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.

项目摘要 mRNA表达的转录控制已被扩展到） 最近对他们的内部修改付款，mRNA“表演组”。 对mRNA的修改在多种分子证明弹簧中具有功能意义 拼接，核输出和稳定性，并在干细胞区分和人类中起重要的生物学作用 开发。 哺乳动物基因组中的元素。 具有功能实现 在他们的宿主mRNA表达上。 小鼠基因组的序列和人类基因组的13.64％。 被认为是“垃圾” DNA，但现在已被广泛接受基因组的一部分在 我们对RNA-硫酸盐测序（BS-Seq）的初步分析 细胞（ESC）确定5-甲基环肽（M5C）在mRNA转录本上富集区域显着 与宿主基因内含子或UTR的嵌入式正弦元素相关。 DNMT1/3A/3B和TET1/2/3的DNA甲基化和脱甲基的代谢，用于表观遗传法规，我们 关于转录后基因调节的RNA M5C的知识非常有限。 ESC的区分可以通过M5C介导的活性核导出和核保留/ 对多能元素的稳定，尊重的人，我们提出了两个目标，以测试这一点 AIM 1。 对于ESC自我更新中的核心。 检查活性核导出多能性mRNA（例如Nanog）是否由M5C读取器Allyref介导。 接下来，我们将测试ALYREF是否抑制M5C修饰的mRNA与PSPC1/Nono异二聚体的亲和力。 我们将使正弦非编码（NC）RNA介导的多能mRNA降解的机理在ESC中 分歧。我们 在视黄酸（RA）的分化中，PSPC1KO ESC的降解受损 正弦NCRNA，与宿主mRNA形成双链（DS）RNA，并募集Nono/PSPC1/ TET2复合物用于MRNA核保留和不稳定。 SEQ在ESC和RA处理中检查正弦NCRNA和宿主mRNA之间的相互作用。 我们将检查TET2是否是由PSPC1募集的mRNA脱甲基化和不可能的。 通过正弦介导的mRNA代谢和TEE建立新型大提琴命运范式测定的新型范式 mRNA M5C读取器，橡皮擦和效应子的功能在多能维持和分化过程中的功能。