Poster abstracts
Poster number 81 submitted by Swetha Rajasekaran
Pumilio regulates Dicer1 protein and miRNA levels in human cells
Swetha Rajasekaran (Department of Molecular Genetics, The Ohio State University), Wayne Miles (Department of Cancer Biology and Genetics, The Ohio State University)
Abstract:
DICER1 is an endoribonuclease involved in processing of mature miRNAs. Maintaining homeostasis of DICER1 protein levels has shown to be important as changes in Dicer1 levels can affect cellular phenotypes, and drive tumor growth. Mutations within the DICER1 gene cause Dicer1 syndrome – an inherited disorder that results in the development of uncommon cancerous and benign tumors. Most DICER1 mutations occur in the coding sequence of the transcript, however around 30% of the Dicer1 syndrome patients do not have a mapped mutation within this region. By analyzing sequences from a large cohort of Dicer1 syndrome patients, our lab has identified an A-to-G mutation in the 3’ untranslated region (3’ UTR) of DICER1. Motif mapping studies have shown that this mutation disrupts the binding site of the Pumilio (PUM) RNA-binding protein (RBP). The PUM family of proteins bind a consensus PUM Regulatory Element (PRE) within the 3’UTR of the mRNA to affect levels of protein translation and mRNA turnover of the substrate. PUM proteins are generally considered to be translational repressors that bind their targets and reduce the translation efficiency and ultimately the protein expression of the transcript. Surprisingly, on depleting PUM proteins using shRNA or CRISPR, we observed a decrease in DICER1 protein levels compared to WT, contrary to the expected model of PUM-mediated translational repression. Luciferase assays with WT and PRE-mutant DICER1 3’UTR fragments showed that this regulation is PRE-mediated and caused by direct binding. To test the importance of PUM-mediated regulation of Dicer1, we made knock-in of PRE-mutations in the endogenous DICER1 3’UTR in human cell lines using CRISPR. We then measured how this mutation affected growth rate, invasion and migration and compared with WT cells. These experiments showed that an isolated mutation in the DICER1-PRE resulted in slower growth, lesser invasion and decreased rate of migration in these cells. DICER1 expression is a double-edged sword in cancer and it is important for the cells to maintain the right amount of DICER1 making it hard to develop therapeutic strategies directly targeting DICER1. Understanding PUM-mediated post-transcriptional regulation of DICER1 may provide opportunities to target DICER1 activity in tumors.
Keywords: Pumilio, DICER1