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Novel insights into RNP granules by employing the trypanosome's microtubule skeleton as a molecular sieve

Please always quote using this URN: urn:nbn:de:bvb:20-opus-126180
  • RNP granules are ribonucleoprotein assemblies that regulate the post-transcriptional fate of mRNAs in all eukaryotes. Their exact function remains poorly understood, one reason for this is that RNP granule purification has not yet been achieved. We have exploited a unique feature of trypanosomes to prepare a cellular fraction highly enriched in starvation stress granules. First, granules remain trapped within the cage-like, subpellicular microtubule array of the trypanosome cytoskeleton while soluble proteins are washed away. Second, theRNP granules are ribonucleoprotein assemblies that regulate the post-transcriptional fate of mRNAs in all eukaryotes. Their exact function remains poorly understood, one reason for this is that RNP granule purification has not yet been achieved. We have exploited a unique feature of trypanosomes to prepare a cellular fraction highly enriched in starvation stress granules. First, granules remain trapped within the cage-like, subpellicular microtubule array of the trypanosome cytoskeleton while soluble proteins are washed away. Second, the microtubules are depolymerized and the granules are released. RNA sequencing combined with single molecule mRNA FISH identified the short and highly abundant mRNAs encoding ribosomal mRNAs as being excluded from granules. By mass spectrometry we have identified 463 stress granule candidate proteins. For 17/49 proteins tested by eYFP tagging we have confirmed the localization to granules, including one phosphatase, one methyltransferase and two proteins with a function in trypanosome life-cycle regulation. The novel method presented here enables the unbiased identification of novel RNP granule components, paving the way towards an understanding of RNP granule function.show moreshow less

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Metadaten
Author: Melanie Fritz, Jens Vanselow, Nadja Sauer, Stephanie Lamer, Carina Goos, T. Nicolai Siegel, Ines Subota, Andreas Schlosser, Mark Carrington, Susanne Kramer
URN:urn:nbn:de:bvb:20-opus-126180
Document Type:Journal article
Faculties:Fakultät für Biologie / Rudolf-Virchow-Zentrum
Language:English
Parent Title (English):Nucleic Acids Research
Year of Completion:2015
Source:Nucleic Acids Research, 2015, Vol. 43, No. 16 8013–8032 doi: 10.1093/nar/gkv731
DOI:https://doi.org/10.1093/nar/gkv731
Dewey Decimal Classification:6 Technik, Medizin, angewandte Wissenschaften / 61 Medizin und Gesundheit / 610 Medizin und Gesundheit
Release Date:2016/01/29
Collections:Open-Access-Publikationsfonds / Förderzeitraum 2015
Licence (German):License LogoCC BY: Creative-Commons-Lizenz: Namensnennung