SINE-mediated Regulation of mRNA Epitranscriptome for Pluripotency Maintenance and Differentiation

项目摘要 mRNA表达的转录调控已被广泛研究，然而， 它们的内部修饰，即mRNA的“表转录组”。最近，越来越多的证据表明， mRNA上的修饰在多种分子过程中具有重要的功能， 剪接，核输出和稳定性，并发挥重要的生物学作用，在干细胞分化和人类 发展转座因子（Transposable elements，TE）包括西内斯、LINES和LTR，是最丰富的DNA 哺乳动物基因组中的元素。与其他TE（如LINE和ERV）不同，西内斯通常 嵌入基因内部的非编码区，如内含子和UTR，具有功能意义 对宿主mRNA表达的影响西内斯基因组拷贝数超过100万，占DNA总拷贝数的8.22 小鼠基因组序列和13.64%的人类基因组序列。TE，包括西内斯，在历史上是 被认为是“垃圾”DNA，但现在人们普遍认为，基因组的这一部分在基因组中起着重要作用， 不同的细胞过程。我们对胚胎干细胞RNA-亚硫酸氢盐测序（BS-seq）的初步分析 细胞（ESCs）发现，mRNA转录本上的5-甲基胞嘧啶（m5 C）富集区显著增加， 与宿主基因内含子或UTR处嵌入的SINE元件相关。尽管有公认的 通过Dnmt 1/3a/3b和Tet 1/2/3代谢DNA甲基化和去甲基化进行表观遗传调节，我们 关于RNA m5 C转录后基因调控的知识相当有限。我们假设自我更新 ESCs的分化可能受m5 C介导的主动核输出和核保留的控制。 分别对携带SINE元件的多能性mRNA的去稳定化。我们提出两个目标来检验这一点 假说.目标1。我们将用嵌入的SINE区域和m5 C来确定多能性mRNA的调节。 在ESC自我更新中进行核出口。我们确定Alyref对于ESC的自我更新是不可或缺的。我们将 检查多能性mRNA（例如Nanog）的活性核输出是否由m5 C读数器Alyref介导。 接下来，我们将测试Alyref是否抑制m5 C修饰的mRNA对Pspc 1/Nono异源二聚体的亲和力。目标二。 我们将详细分析SINE非编码RNA介导的ESC多能性mRNA降解的机制， 分化我们的初步工作表明，m5 C修饰的多能性基因转录本（例如，Nanog）有 在视黄酸（RA）诱导的分化后，Pspc 1 KO ESC的降解受损。RA治疗激活 SINE ncRNA与宿主mRNA反式形成双链（ds）RNA，并募集Nono/Pspc 1/ Tet 2复合物用于mRNA核保留和去稳定化。我们将通过eCLIP探索Pspc 1 RNA靶点- seq在ESC中和用RA处理以检查SINE ncRNA和宿主mRNA之间的相互作用。下一个是 我们将检查Tet 2是否被Pspc 1募集用于mRNA去甲基化和去稳定化。总之，我们将 建立通过SINE介导的mRNA代谢和多能细胞命运决定的新范式 mRNA m5 C阅读器、擦除器和效应器在多能性维持和分化过程中的功能。

PROJECT SUMMARY The transcriptional control of mRNA expression has been extensively investigated; however, less attention has been paid to their internal modifications, the mRNA “epitranscriptome”. Recently, evidence is accumulating that modifications on mRNAs are functionally significant in a variety of molecular processes including pre-mRNA splicing, nuclear export, and stability, and play important biological roles in stem cells differentiation and human development. Transposable elements (TEs), including SINEs, LINEs, and LTRs are the most abundant DNA elements in the mammalian genomes. Unlike the other TEs such as LINEs and ERVs, SINEs are frequently embedded in the non-coding regions inside a gene, such as the introns and UTRs, with functional implications on their host mRNA expression. SINEs have more than a million genomic copies and occupy 8.22% of DNA sequences of the mouse genome and 13.64% of the human genome. TEs, including SINEs, were historically considered as “junk” DNA but now it is widely accepted that this portion of the genome plays a significant role in diverse cellular processes. Our preliminary analysis of RNA-bisulfite sequencing (BS-seq) in embryonic stem cells (ESCs) identified that the 5-methylcytosine (m5C)-enriched regions on mRNA transcripts are significantly associated with the embedded SINE elements at introns or UTRs of the host genes. Despite the well-established metabolism of DNA methylation and demethylation by Dnmt1/3a/3b and Tet1/2/3 for epigenetic regulations, our knowledge on RNA m5C for posttranscriptional gene regulation is quite limited. We hypothesize that self-renewal and differentiation of ESCs may be controlled by m5C-mediated active nuclear export and nuclear retention/ destabilization, respectively, of pluripotency mRNAs bearing SINE elements. We propose two aims to test this hypothesis. Aim 1. We will determine the regulation of pluripotency mRNA with embedded SINE region and m5C for nuclear export in ESC self-renewal. We determined that Alyref is indispensable for ESC self-renewal. We will examine if active nuclear export of pluripotency mRNAs (e.g. Nanog) are mediated by the m5C reader Alyref. Next, we will test if Alyref represses the affinity of m5C-modified mRNAs to the Pspc1/Nono heterodimer. Aim 2. We will dissect the mechanism of SINE non-coding (nc) RNA-mediated pluripotency mRNA degradation in ESC differentiation. Our preliminary work revealed that m5C-modified pluripotency gene transcripts (e.g., Nanog) have impaired degradation in Pspc1KO ESCs upon retinoic acid (RA)-induced differentiation. RA treatment activates SINE ncRNAs, which form double-stranded (ds) RNAs in trans with the host mRNAs and recruit the Nono/Pspc1/ Tet2 complex for mRNA nuclear retention and destabilization. We will explore the Pspc1 RNA targets by eCLIP- seq in ESCs and with RA treatment to examine the interactions between SINE ncRNAs and host mRNAs. Next, we will examine if Tet2 is recruited by Pspc1 for mRNA demethylation and destabilization. In summary, we will establish a novel paradigm of pluripotent cell fate determination via SINE-mediated mRNA metabolism and the functions of mRNA m5C readers, erasers, and effectors in pluripotency maintenance and during differentiation.