BBB Seminar: Gunter Meister
Mechanisms of microRNA-guided gene silencing
Department of Biochemistry I, University of Regensburg, Germany
Small regulatory RNAs such as short interfering RNAs (siRNAs), microRNAs (miRNAs) or Piwi interacting RNAs (piRNAs) have been discovered and it is becoming more and more apparent that these small molecules have key-regulatory functions. Small RNAs are found in all higher eukaryotes and play important roles in cellular processes as diverse as development, stress response or transposon silencing. Members of the Argonaute (Ago) protein family are mediators of gene silencing and bind to small regulatory RNAs. Ago proteins are characterized by PAZ and PIWI domains and facilitate target RNA cleavage in RNA interference (RNAi) or miRNA-guided repression of gene expression. We have generated highly specific monoclonal antibodies against all human Ago proteins and used them for biochemical and functional characterization of human Ago complexes. We find that Ago proteins are embedded into large protein-RNA networks with distinct enzymatic activities. Furthermore, we purified mRNAs from isolated Ago complexes and validated numerous Ago-associated mRNAs as miRNA targets. With this approach we have established a powerful tool allowing for the experimental identification of miRNA targets from any cell line or tissue. Furthermore, cloning and sequencing of the small RNAs associated with isolated Ago protein complexes revealed a novel small RNA pathway in human cells.
Although extensive research has elucidated the major steps in small RNA-guided gene silencing, not much is known about Ago protein regulation. We have performed a comprehensive analysis of Ago2 phosphorylation in human cells. We find that the highly conserved tyrosine Y529, located in the small RNA 5’ end-binding pocket of Ago proteins can be phosphorylated. By substituting Y529 with a negatively charged glutamate mimicking a phosphorylated tyrosine, we show that small RNA-binding is strongly reduced. Our data suggest that a negatively charged phospho-tyrosine generates a repulsive force that prevents efficient binding of the negatively charged 5’ phosphate of the small RNA.
In addition to Y529, we found that Ago2 is phosphorylated at many other residues as well. However, the consequences for Ago2 function are not yet understood. Currently, we are using biochemical as well as cell biological methods to unravel the role of phosphorylation regarding Ago2 activity.
Host: Clive R. Bramham, Department of Biomedicine