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MicroRNA biogenesis and function in single cells
Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.ORCID iD: 0000-0002-6810-1591
2024 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

microRNA are short non-coding RNAs and important post transcriptional gene regulators. miRNAs are found in all animals that have been studied, in numbers that largely correlate with organismal complexity. For instance, nematodes have around 200 miRNA genes, while humans have more than 600 miRNA genes. Mutant animals that are deficient in miRNAs generally exhibit gross developmental defects or embryonic lethality, underlining the importance of these regulators. Given that an estimated ~60% of human mRNAs are targeted by miRNAs in some cellular context, it is not surprising that these regulators are involved in numerous biological processes, ranging from the formation of cell identity to development and human diseases. Even though miRNAs have been systematically studied for over a decade, fundamental questions regarding their biogenesis and function remain unanswered. microRNAs are unevenly distributed between cell types and within homogeneous cell populations, affecting the transcriptomes of individual cells. The vast majority of miRNA studies have been conducted on large pools of cells, and little is known about the biogenesis and function in individual cells. To understand their effect on gene regulation, single-cell measurements are crucial. This thesis introduces techniques that allow us to extend our understanding about microRNA at the resolution of single cells.

In Paper I, we develop and establish agoTRIBE – the first sequencing-based method to measure regulatory interactions between miRNAs and their mRNA targets transcriptome-wide in single cells. We applied Smart-seq3 single-cell RNA sequencing to detect increased editing transcriptome-wide in key miRNA targets and found substantial differential targeting across the cell cycle and in mixed cell populations. This method overcame limitations of current methods and allowed for study of heterogeneity in miRNA targeting across individual cells. In Paper II, we further explored miRNA targeting landscape in single cells using agoTRIBE and revealed differential targeting within homogenous cell populations. We observe two groups of cells with overlapping but distinct targeting patterns and provide evidence that miRNAs act on different groups of genes with distinct biological functions. In paper III, we proposed a method 'micro-imp' to infer miRNA activity from existing single cell RNA-sequencing (scRNA-seq) data, to overcome the limitation of low sensitivity in direct miRNA sequence of single cells. We show significant positive correlation between the inferred activity and the measured miRNA expression suggesting that this approach can be utilised to obtain orthogonal information from existing scRNA-seq datasets. In paper IV, we provide valuable insights from miRNA profiling in single cells and together with integration of scRNA-seq data from the same study system. We highlight heterogeneity in miRNA expression across single cells and link it to the variation and covariation of the target pool. Further, we detect large parts of pri-miRNA transcripts in single cells devoid of biogenesis factors, an aspect previously underexplored. The work presented in this thesis focus on methods and techniques to expand our understanding of miRNA biology at single cell resolution. 

 

Place, publisher, year, edition, pages
Stockholm: Department of Molecular Biosciences, The Wenner-Gren Institute, Stockholm University , 2024. , p. 62
Keywords [en]
microRNA, RNA biology, scRNA-seq, TRIBE, cellular heterogeneity
National Category
Bioinformatics and Systems Biology
Research subject
Molecular Bioscience
Identifiers
URN: urn:nbn:se:su:diva-229027ISBN: 978-91-8014-825-2 (print)ISBN: 978-91-8014-826-9 (electronic)OAI: oai:DiVA.org:su-229027DiVA, id: diva2:1856556
Public defence
2024-08-23, Air & Fire (Gamma 2), SciLifeLab, Tomtebodavägen 23A, Solna, 09:00 (English)
Opponent
Supervisors
Funder
Swedish Research Council, Consolidator Grant no. 2022-03953 ‘InSync’EU, European Research Council, Starting Grant no. 758397, ‘miRCell’Available from: 2024-05-30 Created: 2024-05-07 Last updated: 2024-05-23Bibliographically approved
List of papers
1. Detection of transcriptome-wide microRNA-target interactions in single cells with agoTRIBE
Open this publication in new window or tab >>Detection of transcriptome-wide microRNA-target interactions in single cells with agoTRIBE
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2023 (English)In: Nature Biotechnology, ISSN 1087-0156, E-ISSN 1546-1696Article in journal (Refereed) Epub ahead of print
Abstract [en]

MicroRNAs (miRNAs) exert their gene regulatory effects on numerous biological processes based on their selection of target transcripts. Current experimental methods available to identify miRNA targets are laborious and require millions of cells. Here we have overcome these limitations by fusing the miRNA effector protein Argonaute2 to the RNA editing domain of ADAR2, allowing the detection of miRNA targets transcriptome-wide in single cells. miRNAs guide the fusion protein to their natural target transcripts, causing them to undergo A>I editing, which can be detected by sensitive single-cell RNA sequencing. We show that agoTRIBE identifies functional miRNA targets, which are supported by evolutionary sequence conservation. In one application of the method we study microRNA interactions in single cells and identify substantial differential targeting across the cell cycle. AgoTRIBE also provides transcriptome-wide measurements of RNA abundance and allows the deconvolution of miRNA targeting in complex tissues at the single-cell level.

