Biogenesis & Action of small RNAs

Our group is interested in unraveling the molecular determinants that allow the establishment of a potent RNA interference (RNAi) response in which small RNAs (sRNAs) are used to silence the expression of genes. RNAi pathways that target RNAs from repetitive sequences serve to maintain genome integrity, whereas pathways that target mRNAs allow the regulation of gene expression during development or in adaptation to the environment. We are particularly interested in understanding how selected primary sRNAs are amplified to generate robust RNAi and how this then eventually leads to heterochromatin formation in the nuclear RNAi pathway. We will address these questions by combining various structural biology techniques with quantitative biochemistry. Through our studies, we will thus gain insights into the catalytic activities and regulation and assign the functions to the individual subunits of the large macromolecular multiprotein complexes involved in these pathways. Our research will provide fundamental knowledge of the mechanism of gene silencing and in general on the regulation of gene expression.

Projects

A central step in the biogenesis of endogenous sRNAs in nematodes, fungi, and plants is the amplification of primary sRNAs by RNA-dependent RNA polymerases (RdRPs) to produce highly abundant secondary sRNAs. The secondary sRNAs then associate with Argonaute proteins to form RNA-induced silencing complexes (RISC), which then act as the actual effector complexes. RISC can regulate gene expression by either transcriptional gene silencing in the nucleus or post-transcriptional gene silencing in the cytosol. Our research focuses on two important aspects of the RNAi response. First, we are interested in understanding the amplification step, which is crucial to generate a potent and persistent RNAi response. Second, we study the nuclear RNAi pathway, which leads to epigenetic gene silencing by histone methylation.

Approach

Our group uses an integrated structural biology approach, with structural biological techniques forming the core (X-ray crystallography & single-particle electron microscopy) and complemented by biochemical and biophysical approaches. Our research focuses on the mechanistic characterization of protein complexes, which we obtain by two complementary methods. In the bottom-up approach, we produce and purify individual components or protein subcomplexes, which then can be used for the gradual build-up of increasingly larger assemblies. In the top-down approach, a single subunit of a protein complex carries an affinity tag, which allows purifying stable complexes from endogenous sources that can be utilized for structural studies but also for the identification of new interaction partners by mass spectrometry.