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

Subject Area General Genetics and Functional Genome Biology
Cell Biology
Term from 2011 to 2019
Project identifier Deutsche Forschungsgemeinschaft (DFG) - Project number 208025595
 
Final Report Year 2019

Final Report Abstract

The major aim of my research is to understand, how gene regulation is achieved on the posttranscriptional level. In particular, I am interested in spatial aspects of mRNA metabolism that participate in this regulation. My model system is the African trypanosome Trypanosoma brucei, a unicellular parasite responsible for African sleeping sickness and related cattle diseases. The parasite has the major advantage of (almost) not regulating its transcription. Next to understanding eukaryotic mRNA metabolism in general, I am also interested in identifying differences between trypanosomes and men that can be exploited as drug targets. RNA granules play a key role in regulating gene expression. One aim of this project was the development of a purification protocol for stress granules from trypanosomes. This had not been achieved for any organism at the time. We have employed the subpellicular cytoskeleton of the parasite as a molecular sieve to purify the granules and succeeded to obtain the protein and the RNA content of trypanosome starvation stress granules. The biggest surprise arising from this dataset was the identification of an ApaH like phosphatase among the stress granule proteins. We were able to show that this enzyme was the long-wanted mRNA decapping enzyme of trypanosomes and, thus, to assign the first function to this group of phosphatases that is widespread throughout the eukaryotic kingdom. The further characterisation of this novel enzyme, in particular the understanding of its regulation and evolution has now become a major focus of the lab. Another interesting finding was the complete exclusion of mRNAs encoding ribosomal proteins from stress granules: the reason and mechanism for this is still under investigation. A second RNA granule type is unique to trypanosomes: nuclear periphery granules (NPGs) form around the nucleus when trans-splicing is inhibited and transcription is intact. We were able to develop a purification method for this granule type and to obtain and verify the granule proteome. Moreover, we developed a novel intramolecular multi-colour smFISH method that allows detection of mRNA transcription and decay intermediates. With this method, we were able to show that nuclear export in trypanosomes can be co-transcriptional. Inhibition of trans-splicing traps polycistronic mRNAs within nuclear pores with the 5’ ends reaching into the cytoplasm, forming NPGs. We now want to investigate this unusual nuclear export mechanism in more detail. Several further smaller projects were finished towards publication within this funding period, mostly dealing with the analysis of specific RNA binding proteins. (1) We could show that SCD6 is the core P-body protein in trypanosomes (2) We could show that the trypanosome exosome is in the nucleus and involved in the degradation of unspliced mRNAs (3) We could show that the stabilisation of mRNAs by expression of an inactive DHH1 is due to an AU-rich element in the 3’ UTRs and (4) we could identify the interacting proteins of the two T. brucei Poly(A) binding proteins.

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