Post-Transcriptional RNA Regulons in Stem Cells

 描述（由申请人提供）：诱导多能干细胞（iPSC）可能是治疗多种人类疾病的灵丹妙药。iPSC的产生及其分化为各种谱系都需要基因表达的大量协调变化。当将细胞从成体状态推入多能性时，需要多能性基因的大规模诱导，但也需要在分化的亲本细胞中表达的基因的大规模下调。类似地，更自然的分化过程需要下调多能性因子，同时增加组织特异性基因的表达。这些变化发生在转录水平，但也在转录后，特别是在mRNA衰变的水平。重要的是，转录后事件对实现和维持多能性的贡献的特征很差。我们建议研究三种可能有助于实现和/或维持多能性的转录后RNA调节子。首先，我们确定了大量的锌指蛋白（ZNF）mRNA在iPSC中是稳定的。由于这些ZNF转录因子在发育中起着至关重要的作用，并且可能在控制反转录转座中起着重要的作用，因此了解它们的调节是至关重要的。我们将研究一种潜在的机制，通过这种机制，miRNA靶向ZNF mRNA ORF中的重复结构域，以诱导衰变。我们还将研究为什么这些ZNF mRNA具有异常短的poly（A）尾，并揭示这如何有助于它们的表达。其次，我们发现mRNA的甲基化，特别是ZNF mRNA，与iPSC中稳定性的增加相关。我们将揭示这种修饰影响多能细胞中基因表达的机制。最后，我们已经表明，许多编码对胚胎发育重要的转录因子的mRNA含有富含C的元件，并且在iPSC中显着不稳定。我们将确定负责这种调节的因素，并破译其作用机制。总的来说，这些实验将为干细胞中的转录后控制提供急需的见解，这可能导致改进的重编程和更好地理解多能细胞与其完全分化的亲属之间的差异。

 DESCRIPTION (provided by applicant): Induced pluripotent stem cells (iPSCs) could be a panacea for a wide range of human diseases. Both the generation of iPSCs and their differentiation into various lineages require massive and coordinated changes in gene expression. When cells are pushed from the adult state into pluripotency a large scale induction of pluripotency genes is required, but also a large scale down-regulation of genes expressed in the differentiated parent cells. Similarly, the more natural process of differentiation requires down-regulation of pluripotency factors with concomitant increased expression of tissue-specific genes. These changes occur at the transcriptional level, but also post-transcriptionally, particularly at the level of mRNA decay. Importantly, the contributions of post-transcriptional events to the achievement and maintenance of pluripotency are poorly characterized. We propose to study three post- transcriptional RNA regulons that likely contribute to the achievement and/or maintenance of pluripotency. First, we determined that a large number of Zinc Finger Protein (ZNF) mRNAs are stabilized in iPSCs. As these ZNF transcription factors have vital roles in development, and perhaps in controlling retrotransposition, understanding their regulation is essential. We will examine a potential mechanism by which miRNAs target repeated domains in the ORF of ZNF mRNAs to induce decay. We will also investigate why these ZNF mRNAs have unusually short poly(A) tails and uncover how this contributes to their expression. Second, we found that methylation of mRNAs, and particularly ZNF mRNAs, is correlated with increased stability in iPSCs. We will uncover the mechanisms by which this modification influences gene expression in pluripotent cells. Finally, we have shown that many mRNAs encoding transcription factors important for embryonic development contain C-rich elements and are significantly destabilized in iPSCs. We will identify the factors responsible for this regulation and decipher their mechanism of action. Overall, these experiments will give much needed insights into post- transcriptional control in stem cells that may lead to improved reprogramming and a better understanding of the differences between pluripotent cells and their fully differentiated relatives.