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

SINE介导的mRNA表观转录组多能性维持和分化调节

• 批准号: 10659218
• 负责人: Xin Huang
• 金额: $ 20.56万
• 依托单位: COLUMBIA UNIVERSITY HEALTH SCIENCES
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-07-05 至 2024-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10659218
• 关键词: 3' Untranslated Regions; Affinity; Attention; Binding; Biological; Cell Nucleus; Cell physiology; Cells; Cellular Stress; Chemicals; Complex; DNA; DNA Methylation; DNA Sequence; DNA Transposable Elements; DNA metabolism; Data Set; Development; Disease; Double-Stranded RNA; Elements; Endogenous Retroviruses; Epigenetic Process; Genes; Genetic Transcription; Genome; Genomics; Human Development; Human Genome; Hydroxymethyltransferases; Immunoprecipitation; Impairment; Introns; Junk DNA; Knowledge; Maintenance; Malignant Neoplasms; Mass Spectrum Analysis; Mediating; Messenger RNA; Metabolism; Modeling; Modification; Molecular; Nuclear; Nuclear Export; Play; Pluripotent Stem Cells; Post-Transcriptional Regulation; Process; RNA; RNA Splicing; RNA analysis; RNA-Binding Proteins; Reader; Regulation; Repression; Research; Role; Structure; Testing; Tissues; Transcript; Transcriptional Regulation; Transfer RNA; Tretinoin; Untranslated RNA; Untranslated Regions; Virus Diseases; Work; bisulfite sequencing; cofactor; demethylation; embryonic stem cell; epigenetic regulation; epitranscriptome; mRNA Export; mRNA Expression; mRNA Precursor; mRNA Transcript Degradation; mammalian genome; mouse genome; novel; pluripotency; recruit; self-renewal; stem cell differentiation; stem cell fate; stem cell self renewal; stemness

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.

项目总结 对mrna表达的转录调控进行了广泛的研究；然而，关注较少。 对它们的内部修饰进行了研究，将其称为“表位转录组”。最近，越来越多的证据表明 在包括前信使核糖核酸在内的各种分子过程中，对信使核糖核酸的修饰具有重要的功能 剪接、核输出和稳定性，并在干细胞分化和人类 发展。转座元件(TES)，包括正弦、LINES和LTRS，是最丰富的DNA 哺乳动物基因组中的元素。与LINE和ERV等其他TES不同，SINE经常 嵌入在基因内部的非编码区，如内含子和非编码区，具有功能暗示 对它们的宿主mRNA表达的影响。SINE有100多万个基因组拷贝，占DNA的8.22% 小鼠基因组和13.64%的人类基因组的序列。包括正弦在内的TES在历史上是 被认为是“垃圾”的DNA，但现在人们普遍认为，基因组的这一部分在 不同的细胞过程。胚胎干细胞RNA-亚硫酸氢盐测序(BS-SEQ)的初步分析 细胞(ESCs)鉴定，mRNA转录本上的5-甲基胞嘧啶(M5C)富集区 与宿主基因内含子或UTRs上嵌入的正弦元件相关。尽管历史悠久 Dnmt1/3a/3b和Tet1/2/3对DNA甲基化和去甲基化的代谢作用 关于RNA M5C用于转录后基因调控的知识相当有限。我们假设自我更新 胚胎干细胞的分化可能受M5C介导的主动核输出和核滞留的控制。 含有正弦元件的多能性mRNAs分别失稳。我们提出了两个目标来测试这一点 假设。目的1.我们将通过嵌合正弦区和M5C来确定多能性mRNA的调节 用于核出口的ESC自我更新。我们确定Alyref对于ESC的自我更新是不可或缺的。我们会 检查多能性mRNAs(例如Nanog)的主动核输出是否由M5C阅读器Alyref介导。 接下来，我们将测试Alyref是否抑制M5C修饰的mRNAs对Pspc1/Nono异源二聚体的亲和力。目标2. 我们将剖析Sine Non-Coding(NC)RNA介导的ESC多能性mRNA降解的机制 差异化。我们的初步工作表明，M5C修饰的多能性基因转录本(例如，Nanog)具有 维甲酸(RA)诱导的Pspc1KO胚胎干细胞降解受损。RA治疗激活 Sine ncRNAs，它与宿主mRNAs形成反式双链(DS)RNAs，并招募Non/Pspc1/ TET2复合体对mRNA核滞留和失稳的影响。我们将通过eCLIP探索Pspc1 RNA靶标- 在ESCs和RA处理中的SEQ，以检查Sine ncRNAs和宿主mRNAs之间的相互作用。下一首, 我们将检查TET2是否被Pspc1招募用于mRNA去甲基化和失稳。总而言之，我们将 建立一种新的通过正弦介导的信使核糖核酸代谢决定多能细胞命运的范例 MRNAM5C读取器、擦除器和效应器在多能性维持和分化过程中的功能。