Global identification of target recognition and cleavage by the Microprocessor in human ES cells

Abstract

The Microprocessor plays an essential role in canonical miRNA biogenesis by facilitating cleavage of stem-loop structures in primary transcripts to yield pre-miRNAs. Although miRNA biogenesis has been extensively studied through biochemical and molecular genetic approaches, it has yet to be addressed to what extent the current miRNA biogenesis models hold true in intact cells. To address the issues of in vivo recognition and cleavage by the Microprocessor, we investigate RNAs that are associated with DGCR8 and Drosha by using immunoprecipitation coupled with next-generation sequencing. Here, we present global protein–RNA interactions with unprecedented sensitivity and specificity. Our data indicate that precursors of canonical miRNAs and miRNA-like hairpins are the major substrates of the Microprocessor. As a result of specific enrichment of nascent cleavage products, we are able to pinpoint the Microprocessor-mediated cleavage sites per se at single-nucleotide resolution. Unexpectedly, a 2-nt 3′ overhang invariably exists at the ends of cleaved bases instead of nascent pre-miRNAs. Besides canonical miRNA precursors, we find that two novel miRNA-like structures embedded in mRNAs are cleaved to yield pre-miRNA-like hairpins, uncoupled from miRNA maturation. Our data provide a framework for in vivo Microprocessor-mediated cleavage and a foundation for experimental and computational studies on miRNA biogenesis in living cells.

miRNAs Are Essential for the Regulation of the PI3K/AKT/FOXO Pathway and Receptor Editing during B Cell Maturation

“…Among these ncRNAs are miRNAs that are independent of Dicer, endogenous transposons, small nucleolar RNAs, endogenous small interfering RNAs, mirtrons produced by splicing, and long ncRNAs (Babiarz et al, 2008; Ender et al, 2008; Ruby et al, 2007; Seong et al, 2014). The function, processing, and regulation of many of these classes of ncRNAs in mammals remain to be fully elucidated.…”

Analysis of microRNAs in a knock-in hESC line expressing epitope-tagged AGO2

“…Previously, we validated canonical miRNAs by investigating physical interactions between components of the microprocessor and pre-miRNAs (Seong et al 2014). Interestingly, we found a novel substrate candidate of the Microprocessor (Figure 3(a)).…”

“…Data analysis was conducted as previously described (Seong et al 2014). High-throughput sequencing reads were processed by using functions in a Bioconductor package, ShortRead (Morgan et al 2009).…”

Let‐7 and miR‐125 cooperate to prime progenitors for astrogliogenesis

“…As the predominant role of Dgcr8 is in miRNA biogenesis (Preiss, 2009;Barad et al, 2012;Seong et al, 2014), we next sought to identify specific miRNAs that may underlie the differentiation defect of Dgcr8 D/D cells by simultaneously profiling and functionally screening miRNAs. In a number of lineages, miRNAs that promote differentiation increase upon induction of differentiation and promote downregulation of the progenitor transcriptional program (Yoo et al, 2009;Dugas et al, 2010;Goljanek-Whysall et al, 2011).…”