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

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

A conserved CAF40-binding motif in metazoan NOT4 mediates association with the CCR4-NOT complex

The multisubunit CCR4-NOT mRNA deadenylase complex plays important roles in the posttranscriptional regulation of gene expression. The NOT4 E3 ubiquitin ligase is a stable component of the CCR4-NOT complex in yeast but does not copurify with the human or Drosophila melanogaster complex. Here we show that the C-terminal regions of human and D. melanogaster NOT4 contain a conserved sequence motif that directly binds the CAF40 subunit of the CCR4-NOT complex (CAF40-binding motif [CBM]). In addition, nonconserved sequences flanking the CBM also contact other subunits of the complex. Crystal structures of the CBM-CAF40 complex reveal a mutually exclusive binding surface for NOT4 and Roquin or Bag of marbles mRNA regulatory proteins. Furthermore, CAF40 depletion or structure-guided mutagenesis to disrupt the NOT4-CAF40 interaction impairs the ability of NOT4 to elicit decay of tethered reporter mRNAs in cells. Together with additional sequence analyses, our results reveal the molecular basis for the association of metazoan NOT4 with the CCR4-NOT complex and show that it deviates substantially from yeast. They mark the NOT4 ubiquitin ligase as an ancient but nonconstitutive cofactor of the CCR4-NOT deadenylase with potential recruitment and/or effector functions

Drosophila Bag-of-marbles directly interacts with the CAF40 subunit of the CCR4-NOT complex to elicit repression of mRNA targets

Drosophila melanogaster Bag-of-marbles (Bam) promotes germline stem cell (GSC) differentiation by repressing the expression of mRNAs encoding stem cell maintenance factors. Bam interacts with Benign gonial cell neoplasm (Bgcn) and the CCR4 deadenylase, a catalytic subunit of the CCR4-NOT complex. Bam has been proposed to bind CCR4 and displace it from the CCR4-NOT complex. Here, we investigated the interaction of Bam with the CCR4-NOT complex by using purified recombinant proteins. Unexpectedly, we found that Bam does not interact with CCR4 directly but instead binds to the CAF40 subunit of the complex in a manner mediated by a conserved N-terminal CAF40-binding motif (CBM). The crystal structure of the Bam CBM bound to CAF40 reveals that the CBM peptide adopts an α-helical conformation after binding to the concave surface of the crescent-shaped CAF40 protein. We further show that Bam-mediated mRNA decay and translational repression depend entirely on Bam's interaction with CAF40. Thus, Bam regulates the expression of its mRNA targets by recruiting the CCR4-NOT complex through interaction with CAF40

Cyclooxygenase-2 Silencing for the Treatment of Colitis: A Combined In Vivo Strategy Based on RNA Interference and Engineered Escherichia Coli

Nonpathogenic-invasive Escherichia coli (InvColi) bacteria are suitable for genetic transfer into mammalian cells and may act as a vehicle for RNA Interference (RNAi) in vivo. Cyclooxygenase-2 (COX-2) is overexpressed in ulcerative colitis (UC) and Crohn's disease (CD), two inflammatory conditions of the colon and small intestine grouped as inflammatory bowel disease (IBD). We engineered InvColi strains for anti-COX-2 RNAi (InvColi(shCOX2)), aiming to investigate the in vivo feasibility of a novel COX-2 silencing strategy in a murine model of colitis induced by dextran sulfate sodium (DSS). Enema administrations of InvColi(shCOX2) in DSS-treated mice led to COX-2 downregulation, colonic mucosa preservation, reduced colitis disease activity index (DAI) and increased mice survival. Moreover, DSS/InvColi(shCOX2)-treated mice showed lower levels of circulating pro-inflammatory cytokines and a reduced colitis-associated shift of gut microbiota. Considering its effectiveness and safety, we propose our InvColi(shCOX2) strategy as a promising tool for molecular therapy in intestinal inflammatory diseases