National Category
Bioinformatics and Systems Biology Cell and Molecular Biology
Identifiers
urn:nbn:se:su:diva-223224 (URN)10.1038/s41587-023-01951-0 (DOI)001071129200003 ()37735263 (PubMedID)2-s2.0-85171647219 (Scopus ID)
Available from: 2023-11-06 Created: 2023-11-06 Last updated: 2024-05-07
2. microRNA targeting variability in single cells from a homogenous cell population
Open this publication in new window or tab >>microRNA targeting variability in single cells from a homogenous cell population
(English)Manuscript (preprint) (Other academic)
Abstract [en]

microRNAs (miRNAs) are small non-coding RNAs that regulate gene expression post-transcriptionally. miRNAs have distinct expression profiles across tissues and cell types, and there is even evidence that miRNAs can vary in abundance between individual cells in otherwise homogenous cell populations. However, due to a paucity of single-cell methods to measure miRNA targeting, the functional implications of these variations have not been studied transcriptome-wide. In this study, we re-analyze our published agoTRIBE single-cell targeting data from HEK293T cells, to investigate if single cells from a homogenous population display differences in miRNA targeting. We find that two cell populations display distinct targeting patterns, with one population being targeted specifically by miR-10 and miR-16. Transcripts targeted in one population have functions in biosynthesis and chromatin organization, while transcripts in the other population have functions in various stress responses. In summary, we find that miRNAs appear to have overlapping but also distinct functions in two subpopulations of cells in an otherwise homogenous human cell culture. 

National Category
Bioinformatics and Systems Biology
Research subject
Molecular Biology
Identifiers
urn:nbn:se:su:diva-228940 (URN)
Funder
EU, European Research Council, Starting Grant no. 758397, ‘miRCell’Swedish Research Council, 2019-05320, ‘MioPec’Swedish Research Council, Consolidator Grant no. 2022-03953 ‘InSync’
Available from: 2024-05-06 Created: 2024-05-06 Last updated: 2024-05-07
3. Imputation of miRNA activity in single cells from their transcriptome footprint
Open this publication in new window or tab >>Imputation of miRNA activity in single cells from their transcriptome footprint
(English)Manuscript (preprint) (Other academic)
Abstract [en]

MicroRNAs (miRNAs) are major class of small RNAs that play important role in post-transcriptional gene regulation. There is an increasing interest in profiling miRNAs in single cells for studying their expression heterogeneity; however, all current methods to directly sequence miRNA in single cells have low sensitivity (<5%). Because of Poissonian (sampling) noise, this limits what type of quantitative questions can be addressed using direct sequencing. Here, we investigate if we can - as an alternative approach – indirectly infer activity of miRNAs from their footprint on the transcriptome. Specifically, we want to leverage the high sensitivity (60%) of single-cell RNA-seq data like SmartSeq3 to detect patterns of coordinated repression of the mRNA targets of specific miRNA - thus extracting orthogonal single-cell information on gene regulation from already existing resources. Using our new method micro-imp, we show that activity of miRNAs can be inferred from their footprint of the transcriptome with an accuracy that approaches that of directly sequencing the miRNAs. 

Keywords
microRNAs, transcriptomics, single cells, gene regulation
National Category
Biological Sciences Bioinformatics and Systems Biology
Research subject
Molecular Bioscience
Identifiers
urn:nbn:se:su:diva-228936 (URN)
Funder
Swedish Research Council, 2019-05320, ‘MioPec’EU, European Research Council, Starting Grant 758397, ‘miRCell’Swedish Research Council, Consolidator Grant no. 2022-03953 ‘InSync’
Available from: 2024-05-06 Created: 2024-05-06 Last updated: 2024-05-07
4. Landscape of microRNA and target expression variation and covariation in single mouse embryonic stem cells
Open this publication in new window or tab >>Landscape of microRNA and target expression variation and covariation in single mouse embryonic stem cells
Show others...
(English)Manuscript (preprint) (Other academic)
Abstract [en]

MicroRNAs are small RNA molecules that can repress the expression of protein coding genes post-transcriptionally. Previous studies have shown that microRNAs can also have alternative functions including target noise buffering and co-expression, but these observations have been limited to a few microRNAs. Here we systematically study microRNA alternative functions in mouse embryonic stem cells, by genetically deleting Drosha - leading to global loss of microRNAs. We apply complementary single-cell RNA-seq methods to study the variation of the targets and the microRNAs themselves, and transcriptional inhibition to measure target half-lives. We find that microRNAs form four distinct co-expression groups across single cells. In particular the mir-290 and the mir-182 clusters are abundantly, variably and inversely expressed. Intriguingly, some cells have global biases towards specific miRNAs originating from either end of the hairpin precursor, suggesting the presence of unknown regulatory cofactors. We find that miRNAs generally increase variation and covariation of their targets at the RNA level, but we also find miRNAs such as miR-182 that appear to have opposite functions. In particular, miRNAs that are themselves variable in expression, such as miR-291a, are more likely to induce covariations. In summary, we apply genetic perturbation and multi-omics to give the first global picture of microRNA dynamics at the single cell level. 

Keywords
miRNA, single-cells, biogenesis, gene expression noise, gene expression variation
National Category
Biological Sciences Bioinformatics and Systems Biology
Research subject
Molecular Bioscience
Identifiers
urn:nbn:se:su:diva-228941 (URN)10.1101/2024.03.24.586475 (DOI)
Funder
Swedish Research Council, Grant no. 2019-05320, ‘MioPec’Swedish Research Council, Consolidator Grant no. 2022-03953 ‘InSync’EU, European Research Council, Starting Grant no. 758397, ‘miRCell’
Available from: 2024-05-06 Created: 2024-05-06 Last updated: 2024-05-07

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