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Report

Teaser, summary, work performed and final results

Periodic Reporting for period 1 - miRCell (MicroRNA functions in single cells)

Teaser

Summary

It is now becoming apparent that genes are regulated not only by transcription, but also by thousands of post-transcriptional regulators that can stabilize or degrade mRNAs. Some of the most important regulators are miRNAs, short RNA molecules that are deeply conserved in sequence and are involved in numerous biological processes, including human disease. Surprisingly, transcriptomic and proteomic studies show that most miRNAs only have subtle silencing effects on their targets, suggesting additional important, but yet undiscovered functions. Thus the question is raised: if the main function of miRNAs is not to silence targets, what is it?

I will test two novel hypotheses about miRNA function. The first hypothesis proposes that miRNAs can buffer gene expression noise. The second hypothesis is inspired by my preliminary results and proposes that miRNAs can synchronize expression of genes. If I validate either hypothesis, it would mean that miRNA functions can be investigated in entirely new ways, yielding important new biological insights relevant to both basic research and human health. However, these hypotheses can only be tested in individual cells, and the necessary single-cell technologies and computational tools are only maturing now.

I will apply my expertise in miRNA biology and in combined wet-lab and computational methods to design, develop and apply miRCell-seq to test these two hypotheses in cell cultures and in animals. This new method will for the first time measure miRNAs, their targets, and the interactions between them in single cells and transcriptome-wide. We will use mutant cells devoid of miRNAs and time course experiments to generate sufficient data to develop detailed models of the miRNA impact on their targets. We will then validate our findings with single cell proteomics. This project thus has the potential to reveal novel functions of miRNAs and substantially improve our general understanding of gene regulation.

Work performed

Overall, we have made progress towards implementing the core protocol, miRCell-seq, but have not finalized this objective (Objective 1) yet due to technical challenges (described below). We have backup plans, and in the best-case scenario we could have the protocol ready in early 2020. We have also made substantial progress in later sub-objectives (Objectives 2-3), in particular with regard to the theoretical framework and methods for validation. This progress has resulted in two pre-prints that we will soon submit for publication in high-impact journals, acknowledging the funding from ERC:

https://www.biorxiv.org/content/10.1101/771402v1.full
https://www.biorxiv.org/content/10.1101/749473v3.full

The recruitment of the two wet-lab post docs has been delayed, since it is difficult to attract post docs with the required expertise and level of excellence. Instead, salary costs have covered the work of computational PhD students who have developed theoretical and analysis frameworks and who have also analyzed pilot data of collaborators, for the miRCell project. This is the effort that has pushed Objectives 2-3 and has resulted in the two pre-prints that will soon be submitted.

Final results

The implementation of miRCell-seq (Objective 1.1-3) has overall proven challenging (see original proposed timeline below). As planned, we have created a TRIBE protein that fuses Argonaute and ADAR domains, and we are now analyzing sequence data to see of the protein is functional (Objective 1.1).

We have also applied CATS to sequence miRNAs in single cells, but find that the method is ineffective in profiling miRNAs (Objective 1.2). We will therefore apply CATS in nano-liter volumes or using oligo-assisted sequencing to profile miRNAs effectively.
We have not yet brought these protocols together to form miRCell-seq (Objective 1.3).

However, due to the extra computational manpower, we are ahead of schedule with some objectives. Specifically, we have set up robust theoretical and computational frameworks to measure gene expression noise and synchronicity from sequence data (Objectives 2.1 and 3.1).

Further, over the next few months we will set up methods to genetically reintroduce miRNAs into mouse embryonic stem cells (Objectives 2.2 and 3.2).

With the help of collaborators, we have also established protocols to validate our findings using single-molecule FISH, single-cell qPCR and single-cell oligo-assisted proteomics (Objectives 3.4).

In summary, while we have not yet established the full miRCell-seq protocol, we have made substantial advances towards this, and have implemented important theoretical and analytical framework and validation methods, thus paving a way for fast analysis of the miRCell-seq data when it is generated.