Highly Dynamic and Sex-Specific Expression of microRNAs During Early ES Cell Differentiation

Abstract

Embryonic stem (ES) cells are pluripotent cells derived from the inner cell mass of the mammalian blastocyst. Cellular differentiation entails loss of pluripotency and gain of lineage-specific characteristics. However, the molecular controls that govern the differentiation process remain poorly understood. We have characterized small RNA expression profiles in differentiating ES cells as a model for early mammalian development. High-throughput 454 pyro-sequencing was performed on 19–30 nt RNAs isolated from undifferentiated male and female ES cells, as well as day 2 and 5 differentiating derivatives. A discrete subset of microRNAs (miRNAs) largely dominated the small RNA repertoire, and the dynamics of their accumulation could be readily used to discriminate pluripotency from early differentiation events. Unsupervised partitioning around meloids (PAM) analysis revealed that differentiating ES cell miRNAs can be divided into three expression clusters with highly contrasted accumulation patterns. PAM analysis afforded an unprecedented level of definition in the temporal fluctuations of individual members of several miRNA genomic clusters. Notably, this unravelled highly complex post-transcriptional regulations of the key pluripotency miR-290 locus, and helped identify miR-293 as a clear outlier within this cluster. Accordingly, the miR-293 seed sequence and its predicted cellular targets differed drastically from those of the other abundant cluster members, suggesting that previous conclusions drawn from whole miR-290 over-expression need to be reconsidered. Our analysis in ES cells also uncovered a striking male-specific enrichment of the miR-302 family, which share the same seed sequence with most miR-290 family members. Accordingly, a miR-302 representative was strongly enriched in embryonic germ cells derived from primordial germ cells of male but not female mouse embryos. Identifying the chromatin remodelling and E2F-dependent transcription repressors Ari4a and Arid4b as additional targets of miR-302 and miR-290 supports and possibly expands a model integrating possible overlapping functions of the two miRNA families in mouse cell totipotency during early development. This study demonstrates that small RNA sampling throughout early ES cell differentiation enables the definition of statistically significant expression patterns for most cellular miRNAs. We have further shown that the transience of some of these miRNA patterns provides highly discriminative markers of particular ES cell states during their differentiation, an approach that might be broadly applicable to the study of early mammalian development.

Cnidarian microRNAs frequently regulate targets by cleavage

“…Eighteen miRNAs are male-specific and just one (miR-9459) is female-specific (enrichment in one sex > 10, with Fisher's exact test P-value < 0.01). Sex-specific miRNAs have also been reported in mammals and flatworms and may exist in fish and birds (Wienholds et al 2005;Ciaudo et al 2009;Hao et al 2010;Luo et al 2012;Marco et al 2013).…”

The role of non-coding RNAs in male sex determination and differentiation

“…This view has been challenged by two reports demonstrating sexually dimorphic gene expression in PGCs long before initiation of meiosis in an ovary and mitotic arrest in a testis. The first indication came from a study examining miRNAs expressed during early ES cell differentiation (Ciaudo et al 2009). The authors showed that the miR-302 family of microRNAs was highly expressed in XY, but not XX PGCs at 8.5 and 9.5 dpc, before PGCs arrive at the developing genital ridges (Ciaudo et al 2009).…”

“…The first indication came from a study examining miRNAs expressed during early ES cell differentiation (Ciaudo et al 2009). The authors showed that the miR-302 family of microRNAs was highly expressed in XY, but not XX PGCs at 8.5 and 9.5 dpc, before PGCs arrive at the developing genital ridges (Ciaudo et al 2009). However, it is unclear what regulates this male-enriched expression and what function these microRNAs have at this stage during development.…”

Genome-wide impact of a recently expanded microRNA cluster in mouse

“…To dissect the functions of the Sfmbt2 cluster, we began by examining the represented seeds and their potential targets. Sequencing data revealed the miR-467 family to be the most abundantly expressed miRNAs from the cluster (23)(24)(25). Notably, this family shares the hexamer seed "AAGUGC" with the conserved miR-290-295 and miR-302 clusters, expressed predominantly in ES cells.…”