Defining genetic interactions and dependencies of microRNA biogenesis factors

• Applicant: Professor Dr. Tony Gutschner
• Subject Area: General Genetics and Functional Genome Biology ; Cell Biology
• Term: from 2020 to 2023
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 440716364

The RNase III enzyme Dicer lies at the crux of the biogenesis pathway for almost all microRNAs (miRNAs) in mammalian cells. Its activity is crucial for maintaining a functional miRNA network that controls several cellular processes, e.g. proliferation, migration, apoptosis, and differentiation. While some studies could show that cell-specific ablation of Dicer in murine systems can be tolerated, it is largely unknown how human cell lines respond to a partial or complete loss of miRNAs by Dicer deletion. Only recently, studies using immortalized or transformed cells to knockout human Dicer could show that these cells are viable, yet they presented a strongly reduced proliferation rate. However, the underlying molecular mechanisms of the reduced proliferation are largely obscure. In preliminary experiments, we have applied CRISPR/Cas9-based genome engineering to generate Dicer-deficient human lung cancer cells. These cells also showed a reduced growth rate as well and subsequent transcriptome analyses identified several deregulated genes and signalling pathways. To obtain a deeper understanding of the molecular mechanisms underlying the observed cellular phenotypes and gene expression changes, we aimed at conducting a functional genomics screen thereby mapping the genetic interactions of human Dicer, which have been unknown so far. However, inquiring these interactions in Dicer-deficient cells using pooled, genome-wide small hairpin RNA (shRNA) libraries was not possible due to the fact that shRNAs require Dicer processing for their own maturation and function. Hence, we used the CRISPR/Cas9 system as a complementary genetic tool and performed genome-wide loss-of-function screens in Dicer wild type and knockout cells. This screening approach together with our gene expression data identified a novel regulatory link between Dicer and the TGF-ß pathway. Our data suggest that Dicer, through its critical function in the biogenesis of miRNAs, acts as a rheostat for the TGF-ß pathway and determines the signalling outcome and cell fate. Our finding of TGF-ß switching its pro-tumorigenic function to become tumour suppressive in the absence of Dicer (and miRNAs) could have significant clinical implications. Hence, this proposal aims to elucidate the genetic and molecular details of this interaction and identify critical miRNAs as well as relevant downstream target genes. This might contribute to the development of novel therapeutic strategies for anticancer therapy in the future.

• DFG Programme: Research Grants