Developmental Biology & Stem CellsRegulatory RNA

RNA Methylation: The Next Big Thing in Stem Cell Differentiation and RNA Stability

RNA methylation, stem cell differentiation, and RNA stabilityWhile DNA methylation is probably the longest studied and best understood epigenetic modification, methylation of RNA is gaining increased appreciation, especially recently.  N6-methyladenosine (m6A) in RNA has been observed to be the most frequently occurring epigenetic modification in mRNAs in eukaryotic organisms, but the function of this modification is still poorly understood.  A recent paper by Wang et al. reports that the m6A modification in RNA acts to destabilize transcripts and that RNA methylation is important for keeping embryonic stem cells in their undifferentiated and pluripotent state.  Furthermore, the authors hypothesize that RNA methylation is a critical activity in cell biology and might also play a central role in numerous RNA-mediated cellular processes. The researchers began their study by knocking down the levels of two suspected RNA methyltransferase enzymes (Mettl3 and Mettl14) using RNAi in mouse embryonic stem cells (mESCs) and then investigated the effects on the levels of m6A in RNA, as well as additional downstream phenotypes.  Knock down (KD) of either enzyme resulted in decreased levels of m6A, suggesting that both enzymes can methylate RNA in these mESCs and that both are required for wild type levels of RNA methylation.  Furthermore, the authors found that Mettl3 and Mettl14 proteins physically interacted with each other, and each protein seemed to stabilize the other, leading the researchers to propose that the enzymes function synergistically.  To investigate the role of Mettl3 and Mettl14 in RNA methylation in more detail, the researchers performed m6A RNA immunoprecipitation, followed by next-generation sequencing (meRIP-Seq) on mESC cells with either Mettl3 or Mettl14 levels knocked down.  The targets of Mettl3 and Mettl14 showed extremely high overlap, providing further evidence that these two enzymes work together.  The Mettl3 and Mettl14 targets were primarily mRNAs for genes involved in transcriptional regulation, RNA splicing, chromatin modification and remodeling, suggesting that the RNA methyltransferase enzymes are critical for these biological activities, and that they have specificity for certain types of substrates. In addition to the changes in m6A levels, the authors also observed that the Mettl3 and Mettl14 KD cells exhibited morphological changes relative to the control cells, and they also proliferated at a slower rate.  The authors concluded that the KD cells lost some stem cell-like properties, and this was supported by additional microarray and RT-qPCR gene expression studies showing that the expression of the majority of pluripotency genes was downregulated and developmental genes were upregulated.  Interestingly, the loss of N6-methyladenosine levels in the KD cells was more associated with upregulation of gene expression, suggesting that m6A normally acts to repress gene expression.  Further experiments indicated that m6A-modified transcripts were degraded at an increased rate relative to non-methylated transcripts, providing a mechanistic explanation for how m6A could repress gene expression.  This RNA destabilization phenomenon was investigated further, and the authors observed decreased binding of the RNA stabilization factor HuR when transcripts were methylated.  HuR is a well characterized protein that is generally thought to stabilize specific mRNAs by binding to sequence elements in their 3’ UTR, thus preventing miRNAs from binding to and degrading these transcripts.  Taken together, the authors’ data led them to generate a model that m6A methylation in specific transcripts reduces their stability by decreasing HuR binding, and thus epigenetically downregulating gene expression, possibly by exposing the transcripts to increased miRNA binding in the absence of HuR. It is becoming extremely clear that RNA has many more roles in biology than previously realized, so it isn’t surprising to learn that epigenetic modification of RNA is also important to regulate RNA-mediated cellular mechanisms.  This report characterizes a role for N6-methyladenosine in controlling RNA stability, and demonstrates that this modification is important for the process of stem cell differentiation.  The RNA modification field is rapidly expanding, and it will be exciting to see how this area of research develops in the future.   Wang Y, Li Y, Toth JI, Petroski MD, Zhang Z, & Zhao JC (2014). N6-methyladenosine modification destabilizes developmental regulators in embryonic stem cells. Nature cell biology, 16 (2), 191-8 PMID: 24394384 http://www.ncbi.nlm.nih.gov/pubmed/24394384

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Kevin B.

Kevin B.

Kevin grew up in Northern California and has also spent several years living on the East Coast. When he is not in the lab, Kevin enjoys snowboarding, watching NFL games (and playing fantasy football!), spending time outdoors, and exploring Southern California.