The biogenesis and functions of pre-meiotic small RNAs in male reproductive development in maize

减数分裂前小RNA在玉米雄性生殖发育中的生物发生和功能

• 批准号: 10718996
• 负责人: Kirk Czymmek
• 金额: $ 63.56万
• 依托单位: DONALD DANFORTH PLANT SCIENCE CENTER
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-09-15 至 2027-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10718996
• 关键词: Angiosperms; Animals; Biochemical; Biogenesis; Biological Models; Biology; CRISPR/Cas technology; Cell Differentiation process; Cells; Chromosomes; DNA Methylation; DNA Polymerase II; DNA Transposable Elements; Data; Defect; Dependence; Development; Developmental Process; Distal; Drosophila genus; Drug or chemical Tissue Distribution; Environment; Epidermis; Evolution; Exhibits; Failure; Family; Fertility; Genetic; Genome; Genome Stability; Genomics; Goals; Grain; Image; Individual; Maize; Male Sterility; Male Sterilizations; Mammals; Maps; Mediating; Meiosis; Messenger RNA; MicroRNAs; Minor; Modeling; Molecular; Movement; Mus; Mutation; Nature; Nematoda; Organ; Oryza sativa; Outcomes Research; Paper; Pathway interactions; Phase; Phenotype; Plant Model; Plants; Poaceae; Pollen; Production; Proteins; RNA; RNA Precursors; Reporting; Reproduction; Reproductive Biology; Research; Rice; Role; Signal Transduction; Small Interfering RNA; Small RNA; Somatic Cell; Sterility; Techniques; Testis; Transcript; Untranslated RNA; Work; Zea mays; chromatin modification; experimental study; genome-wide; gonad development; insight; male; male fertility; mutant; novel; piRNA; pluripotency; reproductive; reproductive development; reproductive organ; single-cell RNA sequencing; spatiotemporal; sperm cell; stem cells; transcription factor

PROJECT SUMMARY In numerous flowering plant species, the male reproductive organs (anthers), which nurture pollen grains (containing the sperm, the male germline of plants) and their progenitor cells, accumulate two classes of small RNAs, i.e., the pre-meiotic (21-nt) and meiotic (24-nt) phased, small interfering RNAs (phasiRNAs). Perturbation of the phasiRNA biogenesis pathways in rice or maize causes environmentally-sensitive male sterility. There are many interesting parallels between these plant phasiRNAs and animal PIWI-interacting RNAs (piRNAs), which have also been shown essential for male fertility. However, there is a major gap in our understanding of how the plant reproductive phasiRNAs function to regulate developmental processes important for reproduction. Our long-term goal is to understand mechanistic and functional basis for the dependence on these small RNAs in plant male reproductive development. We and collaborators have described the reproductive phasiRNA pathways in many plant species. We have shown that biogenesis of the 21-nt, pre-meiotic phasiRNAs is initiated in the anther epidermis, but they accumulate in the tapetum, three layers of cells distal, indicating movement across cell layers. Yet, it is largely unknown where the 21-nt phasiRNAs and their biogenesis components localize throughout anther development, as well as what are the targets and thus precise functions of the 21-nt phasiRNAs. Our strong preliminary data has led to our focus on three Specific Aims to characterize 21-nt, pre-meiotic reproductive phasiRNAs in maize, a model for plant reproductive biology, genomics, and genetics. The aims are: (1) Characterize the spatiotemporal localization of key players in pre-meiotic phasiRNA biogenesis, using advanced imaging and single-cell RNA-seq techniques; this aim will generate a spatiotemporal map of 21-nt phasiRNAs, their precursors, microRNA trigger, and biogenesis proteins, in developing maize anthers. (2) Decipher the biogenesis of pre-meiotic phasiRNAs and their developmental roles, via the identification of novel biogenesis factors using biochemical approaches, using publicly available mutants plus CRISPR-Cas9- generated mutants to understand whether the pre-meiotic phasiRNAs are necessary for male reproductive development, and then assign functions to novel components of the pathway. And (3) elucidate the regulatory mechanisms mediated by pre-meiotic phasiRNAs, using established and new molecular approaches to identify the endogenous targets of 21-nt phasiRNAs and examine how they are regulated by the phasiRNAs. The proposed research will have a broad impact on small RNA biology by characterizing the biogenesis and functions of pre-meiotic plant small RNAs in maize anther development. The experiments will provide key insights into where and how they are produced, and what their roles are in anther cell differentiation and male fertility. Comparison of the results of this work with ongoing studies into mammalian piRNAs may shed light on the commonalities in the utilization of small RNA pathways in reproduction and gonad development.

项目摘要 在许多开花植物物种中，雄性生殖器官（花药）， （包含精子，植物的雄性生殖细胞）和它们的祖细胞，积累了两类小的 RNA，即，减数分裂前（21-nt）和减数分裂（24-nt）定相的小干扰RNA（phasiRNA）。 水稻或玉米中phasiRNA生物合成途径的干扰导致环境敏感的雄性 不育这些植物phasiRNA和动物PIWI相互作用之间有许多有趣的相似之处， RNA（piRNA），也被证明对男性生育力至关重要。然而，我们在这方面有一个很大的差距。 了解植物生殖phasiRNAs如何调控发育过程 对繁殖很重要。我们的长期目标是了解 在植物雄性生殖发育中依赖于这些小RNA。 我们和合作者已经描述了许多植物物种中的生殖phasiRNA途径。我们 已经表明，21-nt、减数分裂前phasiRNA的生物发生起始于花药表皮，但是它们 在绒毡层中积累，三层细胞远端，表明跨细胞层的运动。然而，它在很大程度上 未知21-nt phasiRNA及其生物发生组分在整个花药中的位置 发展，以及什么是目标，从而21-nt phasiRNA的精确功能。我们强大 初步的数据使我们集中在三个特定的目的，以表征21-nt，减数分裂前的生殖 玉米中的phasiRNAs，植物生殖生物学、基因组学和遗传学的模型。目标是：（1） 使用以下方法表征减数分裂前phasiRNA生物发生中关键参与者的时空定位： 先进的成像和单细胞RNA-seq技术;这一目标将产生21-nt的时空图 phasiRNA，它们的前体，microRNA触发器和生物发生蛋白，在发育中的玉米花药。（二更） 通过鉴定新的转录因子， 使用生物化学方法的生物发生因子，使用公开可用的突变体加上CRISPR-Cas9- 产生了突变体，以了解减数分裂前的phasiRNA是否是男性生殖所必需的。 发展，然后将功能分配给途径的新组分。（3）阐明监管 通过减数分裂前phasiRNA介导的机制，使用已建立的和新的分子方法来鉴定 21-nt phasiRNA的内源性靶标，并检查它们如何被phasiRNA调节。 这项拟议中的研究将通过表征生物起源对小RNA生物学产生广泛的影响。 以及减数分裂前植物小RNA在玉米花药发育中的作用。这些实验将提供 深入了解它们在哪里以及如何产生，以及它们在花药细胞分化和雄性发育中的作用 生育将这项工作的结果与正在进行的哺乳动物piRNA研究进行比较，可能会揭示 在生殖和性腺发育中利用小RNA途径的共性。