Regulatory non-coding RNAs in pluripotent stem cells

Abstract

The most part of our genome encodes for RNA transcripts are never translated into proteins. These include families of RNA molecules with a regulatory function, which can be arbitrarily subdivided in short (less than 200 nucleotides) and long non-coding RNAs (ncRNAs). MicroRNAs, which act post-transcriptionally to repress the function of target mRNAs, belong to the first group. Included in the second group are multi-exonic and polyadenylated long ncRNAs (lncRNAs), localized either in the nucleus, where they can associate with chromatin remodeling complexes to regulate transcription, or in the cytoplasm, acting as post-transcriptional regulators. Pluripotent stem cells, such as embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs), represent useful systems for modeling normal development and human diseases, as well as promising tools for regenerative medicine. To fully explore their potential, however, a deep understanding of the molecular basis of stemness is crucial. In recent years, increasing evidence of the importance of regulation by ncRNAs in pluripotent cells is accumulating. In this review, we will discuss recent findings pointing to multiple roles played by regulatory ncRNAs in ESC and iPSCs, where they act in concert with signaling pathways, transcriptional regulatory circuitries and epigenetic factors to modulate the balance between pluripotency and differentiation.

Figure 1

Clusters of miRNAs specifically expressed in embryonic stem cells (ESCs). (a) Schematic representations of the two ESC-specific miRNA clusters in mouse and human. miRNAs with the AAGUGC seed sequence within each cluster are underlined. The miR-302/367 cluster is highly conserved in mammals, whereas the other cluster is less conserved; (b) The two clusters are differentially expressed in ESCs during the conversion between the naïve and primed states of pluripotency.

Figure 2

Role of miR-302 during human ESCs (hESC) neural differentiation. Transcription from the miR-302 locus is activated by the ESC core transcriptional regulatory circuitry. miR-302 post-transcriptionally inhibits NR2F2 which in turn is an inhibitor of Oct4. In a negative feedback loop, Oct4 inhibits NR2F2 transcription. NR2F2 expression is necessary for proper activation of neuroectoderm genes. miR-302 also targets inhibitors of both branches of the TGFβ pathway: the Nodal inhibitors, Lefty1 and Lefty 2, and the bone morphogenetic protein (BMP) inhibitors, DAZAP2, SLAIN1 and TOB2. According to the neural default model, inhibition of both branches of the TGFβ pathway leads to neural induction. Thus, by targeting inhibitors of both branches miR-302 has a negative effect on neural induction.

Figure 3

miRNAs target pluripotency genes. The activity of pluripotency genes, including core transcription factors, must be shut down as embryonic stem cells differentiate. Several miRNAs that are induced upon exit from pluripotency directly target these genes in a combinatorial way. Examples are shown in the table. M: mouse; H: human.

Figure 4

let-7 antagonizes pluripotency networks. Let-7 antagonizes indirectly the activity of the ESC core transcriptional regulatory circuitry by targeting multiple genes induced by the core transcription factors (TFs). Moreover, let-7 directly inhibits c-Myc, reducing transcription of its target genes.

Figure 5

Opposing roles for the let-7 and miR-302 families. During ESC differentiation, the levels of pluripotency miRNAs belonging to the miR-302 family are decreased. In parallel, let-7 family members are induced. The miR-302/367 and miR-290-295 clusters are induced by core ESC TFs, and in a positive feedback loop indirectly promote their expression and transcription of their targets. Conversely, let-7 is post-transcriptionally inhibited by Lin-28. In cancer cells c-Myc inhibits let-7 at the transcriptional level. In a negative feedback loop, let-7 targets its inhibitors. Black arrows indicate direct activation; grey arrows indicate indirect activation; dashed lines indicate post-transcriptional inhibition.

Figure 6

Role of miRNAs during reprogramming. The reprogramming process can be divided in three phases. Early events include inhibition of apoptosis and an increase in proliferation. A mesenchymal-to-epithelial transition (MET) occurs in the intermediate phase. Activation of the endogenous pluripotency program occurs as a late event. Several miRNAs have been shown to promote (blue) or inhibit (orange) reprogramming by facilitating or hampering the completion of these events. The miR-93 family includes miR-93 and miR-106b.

Figure 7

Role of lincRNA-RoR during reprogramming. LincRNA-RoR may promote reprogramming by two different mechanisms. It inhibits p53, which in turn negatively affects the initial steps of reprogramming by inducing apoptosis. It also acts as a competing endogenous RNA and releases from repression the endogenous pluripotency genes Oct4, Sox2 and Klf4, which are targeted by miR-145. MiR-145 is also inhibited by Oct4 in a negative feedback loop. Other miRNA may be “sponged” by lincRNA-RoR (not shown).