Biogenesis of ribosomal RNAs in Dictyostelium discoideum

盘基网柄菌核糖体 RNA 的生物发生

• 批准号: 259262141
• 负责人: Professor Dr. Christian Hammann
• 金额: --
• 依托单位: HMU Health and Medical University Potsdam
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2014
• 资助国家: 德国
• 起止时间: 2013-12-31 至 2019-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/259262141?language=en
• 关键词: Biogenesis; ribosomal; RNAs; Dictyostelium; discoideum

Ribosomal RNAs (rRNAs) are encoded in the amoeba Dictyostelium discoideum in approximately 100 copies of an extrachromosomal palindrome, which are thought to be derived from a single chromosomal locus. To analyse this molecularly, we will generate a genetically traceable version of this locus and analyse whether or not the marker can later also be found in the extrachromosomal elements. We will determine whether the content of rDNA copies is constant during development or whether new rDNA is being synthesized upon hatching, or during any other time point in development. We further will analyse whether all 100 extrachromosomal elements are actively transcribed, or only a subset. A likely means of transcriptional silencing of a sub-set of these elements is DNA methylation. This is indirectly supported by our observation of a large fraction of endogenous 21mer RNAs with sequence identity to the transcribed region of the rDNA palindrome by deep sequencing. To analyse whether (RNA-mediated) DNA methylation is operating, we will subject DNA from different genetic backgrounds to treatment with methylation sensitive restriction enzymes and bisulfite sequencing to map the DNA methylation pattern on the extrachromosomal elements. In the spores of the amoeba, precursors of the mature rRNAs are accumulated. Having detailed earlier the sequences of mature rRNAs and their precursors, we wish to analyse here molecularly, what stops rRNA processing during development, and at what point. We will determine the sequences of the stored precursors and determine their sub-cellular localisation. To this end, we have developed a protocol for RNA fluorescence in situ hybridization (FISH) in D. discoideum, as a complementary tool to a wide range of already established techniques in this lab (cRT-PCR, Northern Blot, primer extension, RT-PCR etc.). Unexpectedly, we could observed that the Dicer-protein DrnB is involved in the maturation of rRNAs and we will detail this phenomenon in this project. We will use a bioinformatics to identify further enzymes that are involved in the rRNA maturation and generate gene deletion strains by using a one-step cloning protocol recently established in this lab. Should these strains not viable, we will use a knock-down approach by RNAi to determine the effects of the respective enzyme on rRNA maturation.

核糖体RNA（rRNA）在盘状网柄阿米巴中以大约100个拷贝的染色体外回文序列编码，其被认为来自单个染色体位点。为了从分子上分析这一点，我们将生成该基因座的遗传可追溯版本，并分析该标记随后是否也可以在染色体外元件中发现。我们将确定rDNA拷贝的含量在发育过程中是否是恒定的，或者新的rDNA是否在孵化时或发育过程中的任何其他时间点合成。我们将进一步分析是否所有100个染色体外的元素都被积极转录，或只是一个子集。这些元件的子集的转录沉默的可能手段是DNA甲基化。我们通过深度测序观察到大部分内源性21聚体RNA与rDNA回文序列的转录区具有序列同一性，这间接支持了这一点。为了分析（RNA介导的）DNA甲基化是否起作用，我们将来自不同遗传背景的DNA用甲基化敏感的限制性内切酶和亚硫酸氢盐测序进行处理，以绘制染色体外元件上的DNA甲基化模式。在变形虫的孢子中，积累了成熟rRNA的前体。在前面详细介绍了成熟rRNA及其前体的序列后，我们希望在这里从分子角度分析发育过程中是什么以及在什么时候停止rRNA加工。我们将确定存储的前体的序列，并确定它们的亚细胞定位。为此，我们开发了一种RNA荧光原位杂交（FISH）技术。discoideum，作为该实验室中广泛的已建立的技术（cRT-PCR、北方印迹、引物延伸、RT-PCR等）的补充工具。出乎意料的是，我们可以观察到Dicer蛋白DrnB参与rRNA的成熟，我们将在本项目中详细介绍这一现象。我们将使用生物信息学来鉴定参与rRNA成熟的其他酶，并通过使用本实验室最近建立的一步克隆方案来产生基因缺失菌株。如果这些菌株不能存活，我们将使用RNAi敲低方法来确定相应酶对rRNA成熟的影响。