Abstract
Small RNAs such as microRNAs play important roles in embryonic stem cell maintenance and differentiation. A broad range of microRNAs is expressed in embryonic stem cells while only a fraction of their targets have been identified. We have performed large-scale identification of embryonic stem cell microRNA targets using a murine embryonic stem cell line deficient in the expression of Dgcr8. These cells are heavily depleted for microRNAs, allowing us to reintroduce specific microRNA duplexes and identify refined target sets. We used deep sequencing of small RNAs, mRNA expression profiling and bioinformatics analysis of microRNA seed matches in 3′ UTRs to identify target transcripts. Consequently, we have identified a network of microRNAs that converge on the regulation of several important cellular pathways. Additionally, our experiments have revealed a novel candidate for Dgcr8-independent microRNA genesis and highlighted the challenges currently facing miRNA annotation.
Highlights
MicroRNAs are important mediators of posttranscriptional gene regulation
Two independently derived homozygous mutant cell lines were used in this study and are designated Dgcr8gt1/tm1 and Dgcr8gt2/tm1
Heterozygous cell lines were recovered, in the same electroporation, where the trapped allele was targeted in cis (Dgcr8tm1,gt1/+ and Dgcr8tm1,gt2/+; Figure S2)
Summary
MicroRNAs (miRNAs) are important mediators of posttranscriptional gene regulation They are RNA molecules ,22 nt in length, responsible for guiding the RNA induced silencing complex (RISC) to mRNA molecules, predominantly through complementarity between the 59 end of the miRNA (containing the seed region) and sequences within the 39 UTR of the target molecules. In the nucleus the precursor miRNA (pre-miRNA) hairpin is released by the RNase III enzyme DROSHA, operating in conjunction with the double stranded RNA binding protein, DiGeorge syndrome critical region gene 8 (DGCR8) [5,6,7,8,9] Both components of this microprocessor complex are required for canonical miRNA processing [6,7]. The pre-miRNA hairpin is exported to the cytoplasm where Dicer (DICER1) is responsible for releasing the miRNA from this hairpin [10,11,12]
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