CRAC’n the dynamics of protein-RNA interactions in vivo

May 15, 2017
Versione stampabile

Time: May 15, 2017, h. 02:30 pm
Location: Room B103, Polo scientifico e tecnologico "Fabio Ferrari", building Povo 2, via Sommarive 9, Povo (Trento)

Speaker

Dr. Sander Granneman, Centre for Synthetic and Systems Biology (SynthSys), University of Edinburgh

 Abstract

By rapidly rewiring their transcriptional program to adapt to environmental changes, microbes can thrive even in hostile environments. Typically, the adaptation process is largely attributed to the activity of transcription factors. Yet it is becoming increasingly clear that RNA decay is instrumental in shaping gene expression profiles. Little is known, however, about the contribution of the factors and the signaling pathways that regulate this process.
Our aim is to obtain mechanistic insights into the role of RNA decay factors during stress by identifying the RNA substrates that are targeted for degradation during the stress adaptation response. Such a study is very challenging because RNA decay is fast and occurs already very early during the stress response. Thus, measurements need to be performed on short (minute) time-scales.
UV cross-linking is the preferred choice for identifying protein-RNA interactions as this enables the isolation of RNAs that are directly bound to the protein. With current commercially available equipment this is very inefficient and, depending on wavelength, takes several minutes up to half an hour to complete. Such prolonged UV exposure also induces major cellular stress responses due to DNA damage. These drawbacks make it impossible to capture RNA degradation events that happen during the first few minutes of stress adaptation and can bias the results towards RNA transcripts that are specific for the irradiation conditions.
To make in vivo kinetic studies of direct protein-RNA interactions possible, we have developed a new UV irradiation machine that can cross-link proteins to RNA in living cells in seconds and optimized protocols for cell-collection and cDNA library preparation. With our kinetic cross-linking and cDNA analyses approach (χCRAC) we have been able to, for the first time, quantitatively measure the dynamics of protein-RNA interactions in vivo at minute time-point resolution.
χCRAC-studies on the yeast transcription terminator Nab3 revealed highly dynamic binding during glucose deprivation and a surprising role in dampening expression of stress responsive genes. Our analyses also revealed a novel role for Nab3 in downregulating retrotransposon expression during stress, indicating that this protein plays an important role in maintaining genome integrity during stress.

 About the Speaker

Throughout my career my research has mainly concentrated on studying the assembly of large macromolecular protein-RNA complexes in eukaryotes, such as ribosomes. I have co-authored over thirty publications in this field and have been invited to present our research at seminars and conferences. Since the start of my group in 2011 my group has made significant advances in the yeast ribosome assembly field and developed new research tools that allow us to observe fast and dynamic processes in RNA biology. These technological advances have led me into a new research territory: post-transcriptional regulation of gene expression in microbes. Currently, our research mainly focusses on understanding how microorganisms are able to efficiently adapt to constantly changing environments, such as changes in nutrient availability. This involves a dynamic interplay between RNA transcription and degradation machineries and our goal is to unravel how this is regulated.

Contact person regarding this talk: Gabriella Viero, gabriella.viero [at] cnr.it, Andrea Passerini, andrea.passerini [at] unitn.it