DGCR8’s N-terminus in guiding microRNA biogenesis

An active area of research in biology aims to decipher how the individual cells in our bodies control which of their about 20.000 genes are converted into proteins to meet the requirements in different tissues and organs. One way to influence whether and how much of a particular protein is made is through microRNAs (miRNAs), a type of short, non-protein-coding RNA that have been shown to inhibit gene expression in a specific and regulated manner, thereby impacting almost all biological processes in mammals. To fully understand the function of miRNAs in gene regulation, our longstanding aim is to gain further insight into how miRNAs are made, i.e. how they are converted from a precursor into their biologically active form. We are specifically interested in the first step of this multistage process: Here, each future miRNA - which is characterized by a specific fold embedded in longer RNA molecules - must to be precisely recognized and subsequently cleaved out by a protein complex called the Microprocessor before undergoing further maturation. In the funded project, we will focus on the role of DGCR8, one of the two Microprocessor components involved in this recognition/cleavage step. Preliminary experiments from our laboratory suggest that a significant portion of DGCR8 is dispensable for the generation of some, but critical for other miRNAs. Of note, this part of the protein has not been well studied thus far, likely because it is predicted to lack a defined structure. In the first part of the proposed work, we therefore want to assign a clear biological function to this region of DGCR8, i.e. we want to identify which of its molecular features are required for miRNA generation. To address this, we intend to exchange the functional DGCR8 protein within cultured cells with variants thereof in which defined sections of the protein region have either been modified or removed. By monitoring the effect of these alterations on the pattern of expressed miRNAs, we will be able to pinpoint which parts of the protein are actually relevant for its function. In the second part, we will then decipher how the unstructured part of DGCR8 confers its biological role. Hypothesizing that this is mediated by recruitment of other proteins, we will pursue two independent strategies that aim to precisely characterize which proteins bind to those regions of DGCR8. Follow-up experiments then intend to reveal which of those recruited proteins is relevant for miRNA generation and how their recruitment modulates the Microprocessor. Together, we anticipate that these studies will shed light on the initial stages of miRNA generation, and consequently will further extend our general understanding of miRNAs as gene regulators.