Cell-type specific sequencing of microRNAs from complex animal tissues

Nature Methods
Chiara AlbertiLuisa Cochella

Abstract

MicroRNAs (miRNAs) play an essential role in the post-transcriptional regulation of animal development and physiology. However, in vivo studies aimed at linking miRNA function to the biology of distinct cell types within complex tissues remain challenging, partly because in vivo miRNA-profiling methods lack cellular resolution. We report microRNome by methylation-dependent sequencing (mime-seq), an in vivo enzymatic small-RNA-tagging approach that enables high-throughput sequencing of tissue- and cell-type-specific miRNAs in animals. The method combines cell-type-specific 3'-terminal 2'-O-methylation of animal miRNAs by a genetically encoded, plant-specific methyltransferase (HEN1), with chemoselective small-RNA cloning and high-throughput sequencing. We show that mime-seq uncovers the miRNomes of specific cells within Caenorhabditis elegans and Drosophila at unprecedented specificity and sensitivity, enabling miRNA profiling with single-cell resolution in whole animals. Mime-seq overcomes current challenges in cell-type-specific small-RNA profiling and provides novel entry points for understanding the function of miRNAs in spatially restricted physiological settings.

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Citations

Apr 6, 2018·Annual Review of Neuroscience·Kenneth S Kosik, Tomasz Nowakowski
Jun 28, 2018·Development·Wayo MatsushimaEric A Miska
Aug 7, 2018·Investigational New Drugs·Chongchong WangLi Cheng
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Jun 26, 2020·Frontiers in Cell and Developmental Biology·Philipp J Dexheimer, Luisa Cochella
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Oct 31, 2020·Current Biology : CB·Philipp J DexheimerLuisa Cochella
Jan 12, 2021·Frontiers in Cell and Developmental Biology·Xiaohuan ChaoQingbin Luo
Mar 26, 2021·BioEssays : News and Reviews in Molecular, Cellular and Developmental Biology·Andrea Pozzi, Damian K Dowling
Apr 30, 2021·Scientific Reports·Morten Muhlig Nielsen, Jakob Skou Pedersen
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Aug 17, 2021·Development·Dimitris KatsanosMichalis Barkoulas
Oct 5, 2021·Genomics, Proteomics & Bioinformatics·Tao HuXiaowo Wang

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Methods Mentioned

BETA
dissection
fluorescence-activated cell sorting
transgenic
electrophoresis
immunoprecipitation
immunoprecipitations
PCR
Protein Assay
fluorescence imaging

Software Mentioned

IDT
R script
count
GraphPad
Nextflow
seq
DESeq2
htseq
Prism
Addgene

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