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Exploring the regulation of trypanosome RNA granules with a newly established RNA granule purification protocol.

利用新建立的 RNA 颗粒纯化方案探索锥虫 RNA 颗粒的调控。

基本信息

批准号：
208025595
负责人：
Professorin Dr. Susanne Kramer
金额：
--
依托单位：
Lehrstuhl für Zell- und Entwicklungsbiologie (Zoologie I)
依托单位国家：
德国
项目类别：
Research Grants
财政年份：
2011
资助国家：
德国
起止时间：
2010-12-31 至 2018-12-31
项目状态：
已结题

来源：
https://gepris.dfg.de/gepris/projekt/208025595?language=en
关键词：
Exploring regulation trypanosome RNA granules

项目摘要

Cytoplasmic RNA granules, for example P-bodies and stress granules, are aggregates of RNA and protein that regulate eukaryotic gene expression on the posttranscriptional level. The function and regulation of RNA granules still remains highly elusive: one reason is that RNA granule purification has not yet been achieved in any organism. In the last 2.5 years we have established the first protocol for the biochemical purification of starvation stress granules. The protocol takes advantage of a unique feature of African trypanosomes: we have employed the cage-like subpellicular microtubules skeleton of the parasite as a molecular sieve. We have identified 464 stress granule candidate proteins by mass spectrometry. These include most of the known granule proteins and many expected, but three quarter of the proteins are unrelated to mRNA metabolism, many have putative regulatory functions. Localization to starvation stress granules was confirmed by eYFP tagging for 15 of 32 candidate proteins. The mRNA content of the granules was determined by RNA sequencing. We have identified a very distinct group of mRNAs that is absent from granules: almost all encode for ribosomal proteins. The absence of one such mRNA from RNA granules was confirmed by single mRNA FISH. We are also interested in the purification of a second type of trypanosome RNA granule: these form around the periphery of the nucleus when mRNA maturation is inhibited (nuclear periphery granules). They may have a function in mRNA quality control and show striking similarities to perinuclear germ granules of animals. We have successfully purified nuclei with the granules still attached and currently await mass spectrometry results.In the second funding period, I seek to understand RNA granule regulation. My granule proteome contains many protein-modifying enzymes, such as kinases, phosphatases and methyltransferases. These may regulate granule composition by changing the competence of their target proteins for granule localization, including any mRNAs attached. I want to unravel the underlying signalling pathways. Moreover, I want to understand the mechanisms involved in the exclusion of specific mRNAs from granules. We will also continue our efforts to purify and analyse nuclear periphery granules and aim to purify granules from the pathologically important transmissible short stumpy life cycle stage. The comparison of the granule proteomes and mRNA contents from the different types of granules will identify proteins specific to either type of granule. We hope that our data will contribute to a more comprehensive understanding of RNA granule composition and regulation, in trypanosomes and other eukaryotes.

细胞质RNA颗粒，如P小体和应激颗粒，是RNA和蛋白质的聚集体，在转录后水平上调节真核基因的表达。RNA颗粒的功能和调控仍然很难捉摸：一个原因是目前还没有任何生物体实现RNA颗粒的纯化。在过去的2.5年里，我们已经建立了第一个用于饥饿应激颗粒的生化纯化的方案。该方案利用了非洲锥虫的一个独特特征：我们使用了这种寄生虫的笼状膜下微管骨架作为分子筛子。我们用质谱仪鉴定了464个应激颗粒候选蛋白。这些蛋白质包括大多数已知的颗粒蛋白质和许多预期的蛋白质，但四分之三的蛋白质与mRNA新陈代谢无关，许多蛋白质具有假定的调节功能。32个候选蛋白质中的15个经EYFP标记证实为饥饿应激颗粒。用RNA测序法测定颗粒中的mRNA含量。我们已经确定了一组非常独特的mRNAs，它不存在于颗粒中：几乎所有的mRNAs都编码核糖体蛋白。用单mRNA FISH方法证实了RNA颗粒中不存在这样的一种mRNA。我们还对第二种类型的锥虫RNA颗粒的纯化感兴趣：当mRNA成熟受到抑制时，这些颗粒在细胞核的外围形成(核外围颗粒)。它们可能在mRNA质量控制中起作用，并与动物的核周生殖细胞颗粒显示出惊人的相似性。我们已经成功地提纯了仍附着着颗粒的细胞核，目前正在等待质谱学结果。在第二个资助期，我试图了解RNA颗粒的调节。我的颗粒蛋白质组包含许多蛋白质修饰酶，如激酶、磷酸酶和甲基转移酶。它们可能通过改变它们的目标蛋白对颗粒定位的能力来调节颗粒组成，包括附着的任何mRNAs。我想要解开潜在的信号通路。此外，我想了解将特定的mRNAs从颗粒中排除的机制。我们还将继续努力提纯和分析核外周颗粒，目标是从具有重要病理意义的可传播短桩生命周期阶段提纯颗粒。通过比较不同类型颗粒的蛋白质组和信使核糖核酸含量，可以确定每种类型颗粒所特有的蛋白质。我们希望我们的数据将有助于更全面地了解锥虫和其他真核生物中RNA颗粒的组成和调控。