부산대학교 도서관

Many microRNAs (miRNAs) are expressed with high spatiotemporal specificity during organismal development, with some being limited to rare cell types, often embedded in complex tissues. Yet, most miRNA profiling efforts remain at the tissue and organ levels. To overcome challenges in accessing the microRNomes from tissue-embedded cells, we had previously developed mime-seq (miRNome by methylation-dependent sequencing), a technique in which cell-specific miRNA methylation in C. elegans and Drosophila enabled chemo-selective sequencing without the need for cell sorting or biochemical purification. Here, we present mime-seq 2.0 for profiling miRNAs from specific mouse cell types. We engineered a chimeric RNA methyltransferase that is tethered to Argonaute protein and efficiently methylates miRNAs at their 3′-terminal 2′-OH in mouse and human cell lines. We also generated a transgenic mouse for conditional expression of this methyltransferase, which can be used to direct methylation of miRNAs in a cell type of choice. We validated the use of this mouse model by profiling miRNAs from B cells and bone marrow plasma cells.
Synopsis: Mime-seq 2.0 allows profiling of miRNAs from complex tissues and rare cell populations in the mouse. Cell-specific methylation of miRNAs by the chimeric RNA methyltransferase HENMT1ΔC-T6B enables chemo-selective enrichment by high-throughout sequencing.At-HEN1 efficiently methylates C. elegans and D. melanogaster miRNAs in vivo, but not in mammalian cells.An engineered HENMT1ΔC-T6B chimera methylates miRNAs in vitro in mammalian cell culture and in vivo in mice.Mime-seq 2.0 combines a simple workflow with high sensitivity to enrich miRNAs even from rare cell populations.
A new method based on the expression of a chimeric methyltransferase enables the sequencing of small RNAs from specific cell types in mice.