Abstract
Mouse Embryonic Stem (ES) cells express a unique set of microRNAs (miRNAs), the miR-290-295 cluster. To elucidate the role of these miRNAs and how they integrate into the ES cell regulatory network requires identification of their direct regulatory targets. The difficulty, however, arises from the limited complementarity of metazoan miRNAs to their targets, with the interaction requiring as few as six nucleotides of the miRNA seed sequence. To identify miR-294 targets, we used Dicer1-null ES cells, which lack all endogenous mature miRNAs, and introduced just miR-294 into these ES cells. We then employed two approaches to discover miR-294 targets in mouse ES cells: transcriptome profiling using microarrays and a biochemical approach to isolate mRNA targets associated with the Argonaute2 (Ago2) protein of the RISC (RNA Induced Silencing Complex) effector, followed by RNA–sequencing. In the absence of Dicer1, the RISC complexes are largely devoid of mature miRNAs and should therefore contain only transfected miR-294 and its base-paired targets. Our data suggest that miR-294 may promote pluripotency by regulating a subset of c-Myc target genes and upregulating pluripotency-associated genes such as Lin28.
Highlights
Embryonic stem cells, which are derived from the inner cell mass of the blastocyst, hold great clinical promise because of their unique capacity to both self-renew and differentiate into potentially any cell type
It is important to determine the precise functions of key individual small RNAs in embryonic stem cells
We created embryonic stem cells lacking all miRNAs into which we introduced a single miRNA
Summary
Embryonic stem cells, which are derived from the inner cell mass of the blastocyst, hold great clinical promise because of their unique capacity to both self-renew and differentiate into potentially any cell type. Understanding the molecular controls of pluripotency is key to realising their therapeutic potential. The mature small RNA is incorporated into a protein of the Argonaute family [3,4]. This RNA-protein complex forms the core of the effector complex referred to as the RNA-induced silencing complex (RISC). Within the RISC, the small RNA acts a guide to direct Argonaute proteins to complementary target transcripts to elicit the cleavage, degradation or translational repression of their targets depending on their degree of complementarity [5]
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