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MicroRNA functions in single cells

Periodic Reporting for period 4 - miRCell (MicroRNA functions in single cells)

Reporting period: 2022-09-01 to 2023-02-28

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 repressive 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 repress 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.

Now, at the conclusion of the project, we have achieved most of our objectives and answered the biological questions in part. We have developed agoTRIBE, the first method to detect miRNA targets transcriptome-wide in single cells, and we have also developed a computational method to accurately impute miRNA activities in single cells from their footprint on the transcriptome. We have applied single-cell transcriptomics and genetic perturbation to study miRNA functions in single cells. We find that some miRNA reduce - while other miRNAs induce - gene expression noise at the transcriptional level. This is consistent with some miRNAs having putative functions in noise buffering, while others might facilitate state-switching in single cells. We also provide the first evidence that miRNAs can naturally induce transcriptome-wide synchronicity between targets. This may have functions in ensure stoichiometry between targets that encode proteins that are part of the same protein complexes. Lastly, we have developed a panel of oligo-conjugated antibodies that can be used to test our hypotheses also at the protein level in single cells. With this we can validate our observations also at the protein level. In summary, by developing and applying beyond-the-state-of-the-art single-cell genomics, we provide evidence that miRNAs have many distinct and complementary functions besides repression - including regulation of expression noise and synchronicity.
In the previous period (Period 3) we succeeded in developing the first method to detect microRNA targets transcriptome-wide in single cells. We call our method agoTRIBE. Our study was submitted to the prestigious Nature Biotechnology journal and received favorable reviews, however more experiments were requested. In this period (Period 4) we have focused on conducted these fairly extensive experiments in single cells and in pooled cell populations, and we are now very close to resubmitting our revised manuscript to the journal. We consider our agoTRIBE method to be clearly beyond-the-state-of-the-art and also as the main deliverable of the project. Together with our new method to impute microRNA activities from their transcriptome footprint in single cells (being written up as a manuscript) we have entirely fulfilled Objective 1 of the project.

In this period, we have also worked on a method to profile microRNA-related proteins in single cells. We are now very close to getting the method to work, and this will be used in our future studies and of course the ERC project will be properly acknowledged. This method should be used for validation studies related to Objective 2 and 3 of the project, validating the important findings we have made at the transcriptome level related to Objective 2 (microRNAs can reduce and induce noise on their targets) and Objective 3 (microRNAs can synchronize expression of their targets).

We have not performed functional validation in nematode worms (Objective 4) since we have decided to prioritize studies in mammalian cells.

Overall, our studies have resulted in a number of main publications and spin-off publications (listed under ‘Publications’ in the report) and several manuscripts that are close to submission.
As part of the project, we have developed the agoTRIBE method, which is clearly beyond the state-of-the-art. The current state of the art for identifying miRNA targets are CLIP-seq, which is a functional genomics method to find targets transcriptome-wide, using immunoprecipitation and sequencing. This method however requires millions of cells as input. On the other hand, using imaging it is possible to detect miRNA targeting in single cells, however imaging is typically limited to studying a single artificial target in one experiment. The agoTRIBE method in a sense combines the best features of the two state-of-the-art methods: it allows the transcriptome-wide mapping of miRNA targets in single cells. Besides the development of this experimental method, we have developed a computational method to accurately impute miRNA activities in single cells from their footprint on the transcriptome, and we have developed a panel of oligo-conjugated antibodies for studying/validating miRNA functions at the protein level in single cells.

Importantly, we have pushed the state-of-the-art of our understanding of miRNA functions, in that we provide evidence that miRNAs can both induce and reduce gene expression noise in single cells, and can naturally induce gene expression synchronicity between targets transcriptome-wide in single cells.
agoTRIBE compared to the two state-of-the-art methods
Outline of the agoTRIBE method