mediates its effects by recruiting the eIF4E-binding translation inhibitor Cup and the CCR4-NOT deadenylase complex (Götze et al, 2017;Jeske et al, 2011;Nelson et al, 2004;Semotok et al, 2005;Zaessinger et al, 2006;Pekovic et al, 2022).…”

Section: Introductionmentioning

confidence: 99%

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Structural basis for binding of Smaug to the GPCR Smoothened and to the germline inducer Oskar

Kubíková

Ubartaitė

Metz

et al. 2023

Preprint

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Drosophila Smaug and its orthologs comprise a family of mRNA repressor proteins that exhibit various functions during animal development. Smaug proteins contain a characteristic RNA-binding sterile-α motif (SAM) domain and a conserved but uncharacterized N-terminal domain (NTD). Here, we resolved the crystal structure of the NTD of the human SAM domain-containing protein 4A (SAMD4A, a.k.a. Smaug1) to 2.0 Å resolution, which revealed its composition of a homodimerization D-subdomain and a subdomain with similarity to a PHAT domain. Furthermore, we show that Drosophila Smaug directly interacts with the Drosophila germline inducer Oskar and with the Hedgehog signaling transducer Smoothened through its D-PHAT domain. We determined the crystal structure of the D-PHAT domain of Smaug in complex with a Smoothened α-helical peptide to 1.61 Å resolution. The peptide binds within a groove that is formed by both the D- and PHAT subdomains. Structural modeling supported by experimental data suggested that an α-helix within the disordered region of Oskar binds to the D-PHAT domain in a mode similar to Smoothened. Together, our data uncover the N-terminal D-PHAT domain of Smaug as peptide-binding domain.

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

Cited by 5 publications

References 137 publications

Cup is essential foroskarmRNA translational repression during earlyDrosophilaoogenesis

Cup is essential foroskarmRNA translational repression during earlyDrosophilaoogenesis

Structural basis for binding of Smaug to the GPCR Smoothened and to the germline inducer Oskar

Widespread regulation of the maternal transcriptome by Nanos in Drosophila

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