title: Novel insights into RNP granules by employing the trypanosome's microtubule skeleton as a molecular sieve.
abstract: 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, 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.
authors.affiliation: Biocenter, University of Würzburg, Am Hubland, 97074 Würzburg, Germany.
authors.forename: Melanie
authors.initials: M
authors.lastname: Fritz
authors.affiliation: Rudolf Virchow Center, University of Würzburg, Josef-Schneider-Str. 2, 97080 Würzburg, Germany.
authors.forename: Jens
authors.initials: J
authors.lastname: Vanselow
authors.affiliation: Biocenter, University of Würzburg, Am Hubland, 97074 Würzburg, Germany.
authors.forename: Nadja
authors.initials: N
authors.lastname: Sauer
authors.affiliation: Rudolf Virchow Center, University of Würzburg, Josef-Schneider-Str. 2, 97080 Würzburg, Germany.
authors.forename: Stephanie
authors.initials: S
authors.lastname: Lamer
authors.affiliation: Biocenter, University of Würzburg, Am Hubland, 97074 Würzburg, Germany.
authors.forename: Carina
authors.initials: C
authors.lastname: Goos
authors.affiliation: Research Center for Infectious Diseases, University of Würzburg, Josef-Schneider-Str. 2, 97080 Würzburg, Germany.
authors.forename: T Nicolai
authors.initials: TN
authors.lastname: Siegel
authors.affiliation: Biocenter, University of Würzburg, Am Hubland, 97074 Würzburg, Germany.
authors.forename: Ines
authors.initials: I
authors.lastname: Subota
authors.affiliation: Rudolf Virchow Center, University of Würzburg, Josef-Schneider-Str. 2, 97080 Würzburg, Germany.
authors.forename: Andreas
authors.initials: A
authors.lastname: Schlosser
authors.affiliation: Department of Biochemistry, Tennis Court Road, Cambridge CB2 1QW, UK.
authors.forename: Mark
authors.initials: M
authors.lastname: Carrington
authors.affiliation: Biocenter, University of Würzburg, Am Hubland, 97074 Würzburg, Germany susanne.kramer@uni-wuerzburg.de.
authors.forename: Susanne
authors.initials: S
authors.lastname: Kramer
mesh_terms: D002458:Cell Fractionation; D003594:Cytoplasmic Granules / Q000737:chemistry*; D015852:Eukaryotic Initiation Factor-2 / Q000378:metabolism; D008870:Microtubules; D015800:Protozoan Proteins / Q000032:analysis* / Q000235:genetics; D012333:RNA, Messenger / Q000032:analysis; D012261:Ribonucleoproteins / Q000032:analysis*; D012269:Ribosomal Proteins / Q000235:genetics; D013347:Subcellular Fractions; D014346:Trypanosoma brucei brucei / Q000737:chemistry / Q000235:genetics
chemical_list: D015852:Eukaryotic Initiation Factor-2; D015800:Protozoan Proteins; D012333:RNA, Messenger; D012261:Ribonucleoproteins; D012269:Ribosomal Proteins