Drosophila melanogaster Roquin and Bam share a CAF40 binding motif to recruit the CCR4-NOT deadenylase complex and repress target mRNAs

Abstract

Messenger RNA (mRNA) encodes the information for cellular protein biosynthesis. To maintain correct protein levels, which is essential for cell function, strict control over mRNA levels is crucial. The CCR4-NOT complex is a key regulator of gene expression and uses transcriptional as well as posttranscriptional mechanisms to fine-tune the expression of mRNAs in diverse biological contexts. Its best characterized function is to trigger cytoplasmic mRNA decay: The complex deadenylates bulk and targeted mRNAs, which ultimately leads to their degradation. Over the past years, research in the RNA decay field has focused on understanding how the multisubunit CCR4-NOT complex assembles, and on identifying regulatory proteins that specifically modulate the repressive activity of the complex towards individual transcripts. RNA-associated proteins that recruit the CCR4-NOT complex to mRNA targets provide a unique opportunity to specifically control mRNA translation and decay. However, no general principle underlying this recruitment has been found. In fact, it appears that every RNA-associated protein uses an individual mode to interact with the different subunits of the complex. Thus, to understand the function of these regulatory proteins, it is crucial to reveal the molecular mechanisms they use to recruit CCR4-NOT. During my doctoral studies, I aimed to shed light on the recruitment of the CCR4-NOT complex by RNA-associated proteins to target mRNAs. I characterized at the molecular level how the Drosophila melanogaster (Dm) proteins Roquin and Bag-of-marbles (Bam) interact with the CCR4-NOT complex to induce repression of their targets. Roquin carries at least two distinct motifs, which both contribute to a direct interaction with the CCR4-NOT complex. These are a NOT module binding motif (NBM) and a CAF40 binding motif (CBMR; where the subscript R refers to Roquin). In contrast to Roquin, Bam harbors only one motif, a CBMB, that directly recruits the fully assembled CCR4-NOT complex. Subsequently, my work on Roquin and Bam led to the identification of a CBMN in Dm NOT4, a protein that is a stable subunit of CCR4-NOT in yeast. In conclusion, my studies identified a previously unknown peptide-binding surface on the CAF40 subunit of the CCR4-NOT complex, which is bound by at least three distinct proteins via their CBMs, suggesting mutually exclusive binding. Surprisingly, Roquin, Bam and NOT4 CBMs show a similar way of interacting with CAF40 despite the lack of sequence conservation. This implies convergent evolution of the CBMs and suggests that CAF40 provides a binding platform within the CCR4-NOT complex for additional, yet unidentified proteins. The CBM-containing proteins presented in this study add to a growing body of peptide-mediated associations in the highly complex network of CCR4-NOT-mediated mRNA regulation.