SciLifeLab researchers develop agoTRIBE – the first method to detect microRNA targets in single cells
Researchers at SciLifeLab and Stockholm University (SU) have developed the first method to experimentally map microRNA targets transcriptome-wide in single cells. This is a huge improvement over existing state-of-the-art methods that require millions of cells and will facilitate the entry of the microRNA field into the single-cell era. The study was published in the journal Nature Biotechnology.
MicroRNAs are ubiquitous small transcripts that repress gene activity in basically all multicellular animals and plants. They bind to Argonaute (AGO) proteins and guide these to specific mRNA targets, causing the targets to be destroyed by the protein. More than 60% of all human genes are estimated to be regulated by microRNAs – therefore it is not surprising that they have been found to be involved in numerous biological processes including formation of cell identify, development and diseases including cancer. To discover the function of a microRNA, it is necessary to find out exactly which mRNAs are targeted by it.
Current state-of-the-art methods uses antibodies to isolate the Argonaute (AGO) proteins and then sequence the bound mRNA targets. However, these methods require a lot of material – typically in order of millions of cells – to work.
Now researchers at SciLifeLab and SU have developed a new method – agoTRIBE – to detect microRNA targets at the level of single cells. They have fused an Argonaute protein to the deaminase domain of the ADAR2 editing enzyme. When microRNAs guide the fusion protein to mRNAs, these targets undergo RNA editing events that can be detected by sensitive single-cell sequencing as A>G nucleotide conversions. With their new method, the researchers can follow microRNA targeting of thousands of RNAs during biological processes such as the cell cycle or cell differentiation.
In these processes, the researchers find that microRNAs – surprisingly – perform quite different tasks in each cell. In the future, it will be possible to also apply this method to study microRNA targeting in whole tissues, to find out exactly what is happening in each of the many cell types that comprise complex organs such as brains.
Dr. Marc Friedländer, SciLifeLab Fellow Alumnus and associate professor at Stockholm University, says: “The microRNA field has for many years been hampered by the lack of methods that can be applied to individual cells – we hope that our new method will help lift the field into the single-cell era.”
The work was spearheaded by Dr. Inna Biryukova, who took a leading role in developing the laboratory method, and by PhD student Vaishnovi Sekar, who performed the bulk of the advanced computational analyses. Vaishnovi Sekar highlights the challenges of the project: “In terms of complexity of the computational work, this is uncharted territory, and we lacked reference points and thresholds. We had to explore a myriad of approaches to devise a methodology that not only works but also yields biologically meaningful observations.”
The study was supported by ERC Starting Grant “miRCell” and by Vetenskapsrådet and has been published in the journal Nature Biotechnology.
