Mechanistic and functional characterization of CCR4-NOT-mediated mRNA decay

• Applicant: Dr. Filip Pekovic
• Subject Area: General Genetics and Functional Genome Biology ; Biochemistry ; Structural Biology
• Term: since 2023
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 531520533

Almost all human mRNAs carry a polyadenosine tail at 3’ ends. This poly(A) tail has a critical function in the stability and translatability of the mRNA. The CCR4-NOT complex is an essential and highly conserved eukaryotic trans-acting effector responsible for the enzymatic shortening of the poly(A) tails of a large fraction of human mRNAs. The CCR4-NOT complex is recruited to specific mRNAs via RNA-binding proteins (RBPs). This mechanism, known as targeted deadenylation, acts as a means for the cell to effect rapid and gene-specific silencing of expression in response to cellular cues or extracellular signals. Defects in RBP-mediated targeted deadenylation are often associated with diseases such as cancer and inflammation and manifest as developmental defects. In the proposed research, I will focus on the RBP Tristetraprolin (TTP) as a model for studying RBP-targeted deadenylation by the CCR4-NOT. TTP recognizes AU-rich elements in the 3’ untranslated regions of many mRNAs encoding factors involved in the immune response and inflammation. I will conduct an interdisciplinary study involving multiple collaborations to advance the project's goals. First, I will perform structural studies using high-resolution single-particle cryoelectron microscopy to determine the structure of the human CCR4-NOT alone, in a complex with RNA substrate, and in a complex with TTP. The identified interfaces and key stabilizing residues will be characterized by biophysical methods to determine the binding affinities and dissociation rates. The loss-of-function mutations will be assessed in established in vitro RNA degradation assays and with mRNA reporters in cells. I will then aim to decipher whether the molecular principles governing the TTP-targeted deadenylation via the CCR4-NOT are generalizable to other RBPs. I will apply mass spectrometry combined with chemical cross-linking to identify novel RBPs that function to regulate mRNA targets via the CCR4-NOT in human cells. This will reveal if interaction sites and mechanisms are conserved among RBPs, which I will correlate to disease mutations. Besides identifying novel interactions with RBPs, I aim to utilize crosslinking and immunoprecipitation (CLIP) to identify direct interactions between CCR4-NOT and mRNAs to investigate the mechanism of RBP-independent targeted deadenylation. The proposed research seeks to advance our understanding of the mechanism of CCR4-NOT-mediated deadenylation, which will be of significant interest to a broad scientific community interested in gene expression. The molecular focus of the research has translational potential and will serve as an immediate platform for studies aimed at optimizing mRNA-based therapeutics and inhibitors of RBP-mediated interactions to identify new treatments for cancer and inflammation.

• DFG Programme: WBP Fellowship
• International Connection: USA
• Host: Eugene Valkov, Ph.D.