Meiotic Silencing by Unpaired DNA

不配对 DNA 的减数分裂沉默

• 批准号: 0533093
• 负责人: Stan Metzenberg
• 金额: --
• 依托单位: The University Corporation, Northridge
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-03-01 至 2009-09-30
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=0533093&HistoricalAwards=false
• 关键词: Meiotic; Silencing; Unpaired; DNA

Viruses, retrotransposons, and insertion sequences put the genomic integrity of all organisms at risk. Fungi are arguably under even greater jeopardy than plants and animals because they are organized coenocytically: a macroscopic "individual" may contain millions of nuclei sharing a continuous cytoplasm that is undivided by septa or cell membranes. Unsurprisingly, fungi have at least three sophisticated mechanisms for fighting back: quelling, RIP, and meiotic silencing by unpaired DNA (MSUD). The research that is the subject of this project is aimed at understanding the mechanism of MSUD and its role in the overall biology of one experimental organism, Neurospora crassa. Earlier work showed that during the very brief diploid phase of this organism, presumably when homologs are paired in the pachytene stage of meiosis, any sizeable stretch of unpaired DNA is "seen" by the organism as a foreign sequence, one that was not present in both parents at the same locus, and hence having the capacity for mischief. A signal in the form of double-stranded RNA is generated. Ultimately, this silences the expression not only of the unpaired DNA, but also any other DNA of the same sequence, whether paired or unpaired. Selecting and screening for suppressor mutations which fail in this silencing process has already been rewarding in terms of insight gained; one called Sad-1 (suppressor of ascus dominance) encodes a sexual-phase specific RNA-directed RNA polymerase, and other Sad mutants will be sought and studied. A major focus of the research will be on the role of two paralogous genes called dicer-1 and dicer-2 thought to be involved in quelling. In many organisms, and in the vegetative phase of fungal growth, these aptly-names genes are thought to dice up long double-stranded RNA molecules into short, double-stranded pieces which, in turn, give rise to single-stranded molecules. These then direct the destruction of mRNA homologous to them, resulting in "silencing". It is not clear that they have a similar role in MSUD, and a sizeable effort will be expended in trying to illuminate this issue. Finally, it has become clear that functional Sad genes are necessary for the completion of meiosis. This suggests that some of the machinery of silencing, presumably involving double-stranded RNA and its cleavage, is shared with the machinery of meiosis. A search for the key molecules is an additional aim of this research. All organisms are under continuous challenge by "selfish" DNA sequences or molecular parasites that are able to reproduce themselves at the expense of the host organism. Often the host has sophisticated machinery for fighting back, either by attacking and mutating the parasitic DNA to a harmless condition, or by preventing the parasitic DNA from making molecules necessary for its multiplication. An understanding of how these silencing mechanisms work would make it possible to prevent not only the reproduction of viruses, but also to prevent other cellular genes from making undesired products, or from making benign products in excessive amounts.

病毒、反转录转座子和插入序列使所有生物的基因组完整性处于危险之中。真菌可以说比植物和动物面临更大的危险，因为它们是按共核细胞组织的：一个宏观的“个体”可能包含数百万个细胞核，共享一个连续的细胞质，该细胞质没有被隔膜或细胞膜分割。毫不奇怪，真菌至少有三种复杂的反击机制：平息、RIP和未配对DNA(MSUD)对减数分裂的沉默。这项研究是这个项目的主题，目的是了解MSUD的机制及其在一种实验有机体--粗糙脉孢菌的整体生物学中的作用。早期的工作表明，在这种生物体非常短暂的二倍体阶段，大概当同源物在减数分裂的粗线期配对时，任何相当大的未配对的DNA片段都会被有机体视为外来序列，即不存在于双亲中同一基因座的外来序列，因此具有恶作剧的能力。产生双链RNA形式的信号。最终，这不仅会沉默未配对的DNA的表达，还会抑制同一序列的任何其他DNA的表达，无论是配对的还是未配对的。选择和筛选在这一沉默过程中失败的抑制突变已经在洞察力方面获得了回报；一种名为SAD-1(ASCUS显性抑制因子)的突变编码了有性时期特定的RNA指导的RNA聚合酶，其他SAD突变将被寻找和研究。这项研究的一个主要焦点将是两个类似的基因DICER-1和DICER-2的作用，这两个基因被认为参与了抑制。在许多生物体中，在真菌生长的营养阶段，这些恰当的名字被认为是将长的双链RNA分子切成短的双链片段，进而产生单链分子。然后，这些基因直接破坏与它们同源的信使核糖核酸，导致“沉默”。目前还不清楚它们在MSUD中是否有类似的作用，将花费相当大的努力来试图阐明这个问题。最后，功能上的SAD基因是完成减数分裂所必需的，这一点已经很清楚了。这表明，一些沉默的机制，可能涉及双链RNA及其切割，与减数分裂机制是相同的。寻找关键分子是这项研究的另一个目的。所有生物体都不断受到“自私的”DNA序列或分子寄生虫的挑战，这些DNA序列或分子寄生虫能够以牺牲宿主生物体为代价进行繁殖。通常情况下，宿主拥有复杂的反击机制，要么通过攻击和突变寄生的DNA使其处于无害的状态，要么通过阻止寄生的DNA制造繁殖所需的分子。了解这些沉默机制是如何工作的，不仅可以防止病毒的繁殖，还可以防止其他细胞基因产生不需要的产物，或过量产生良性产物。