Biogenesis of ribosomal RNAs in Dictyostelium discoideum

核糖体rna （rrna）编码于变形虫盘状盘状体中，大约有100个染色体外回文拷贝，被认为来自于单个染色体位点。为了对其进行分子分析，我们将生成该位点的遗传可追溯版本，并分析该标记随后是否也可以在染色体外元素中找到。我们将确定在发育过程中rDNA拷贝的内容是否恒定，或者在孵化时是否正在合成新的rDNA，或者在发育过程中的任何其他时间点。我们将进一步分析是否所有100染色体外元件都被积极转录，还是只有一个子集。这些元素的一个子集的转录沉默的一个可能的手段是DNA甲基化。我们通过深度测序观察到大部分内源性21mer rna与rDNA回文序列的转录区序列相同，间接支持了这一点。为了分析（rna介导的）DNA甲基化是否在起作用，我们将对来自不同遗传背景的DNA进行甲基化敏感限制性内切酶处理和亚硫酸酯测序，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，成熟rna的前体被积累起来。在详细介绍了成熟rRNA及其前体的序列之后，我们希望从分子角度分析在发育过程中是什么阻止了rRNA的加工，以及在什么时候停止。我们将确定存储的前体序列并确定其亚细胞定位。为此，我们开发了一种RNA荧光原位杂交（FISH）方案，作为本实验室已建立的广泛技术（RT-PCR, Northern Blot，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参与了rnas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进一步参与rRNA成熟的酶，并通过使用本实验室最近建立的一步克隆方案产生基因缺失菌株。如果这些菌株不能存活，我们将使用RNAi的敲除方法来确定相应酶对rRNA成熟的影响。

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.