Exploring the regulation of trypanosome RNA granules with a newly established RNA granule purification protocol.
Cell Biology
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.
Publications
- (2013). SCD6 induces ribonucleoprotein granule formation in trypanosomes in a translation-independent manner, regulated by its Lsm and RGG domains. Mol Biol Cell 24: 2098-2111
Krüger T, Hofweber M, Kramer S
(See online at https://doi.org/10.1091/mbc.E13-01-0068) - (2014). An AU-rich instability element in the 3’UTR mediates an increase in mRNA stability in response to expression of a dhh1 ATPase mutant. Translation 2: e28587
Kramer S, Carrington M
(See online at https://doi.org/10.4161/trla.28587) - (2014). RNA in development: how ribonucleoprotein granules regulate the life cycles of pathogenic protozoa. Wiley interdisciplinary reviews RNA 5: 263-284
Kramer S
(See online at https://doi.org/10.1002/wrna.1207) - (2015) Novel insights into RNP granules by employing the trypanosome’s microtubule skeleton as a molecular sieve. Nucleic Acids Res 43: 8013–8032
Fritz M , Vanselow J, Sauer N, Lamer S, Goos C, Siegel TN, Subota I, Schlosser A, Carrington M and Kramer S
(See online at https://doi.org/10.1093/nar/gkv731) - (2016). Polycistronic trypanosome mRNAs are a target for the exosome. Mol Biochem Parasitol 205: 1–5
Kramer S, Piper S, Estevez A, Carrington M
(See online at https://doi.org/10.1016/j.molbiopara.2016.02.009) - (2017). Simultaneous detection of mRNA transcription and decay intermediates by dual colour single mRNA FISH on subcellular resolution. Nucleic Acids Res 45:e49
Kramer S
(See online at https://doi.org/10.1093/nar/gkw1245) - (2017). The ApaH-like phosphatase ALPH1 is the major mRNA decapping enzyme of trypanosomes. PLoS Pathog. 13:e1006456
Kramer S
(See online at https://doi.org/10.1371/journal.ppat.1006456) - (2017). The nuclear proteome of Trypanosoma brucei. PLoS ONE, 12:e0181884
Goos C, Dejung M, Janzen CJ, Butter F, Kramer S
(See online at https://doi.org/10.1371/journal.pone.0181884) - (2018) The complex enzymology of mRNA decapping: enzymes of four classes cleave pyrophosphate bounds. WIREs RNA 10, e1511
Kramer S and McLennan AG
(See online at https://doi.org/10.1002/wrna.1511) - (2018). Comparative proteomics of the two T. brucei PABPs suggests that PABP2 controls bulk mRNA. PLoS Negl Trop Dis.12:e0006679
Zoltner M, Krienitz N, Field MC, Kramer S
(See online at https://doi.org/10.1371/journal.pntd.0006679) - (2019) Trypanosomes can initiate nuclear export cotranscriptionally. Nucleic Acids Res 47, 266-282
Goos C, Dejung M, Wehman AM, Meyer-Natus E, Schmidt J, Sunter J, Engstler M, Butter F and Kramer S
(See online at https://doi.org/10.1093/nar/gky1136)
