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bioRxiv · 10.1101/2022.05.11.491288

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

Pekovic, F.·Rammelt, C.·Kubikova, J.·Metz, J.·Jeske, M.·Wahle, E.
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Abstract

Posttranscriptional regulation of the maternal nanos mRNA is essential for the development of the anterior - posterior axis of the Drosophila embryo. The nanos RNA is regulated by the protein Smaug. Binding to Smaug recognition elements (SREs) in the nanos 3-UTR, Smaug nucleates the assembly of a larger repressor complex including the eIF4E-T paralog Cup and five additional proteins. The Smaug-dependent complex represses translation of nanos and induces its deadenylation by the CCR4-NOT deadenylase. Here we report an in vitro reconstitution of the Drosophila CCR4-NOT complex and Smaug-dependent deadenylation. We find that Smaug by itself is sufficient to cause deadenylation by the Drosophila or human CCR4-NOT complexes in an SRE-dependent manner. CCR4-NOT subunits NOT10 and NOT11 are dispensable, but the NOT module, consisting of NOT2, NOT3 and the C-terminal part of NOT1, is required. Smaug interacts with the C-terminal domain of NOT3. Both catalytic subunits of CCR4-NOT contribute to Smaug-dependent deadenylation. Whereas the CCR4-NOT complex itself acts distributively, Smaug induces a processive behavior. The cytoplasmic poly(A) binding protein (PABPC) has but a minor effect on Smaug-dependent deadenylation. Among the additional constituents of the Smaug-dependent repressor complex, Cup also facilitates CCR4-NOT-dependent deadenylation, both independently and in cooperation with Smaug.

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Pekovic, F., Rammelt, C., Kubikova, J., Metz, J., Jeske, M., Wahle, E.. 2022-05-11. RNA binding proteins Smaug and Cup induce CCR4-NOT-dependent deadenylation of the nanos mRNA in a reconstituted system. https://doi.org/10.1101/2022.05.11.491288

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