A noncanonical way out of pluripotency

Mouse embryonic stem cells (mESCs) deficient for DGCR8, a key component of the microprocessor complex, present strong differentiation defects. However, the exact reasons impairing their commitment remain elusive. An analysis of newly generated mutant mESCs conducted by the group of Constance Ciaudo (ETH Zurich) revealed that DGCR8 is essential for the exit from the pluripotency state. The group suggests a new noncanonical function of DGCR8 in addition to its established function in microRNA biogenesis.

As in real life, in the cell machinery too there are canonical and noncanonical ways of accomplishing things. The correct functioning of a machinery – and thus life in general – usually depends on the interplay of both, whereas from a scientist’s point of view the latter is probably more interesting. The group of Constance Ciaudo from the Institute for Molecular Health Sciences of the ETH Zurich has shown in a paper recently published in the Journal of Cell Biology how a noncanonical function of DGCR8 proteins controls the mouse embryonic stem cells (mESCs) exit from pluripotency.

But first to the orthodox side of things: The canonical miRNA pathway is crucial for stem cell biology, regulating features such as pluripotency factors and cell fate commitment; no surprise therefore that its misregulation contributes to human diseases. miRNAs are processed from primary transcripts in the nucleus by the microprocessor complex. This protein complex in the cell nucleus is composed of the ribonuclease enzyme Drosha and the RNA-binding protein DGCR8 and cleaves primary miRNA substrates to pre-miRNA. These are exported into the cytoplasm, where the enzyme DICER processes them into mature miRNAs, to be then incorporated into the RNA-induced silencing complex, leading to the destabilization or translational repression of their mRNA targets.

Ciaudo and her group were intrigued by recent scientific work about the identification of posttranslational modifications (phosphorylation) of the DGCR8 protein and especially by the discovery of noncanonical functions for DGCR8 in specific biological systems. In their paper, they now present a new role for the DGCR8 protein, independent of DRO- SHA, regulating the exit from pluripotency of mESCs. Interestingly they have found that the impaired differentiation of Dgcr8_ KO mESCs is independent of its function in miRNA biogenesis. The crucial newly found mechanism involves Tcf7l1 pre-mRNA, a core component of the pluripotency network. It turned out that proper phosphorylation of the DGCR8 protein is required for its binding to Tcf7l1.

To understand the molecular mechanisms causing the differentiation defects in Dgcr8 mutant mESCs the group generated new mutant cells deleted for the Dgcr8 gene using CRISPR/Cas9 genome engineering. Several molecular analyses indicated that these mutant mESCs complemented with impaired phosphorylated form of DGCR8 are not able to exit the pluripotency state, despite a restoration of miRNAs expression, cell proliferation, and proper cell cycle distribution. So the group came up with the hypothesis that the exit from pluripotency impairment might actually be independent of the role of DGCR8 in the miRNA biogenesis pathway and that the phosphorylation of DGCR8 could represent another mechanism ensuring tight control of the exit from pluripotency in mESCs.

Using RIP (RNA immunoprecipitation) experiments, they were able to demonstrate the direct interaction of DGCR8 protein with the Tcf7l1 mRNA in wildtype and Drosha_KO mESCs, indicating that this interaction is independent of the microprocessor complex. There are two isoforms of Tcf7l1, and DGCR8 facilitated the splicing of the long Tcf7l1, an event necessary for the differentiation of mESCs. To finally demonstrate the importance of the two Tcf7l1 isoforms in mESC commitment, they forced the expression of the short isoform in WT and Dgcr8_KO mESCs in an inducible manner or down-regulate the long isoform and thereby demonstrate that the up-regulation of the short Tc- f7l1 isoform and the down-regulation of the long Tcf7l1 isoform promote the exit from pluripotency and differentiation of mESCs. With all this collected experimental data, Ciaudo and her colleagues believe they can now explain the previously observed impaired differentiation process of Dgcr8_KO mESCs. More interestingly, the results reveal a new noncanonical function of DGCR8 essential for the exit from pluripotency of mESCs.

Cirera-Salinas D. et al. (2017) Journal of Cell Biology 216(2), 355-366

By Roland Fischer