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

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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.