Postdoctoral Fellow

Joana Vidigal, Bethesda, MD and surrounding area Scientific focus area Cancer Biology,Cell Biology,Developmental Biology,Genetics and Genomics,Immunology,Molecular Biology and Biochemistry,RNA Biology,Stem Cell Biology The Vidigal lab seeking a postdoctoral researcher interested in dissecting small RNA biology using mouse models. About the position Our lab combines mouse genetics with computational, biochemical, imaging, and genomic approaches to gain a comprehensive understanding of the regulatory functions of small RNA in vivo. Current projects focus on the physiological roles of AGO-mediated RNA cleavage. Although this is a highly conserved process—also known as RNAi—its functions in mammals remain poorly understood. About the project: You will be working with mouse models and human cells to understand what roles AGO2 catalytic activity plays in mammals or how AGO2 functions are regulated in vivo. We put a high emphasis on mechanisms that are physiologically-relevant and have a potential to impact human health. This research will build on previous work from the lab (Perez, Nat. Comm. 2021; Sala, Nat. Struct. Mol. Bio. 2023; Prajapat, RNA 2024; Prajapat, NAR, 2024). You will work on projects supported by strong preliminary data that can be used to apply for fellowships and strengthen your CV. Once you end your research tenure with us, you will have full support to start the next step of your career including help to establish an independent research program if your goal is to start an independent lab. Read more about current research in the Vidigal Lab: 1. Dissecting AGO2 nuclear functions in vivo. Argonautes of the AGO clade are prototypically involved in gene regulation via the miRNA pathway, which requires their association with TNRC6/GW182 proteins in the cytoplasm, assembly of a large ribonucleoprotein RISC complex, and ultimately repression of gene expression through mRNA destabilization and/or inhibition of translation. This pathway is thought to play largely ubiquitous roles in mammals, with well-established functions during mammalian development, tumorigenesis, and tissue homeostasis. Yet—in collaboration with the laboratory of Craig Thompson (MSKCC)—we had previously found that AGO2 is not associated with high-molecular RISC complexes in quiescent B and T cells freshly isolated from mouse spleens (La Rocca et al. 2015). This contrasted data from developing embryos and established cancer lines. Because assembly of a high-molecular weight RISC is essential for gene repression by the miRNA pathway, these data suggested that AGO2’s roles in the pathway might be impaired in the context of quiescent splenocytes. It also raised the possibility that, in the absence of canonical RISC assembly, AGO2’s functions could be diverted to other regulatory mechanisms. We have started addressing these questions using a new genetic mouse model that carries a small epitope tag at the endogenous Ago2 locus. This work led to the discovery that during immune quiescence, low PI3K-AKT-mTOR signaling leads to poor translation of TNRC6, AGO2’s cytoplasmic binding partner, and other components of the miRNA pathway. Low levels of TNRC6 allow the majority of AGO2 to accumulate in the nucleus—through a yet unknown mechanism—where it associates with loci of transposable elements (TE) that are still mobile in mammalian genomes in an RNA-dependent manner. Using conditional loss-of-function mouse models we further showed that in these cells AGO2 represses TE expression via its conserved but poorly understood catalytic domain (Sala et al. 2023). These data support the surprising model that RNAi in mammals is not limited to the murine female germline but is also present in the soma. These findings opened numerous new research avenues that we are following up on, including: i) how AGO2 localization is regulated; ii) how is AGO2 targeted to transposons; and iii) what is the mechanism of AGO2-mediated TE repression. 2. Regulation of embryonic development by AGO catalytic activity. The ability of Argonaute proteins to cleave target nucleic acids is conserved from Archaea to humans. Yet, despite the deep conservation, how it is deployed to regulate mammalian physiology is not fully understood. We are tackling two unanswered questions regarding AGO catalytic functions in mammals which together with the work above, will further expand our view of the impact of AGO-mediated RNA cleavage to mouse physiology: Question 1) What are the functions of AGO2 catalysis during embryogenesis? AGO2’s catalytic activity is known to be required for neonatal viability. Yet, the reasons behind animal lethality are not known. Our preliminary data suggest that animals homozygous for an Ago2 catalytic dead allele die due to endothelial cell dysfunction caused by de-repression of an imprinted domesticated retrotransposon. We are building genetic models to test this hypothesis and to study how loss of cleavage of this transcript contributes to human disease. Question 2) What are the physiological requirements for AGO3’s catalytic domain? AGO catalytic competence depends on the DEDH tetrad present in the PIWI domain of the protein. This tetrad is also present in AGO3, but motifs in its N-terminus are thought to hinder cleavage. Recent work by other labs suggests that in vitro, the N-terminal block can be circumvented yielding a catalytic competent protein. We propose to test if catalytic activation of AGO3 can also occur in vivo, and if so whether it serves important physiological functions. Question 3) How does AGO catalytic activity impact tumorigenesis? We have preliminary data suggesting that AGO catalytic competence may be critical in some tumors. If true, this would be the first instance showing that cleavage by AGO proteins plays a role in human disease. International scientists and U.S. citizens are equally eligible. We are looking for researchers within 1 year of their PhD thesis defense that have experience in molecular biology. Expertise in mouse genetics, RNA biology, cancer, development, or immunology is a plus. #J-18808-Ljbffr Office of Intramural Training & Education