The overall aim of our research is to understand RNA based mechanisms involved in the control of cellular processes, in particular those that are related to disease and affecting the efficiency of cell based production systems. Moreover, we want to contribute to the further development of RNA based therapies that utilize our knowledge of RNA based mechanisms.
Sequencing based RNA probing.
The transcription of genomic information into RNA is central for all living organisms. Part of this information codes for proteins, but it is now also clear that many RNA molecules function in other ways, which depend on specific structure formed by internal base pairing within the RNA. In fact, all RNA molecules, including mRNAs coding for proteins, will be able to form RNA structures and how this “hidden” layer of genomic information affect cellular function remains largely unexplored. It is likely that RNA based regulation affects most cellular processes and is involved in many different diseases, but prediction of RNA structures from the primary sequence alone is very difficult and the structural RNA changes occurring inside cells have so far been impossible to study.
Over the last years, our group has contributed to the further development of well-known RNA probing methods such as the SHAPE method, which probes RNA secondary structure (basepairing) and Hydroxyl Radical Footprinting, which provides information on RNA tertiary structure (Kielpinski and Vinther, 2014). Historically, these methods have been restricted to the analysis of one RNA molecule at the time, but by adapting the experiments for modern sequencing technology and developing novel computational tools (Kielpinski et al. 2015), we have increased the throughput of such experiments by several orders of magnitude to make transcriptome wide analysis possible. Moreover, we have developed new reagents for SHAPE based RNA structure probing that allows us to select probed RNA positions from the background. The selection technology (SHAPES) allows us to increase the probing signal and makes the use of a nonprobed control unnecessary (Poulsen et al., 2015, WO2015-021990-A1). Our methods give us a unique possibility to identify RNA structural and regulatory changes inside cells.
SHAPE Selection strategy for reducing backgroung in SHAPE probing experiments.
Going forward, we want to use RNA probing to decode the RNA structure within the cellular environment and in this way identify:
- RNA structures that change in response to cellular stimuli.
- Human genetic variants and cancer driver mutations that affect RNA structure.
- Regions in the human transcriptome that is accessible to antisense therapeutics.
- Functionally relevant RNA structures in bacteria and positive-stranded RNA viruses.
Investigation of RNA regulation using Library Sequencing
Short stretches of RNA can regulate gene expression by interacting with proteins, other other RNAs or DNAs and antisense oligonucleotides. To investigate such RNA sequences, we have developed LibSeq (short for Library Sequencing), which is an accurate massive parallel sequencing-based method for completely characterizing the regulatory potential of thousands of short RNA sequences in one experiment. Recently, we demonstrated that LibSeq can be used to detect functional motifs for endogenous regulators such as RNA binding proteins and miRNAs and can quantify the regulatory impact of exogenous factors such as chemically modified DNA oligonucleotides designed to recruit RNase H (Rukov et al., 2015). The LibSeq strategy has many potential applications and going forward, we want to use this powerful methodology to dissect other regulatory mechanisms based on RNA.