Epitranscriptomic control of mRNA and noncoding RNAs in spermatogenesis

精子发生中 mRNA 和非编码 RNA 的表观转录组控制

• 批准号: 10615702
• 负责人: SAMIE R JAFFREY
• 金额: $ 29.97万
• 依托单位: CORNELL UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-05-01 至 2026-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10615702
• 关键词: 3' Untranslated Regions; Address; Adenosine; Affect; Affinity; Animals; Binding; Binding Proteins; Cells; Characteristics; Chromatin; Cytidine; Cytidine Deaminase; Data; Deamination; Defect; Development; Distal; Enzymes; Epigenetic Process; Exhibits; Gene Expression Regulation; Genetic Transcription; Genomics; Germ Cells; Human; Impairment; Induction of Apoptosis; Infertility; Knock-out; Length; Maps; Mediating; Meiosis; Messenger RNA; Methods; Methyltransferase; Modification; Mus; Mutation; Nucleotides; Patients; Pattern; Pharmaceutical Preparations; Poly A; Polyadenylation; Positioning Attribute; Process; Proteins; RNA; RNA Binding; RNA Splicing; Reader; Regulation; Role; Sampling; Series; Site; Sperm Motility; Spermatids; Spermatocytes; Spermatogenesis; Testis; Time; Translational Regulation; Translations; Untranslated RNA; Zygonema; cell type; epigenetic regulation; epigenetic silencing; epigenome; epitranscriptome; epitranscriptomics; experimental study; genomic locus; insight; link protein; mRNA Stability; mRNA Transcript Degradation; mRNA Translation; mouse development; recruit; ribosome profiling; small molecule; sperm cell; sperm morphology; stem cell differentiation; stoichiometry; transcriptome

Spermatogenesis is a carefully orchestrated process in which spermatogonial stem cells differentiate into spermatids and eventually motile sperm. This process requires a series of changes in epigenetic changes, chromatin reorganization, dynamic changes in mRNA 3’UTR length, and temporally regulated patterns of translation. Emerging evidence suggests that precise stage-specific alterations in the epitranscriptome are also required for proper spermatogenesis. For example, spermatocyte-specific depletion of “readers,” “writers,” or “erasers,” of N6-methyladenosine, a modified nucleotide that impacts long noncoding RNA (lncRNA) function and mRNA stability, translation, and splicing, are all associated with stage-specific arrests in spermatogenesis. Additional data suggests that spermatogenesis is also affected by N6, 2’-O- dimethyladenosine (m6Am), a modified adenosine that is found exclusively at the first transcribed nucleotide position of certain mRNAs. Based on these studies, it is clear that epitranscriptomic modifications are required for spermatogenesis. However the mechanisms by which m6A and m6Am regulate spermatogenesis remain unclear. In order to decipher the role of the epitranscriptome in spermatogenesis, the specific aims of this project are: (1) To map m6A in a cell-type specific and quantitative manner during spermatogenesis. We will develop new methods to selectively map m6A in animals, and determine if dynamic changes in m6A control mRNA 3’UTR length. These methods will reveal the dynamics of m6A levels throughout spermatogenesis and if m6A function is involved in orchestrating 3’UTR length dynamics that is characteristic of spermatogenesis. (2) To determine the role of m6A in controlling the epigenome during spermatogenesis. m6A is often enriched in lncRNAs, and it can affect their ability to induce epigenetic gene silencing. We will identify chromatin- associated lncRNAs that contain m6A and determine how m6A affects epigenetic dynamics during spermatogenesis. (3) To determine how YTHDC2 and m6Am regulate spermatogenesis. YTHDC2 is required for meiotic progression by binding and regulating m6A mRNAs. However, YTHDC2 shows relatively weak binding to m6A. We find that YTHDC2 shows high-affinity binding to m6Am. We will map m6Am during spermatogenesis, and determine the function of m6Am by depleting its biosynthetic methyltransferase. We will also determine if the function of YTHDC2 is to regulate the stability or translation of m6Am mRNAs during spermatogenesis. Together, these experiments will reveal the cell-type specific dynamics of the epitranscriptome and will reveal how these epitranscriptomic modifications affect epigenetic states, mRNA stability, mRNA 3’UTR processing and mRNA translation during spermatogenesis.

精子发生是精心策划的过程，其中精子干细胞分化为 精子，有时是运动精子。这个过程需要一系列表观遗传变化的变化， 染色质重组，mRNA 3'UTR长度的动态变化以及暂时调节的模式 翻译。新兴的证据表明，表演组的精确阶段特异性变化是 适当的精子发生也需要。例如，“读者”的精子特异性耗竭 N6-甲基趋化胺的“作家”或“橡皮”，这是一种影响长的非编码RNA的修饰核苷酸 （lncRNA）功能和mRNA稳定性，翻译和拼接都与特定于阶段的逮捕有关 精子发生。其他数据表明，精子发生也受N6、2'-O-的影响 二甲基腺苷（M6AM），一种修饰的腺苷，仅在第一个转录的核苷酸上发现 某些mRNA的位置。基于这些研究，很明显，表面翻译的修饰是 精子发生所需。但是，M6A和M6AM调节精子发生的机制 保持不清楚。为了破译同源体在精子发生中的作用， 该项目是：（1）在精子发生过程中以细胞类型的特异性和定量方式映射M6A。我们 将开发新方法以选择性地映射动物中的M6a，并确定M6A控制中的动态变化是否 mRNA 3’UTR长度。这些方法将揭示整个精子发生和 如果M6A功能参与了精子发生特征的3'UTR长度动力学。 （2）确定M6A在控制精子发生过程中控制表观基因组中的作用。 M6A经常被丰富 LNCRNA，并且可能会影响他们诱导表观遗传基因沉默的能力。我们将确定染色质 - 包含M6A并确定M6A如何影响表观遗传动力学的相关lncRNA 精子发生。 （3）确定YTHDC2和M6AM如何调节精子发生。需要ythdc2 通过结合和控制M6A mRNA来进行减数分裂进程。但是，ythdc2显示相对较弱 与M6A结合。我们发现YTHDC2显示出高亲和力与M6AM的结合。我们将在 精子发生，并通过耗尽其生物合成甲基转移酶来确定M6AM的功能。我们将 还确定YTHDC2的功能是否是调节M6AM mRNA的稳定性或翻译 精子发生。总之，这些实验将揭示细胞类型的特定动力学 同意组，并将揭示这些表面参考的修饰如何影响表观遗传状态，mRNA 稳定性，mRNA 3'UTR加工和精子发生过程中的mRNA翻译。