Regulatory RNA Key Player in p53-Mediated Apoptosis in Embryonic Stem Cells
Regulatory RNA Apela in DNA damage response of embryonic stem cells (ESCs)
Embryonic stem cells (ESCs) must maintain the integrity of their genomes or risk passing potentially deleterious mutations on to numerous tissues. Thus, ESCs have a unique genome surveillance system and easily undergo apoptosis or differentiation when DNA damage is detected. The protein p53 is known to promote differentiation in mouse ESCs (mESCs), but its role in DNA damage-induced apoptosis (DIA) is unclear. p53 may have a pro-apoptotic function since it can regulate apoptotic genes in embryonal cells. Given that ESCs have a distinct transcriptional program, Jing Huang, Ph.D., of CCR’s Laboratory of Cancer Biology and Genetics, and his colleagues wondered whether p53 might regulate DIA in ESCs by utilizing the ESC-specific expression program.
First, the researchers verified that DIA in ESCs depends on p53. They treated ESCs from p53 wild type and knockout mice with the DNA damaging agent, Adriamycin. One day later, a majority of the wild type mESCs had undergone apoptosis compared to very few of the knockout cells.
To identify p53 targets that might participate in DIA, the investigators used a genome-wide sequencing approach to compare gene expression from Adriamycin-treated p53 wild type and knockout mESCs. They reasoned that targets enriched in mESCs are more likely to play a role in the function of p53 in these cells. Likewise, genes repressed by p53 have a higher probability of acting in an ESC-specific manner. Of the 22 p53-repressed, ESC-enriched genes they identified, the scientists decided to focus on Apela, which was thought to be a long non-coding RNA; however, its zebrafish homolog was recently shown to encode a secretory peptide. They found that p53 regulated Apela expression by binding to the gene’s enhancer domain.
The researchers then knocked down Apela in mESCs. Loss of Apela did not alter mESC self-renewal but reduced DIA in a p53-dependent manner. They found that Apela knockdown had no consistent effect on the expression of p53-regulated apoptotic genes when mESCs were treated with Adriamycin. Instead, the fraction of p53 in the mitochondria decreased, suggesting Apela regulates p53-mediated apoptosis by altering its subcellular localization.
Intriguingly, the investigators showed that the receptor for the Apela-encoded peptide was not expressed in mESCs with or without exposure to DNA damage. Because they were unable to identify any other proteins that resemble the Apela receptor expressed by mESCs, the scientists wondered whether Apela acts as a regulatory RNA independent of its coding region. In support of this idea, they found that modified Apela sequences, including loss of the first start codon, loss of the first and second start codons, or deletion of the entire coding region, could rescue Apela knockdown to a similar extent as the unmodified sequence, demonstrating that the coding region is dispensable for Apela’s pro-apoptotic function.
Since regulatory RNAs act through associations with target proteins, the researchers performed RNA pull-down assays to identify Apela-interacting proteins. Apela failed to bind to known ESC master regulators or p53 but pulled-down two splice variants of heteronuclear riboneucleoprotein L (hnRNPL). The interaction between hnRNPL and Apela was direct, independent of p53, and required the first three RNA recognition motifs of hnRNPL and the 3’ untranslated region (UTR) of Apela. Mutating the Apela 3’ UTR disrupted the interaction with hnRNPL and failed to increase apoptosis from DNA damage in Apela knockdown mESCs. Thus, the binding of Apela and hnRNPL is required for Apela’s role in p53-mediated DIA.
To understand how hnRNPL is involved in p53-mediated DIA in mESCs, the investigators knocked down hnRNPL in ESCs from p53 wild type and knock-out mice. Loss of hnRNPL induced p53 activation in the absence and presence of DNA damage, prolonged the half-life of p53, and increased the mitochondrial localization of p53. hnRNPL knockdown also led to increased apoptosis in p53-expressing mESCs. These results suggest that hnRNPL prevents apoptosis in mESC by inhibiting p53 activation.
Since both Apela and hnRNPL were involved in p53-mediated DIA and bound to each other, the scientists needed to tease apart the hierarchy of these interactions. They knocked down both Apela and hnRNPL and found that the double knockdown had a similar outcome to hnRNPL knockdown alone, indicating that Apela lies upstream of hnRNPL. The researchers also observed a direct interaction between the first two RNA recognition motifs of hnRNPL and the tail of p53. In the presence of DNA damage, loss of Apela increased the interaction of p53 and hnRNPL, demonstrating that Apela negatively regulates this association.
Together these studies have identified a new negative feedback loop where, in response to DNA damage in mESCs, the RNA Apela binds to hnRNPL, allowing p53 to migrate to the mitochondria where it can be activated. The investigators have also revealed a new layer of complexity for RNA in which RNAs encoding proteins can also have non-coding functions.
Summary Posted: Wed, 07/01/2015
Li M, Gou H, Tripathi BK, Huang J, Jiang S, Dubois W, Waybright T, Lei M, Shi J, Zhou M, and Huang J. An Apela RNA-Containing Negative Feedback Loop Regulates p53-Mediated Apoptosis in Embryonic Stem Cells. Cell Stem Cell. June 4, 2015 PubMed Link