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Nucleic Acids Res. 2015 Sep 18;43(16):8013-32. doi: 10.1093/nar/gkv731. Epub 2015 Jul 17.

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

Author information

1
Biocenter, University of Würzburg, Am Hubland, 97074 Würzburg, Germany.
2
Rudolf Virchow Center, University of Würzburg, Josef-Schneider-Str. 2, 97080 Würzburg, Germany.
3
Research Center for Infectious Diseases, University of Würzburg, Josef-Schneider-Str. 2, 97080 Würzburg, Germany.
4
Department of Biochemistry, Tennis Court Road, Cambridge CB2 1QW, UK.
5
Biocenter, University of Würzburg, Am Hubland, 97074 Würzburg, Germany susanne.kramer@uni-wuerzburg.de.

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.

PMID:
26187993
PMCID:
PMC4652759
DOI:
10.1093/nar/gkv731
[Indexed for MEDLINE]
Free PMC Article

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