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

细胞质RNA颗粒，例如P体和应激颗粒，是在转录后水平上调节真核基因表达的RNA和蛋白质的聚集体。RNA颗粒的功能和调节仍然非常难以捉摸：一个原因是RNA颗粒的纯化尚未在任何生物体中实现。在过去的2.5年中，我们已经建立了第一个协议的生化纯化饥饿应激颗粒。该协议利用非洲锥虫的一个独特的功能：我们采用了笼状的膜下微管骨架的寄生虫作为分子筛。我们已经确定了464应激颗粒的候选蛋白质的质谱。这些蛋白质包括大多数已知的颗粒蛋白和许多预期的蛋白质，但四分之三的蛋白质与mRNA代谢无关，许多蛋白质具有推定的调节功能。通过eYFP标记32个候选蛋白中的15个，确认了饥饿应激颗粒的定位。通过RNA测序测定颗粒的mRNA含量。我们已经确定了一组非常独特的mRNA，这是缺乏颗粒：几乎所有的核糖体蛋白编码。通过单个mRNA FISH证实了RNA颗粒中没有这样的mRNA。我们还感兴趣的第二种类型的锥虫RNA颗粒的纯化：这些形式周围的细胞核的mRNA成熟时，被抑制（核周边颗粒）。它们可能具有mRNA质量控制的功能，并显示出与动物核周生殖颗粒惊人的相似性。我们已经成功地纯化了仍然附着颗粒的细胞核，目前正在等待质谱分析结果。在第二个资助期，我试图了解RNA颗粒的调控。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。这些可以通过改变其靶蛋白的颗粒定位能力来调节颗粒组成，包括任何附着的mRNA。我想解开潜在的信号通路。此外，我想了解颗粒中排除特定mRNA的机制。我们还将继续努力纯化和分析核外周颗粒，并旨在从病理学上重要的可传播短生命周期阶段纯化颗粒。颗粒蛋白质组和mRNA含量的比较，从不同类型的颗粒将确定蛋白质的特定于任何类型的颗粒。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。

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.