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Cell-type specific sequencing of microRNAs from complex animal tissues II

ABSTRACT: MicroRNAs (miRNAs) play an essential role in the post-transcriptional regulation of animal development and physiology. However, in vivo studies linking miRNA-function to the biology of distinct cell types within complex tissues remain challenging, partly due to the difficulties in achieving cellular resolution using available miRNA-profiling methods. Here, we report an in vivo small RNA-tagging approach that enables high-throughput sequencing of tissue- and cell type-specific miRNAs in animals, which we call microRNome by methylation-dependent sequencing, or mime-seq. The method combines orthogonal, cell-type-specific 3´ terminal 2’-O-methylation of animal miRNAs by the plant-specific methyltransferase HEN1, with oxidation-sensitive small RNA cloning and high-throughput sequencing. We show that mime-seq uncovers the miRNomes of rare cells within C. elegans and Drosophila at unprecedented specificity and sensitivity, enabling miRNA profiling with single-cell resolution in whole animals. Mime-seq overcomes the current challenges in tissue- and cell-type-specific small RNA profiling and provides novel entry points for understanding the function of miRNAs in spatially restricted physiological settings.

OTHER RELATED OMICS DATASETS IN: PRJNA412782PRJNA412779PRJNA412778PRJNA412474PRJNA412780PRJNA419385

ORGANISM(S): Mus musculus Drosophila melanogaster

PROVIDER: GSE104466 | GEO | 2018/02/14

REPOSITORIES: GEO

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Project description:miRNA levels depend on both biogenesis and turnover. The methyltransferase HEN1 stabilizes plant miRNAs, animal piRNAs, and siRNAs in both kingdoms via 3' terminal methylation. Loss of HEN1 in plants results in non-templated oligo-uridylation and accelerated degradation of miRNAs. In hen1 mutants from Arabidopsis and rice, we found that the patterns of miRNA truncation and uridylation differ substantially among miRNA families, but such patterns for the same miRNA are conserved between species. miR166 and miR163 are truncated predominantly to ~17 and ~16 nt, and subsequently recover via uridylation to approximately their original sizes, 21 and 24 nt, suggesting that in these cases miRNA truncation triggers uridylation. miR171 is untruncated but uridylated to 22 nt in hen1 mutants, gaining the ability to trigger production of phased, secondary siRNAs. Truncated and tailed variants were bound by ARGONAUTE1 (AGO1) in hen1, implying that these events occur while miRNAs are still bound by AGO1. Unexpectedly, a portion of miR158 in wildtype remains unmethylated and thus subject to uridylation and destabilization, suggesting that plants naturally utilize miRNA methylation to modulate miRNA accumulation. Our results suggest that the AGO1-containing RISC complex may undergo programming to reflect each bound miRNA, determining a defined, distinct decay destiny. In this analysis, we sequenced sRNAs from two hen1 mutant alleles in Arabidopsis and three hen1 alleles in rice. In Arabidopsis, the strong hen1-1 allele in the Landsberg erecta (Ler) ecotype is the first hen1 mutant and emerged from an enhancer screen in the hua1-1/hua2-1 background, and hen1-8 in the Columbia (Col) background is a weak allele. In rice, WAVY LEAF1 (WAF1) is the ortholog of Arabidopsis HEN1, and two mutant alleles waf1-1 and waf1-2 each bear a single-base substitution leading to a premature stop codon in the second exon and a non-functional splicing site of the fourth intron, respectively. We identified a third mutant allele of the rice HEN1 gene (Oshen1-3 from the Korean (POSTEC) rice T-DNA mutant population).

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Cell-type specific sequencing of microRNAs from complex animal tissues II

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