Final Report Summary - MIRFUNCTION (Dissecting the mechanisms of miRNA function) 1. Publishable summary miRNAs are crucial regulators of gene expression that target more than half of eukaryotic genes and play a role in human diseases, including cancer and neurological disorders. Therefore, it is important to understand the molecular mechanisms underlying miRNA-mediated repression. miRNAs act as guides recognizing their target mRNAs by complementarity and recruiting a complex of proteins, including Argonaute and GW182/TNRC6 family, that represses mRNA expression. Chekulaeva and colleagues have shown that mammalian GW182 recruits deadenylation complexes via novel protein motifs (W-motifs), leading to translational repression and mRNA deadenylation (Chekulaeva, Mathys, NSMB 2011). As a part of the current project, the Chekulaeva lab showed the mechanism of miRNA silencing via the CCR4-NOT-W-motif complex is conserved for at least 600 million years and was already present in ancient animals (Mauri et al. Chekulaeva, NAR 2016). The role of miRNAs in the repression of protein production has been well demonstrated. However, for a cell it is important not only how much of a given protein it produces, but also where in the cell this protein is produced. This is often achieved by localizing mRNAs to specific sites of the cell and repressing their translation prior to localization. These processes underlie establishment of body axis, cell migration, synaptic plasticity and other important events. Moreover, a number of human pathologies, including neurological disorders such as the Fragile X syndrome, are associated with a failure to localize specific mRNAs to specific subcellular compartments. miRNAs have been shown to play a role in localized translation in synapses, and in the Drosophila embryos. However, no systematic analysis has been done to dissect the roles of miRNAs in subcellular mRNA localization and localized translation on a genome-wide level. To bridge this gap, Chekulaeva lab developed a neurite/soma separation scheme in combination with mass spectrometry, RNAseq, Riboseq and bioinformatic analyses (Zappulo et al. Chekulaeva, Nature Com. 2017). This work lead to identification of proteins and RNAs that are differentially localized and translated between neurites and soma of neuronal cells. Using this approach, Chekulaeva lab quantified 7323 proteins and the entire transcriptome and identified hundreds of neurite-localized proteins and locally translated mRNAs. These results demonstrate that mRNA localization is the primary mechanism for protein localization in neurites and may account for more than a half of the neurite-localized proteome. Moreover, Chekulaeva lab identified multiple neurite-targeted non-coding RNAs and RNA-binding proteins with potential regulatory roles. This work established the tools for dissection of the roles of neurite-targeted miRNAs in establishment of neuronal polarity. The research group of Marina Chekulaeva at the Max Delbrück Center for Molecular Medicine in the Helmholtz Association comprises five PhD students, one Master student, two interns and one postdoc. As the Principal Investigator, Marina Chekulaeva has been awarded several research grants (EU-JPND multi-national consortium “localMND: Common architecture of local proteome, transcriptome and translatome across Motor Neuron disorders”; DFG SPP1935 “Deciphering the mRNP code”; Einstein Foundation Berlin to the collaborative network “Single Molecule RNA Biology”), and secured external funds for her PhD students (Engelhorn Stiftung, DAAD). This provides Marina Chekulaeva with a long-term perspective at the Max Delbrück Center (MDC) and allows for continuation of her research on the mechanisms of post-transcriptional gene regulation and roles of miRNAs in this process.
