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Inhibition of mRNA maturation in trypanosomes causes the formation of novel foci at the nuclear periphery containing cytoplasmic regulators of mRNA fate.

ABSTRACT: Maturation of all cytoplasmic mRNAs in trypanosomes involves trans-splicing of a short exon at the 5' end. Inhibition of trans-splicing results in an accumulation of partially processed oligocistronic mRNAs. Here, we show that the accumulation of newly synthesised partially processed mRNAs results in the formation of foci around the periphery of the nucleus. These nuclear periphery granules (NPGs) contain the full complement of P-body proteins identified in trypanosomes to date, as well as poly(A)-binding protein 2 and the trypanosome homologue of the RNA helicase VASA. NPGs resemble perinuclear germ granules from metazoa more than P-bodies because they: (1) are localised around the nuclear periphery; (2) are dependent on active transcription; (3) are not dissipated by cycloheximide; (4) contain VASA; and (5) depend on nuclear integrity. In addition, NPGs can be induced in cells depleted of the P-body core component SCD6. The description of NPGs in trypanosomes provides evidence that there is a perinuclear compartment that can determine the fate of newly transcribed mRNAs and that germ granules could be a specialised derivative.

SUBMITTER: Kramer S

PROVIDER: S-EPMC3434824 | biostudies-literature | 2012 Jun

REPOSITORIES: biostudies-literature

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Inhibition of mRNA maturation in trypanosomes causes the formation of novel foci at the nuclear periphery containing cytoplasmic regulators of mRNA fate.

Kramer Susanne S Marnef Aline A Standart Nancy N Carrington Mark M

Journal of cell science 20120224 Pt 12

Maturation of all cytoplasmic mRNAs in trypanosomes involves trans-splicing of a short exon at the 5' end. Inhibition of trans-splicing results in an accumulation of partially processed oligocistronic mRNAs. Here, we show that the accumulation of newly synthesised partially processed mRNAs results in the formation of foci around the periphery of the nucleus. These nuclear periphery granules (NPGs) contain the full complement of P-body proteins identified in trypanosomes to date, as well as poly( ...[more]

PMID: 22366449

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Project description:In higher eukaryotes, mRNA degradation and RNA-based gene silencing occur in cytoplasmic foci referred to as processing bodies (P-bodies). In protozoan parasites, the presence of P-bodies and their putative role in mRNA decay have yet to be comprehensively addressed. Identification of P-bodies might provide information on how mRNA degradation machineries evolved in lower eukaryotes. Here, we used immunofluorescence and confocal microscopy assays to investigate the cellular localization of mRNA degradation proteins in the human intestinal parasite Entamoeba histolytica and found evidence of the existence of P-bodies. Two mRNA decay factors, namely the EhXRN2 exoribonuclease and the EhDCP2 decapping enzyme, were localized in cytoplasmic foci in a pattern resembling P-body organization. Given that amoebic foci appear to be smaller and less rounded than those described in higher eukaryotes, we have named them "P-body-like structures". These foci contain additional mRNA degradation factors, including the EhCAF1 deadenylase and the EhAGO2-2 protein involved in RNA interference. Biochemical analysis revealed that EhCAF1 co-immunoprecipitated with EhXRN2 but not with EhDCP2 or EhAGO2-2, thus linking deadenylation to 5'-to-3' mRNA decay. The number of EhCAF1-containing foci significantly decreased after inhibition of transcription and translation with actinomycin D and cycloheximide, respectively. Furthermore, results of RNA-FISH assays showed that (i) EhCAF1 colocalized with poly(A)(+) RNA and (ii) during silencing of the Ehpc4 gene by RNA interference, EhAGO2-2 colocalized with small interfering RNAs in cytoplasmic foci. Our observation of decapping, deadenylation and RNA interference proteins within P-body-like foci suggests that these structures have been conserved after originating in the early evolution of eukaryotic lineages. To the best of our knowledge, this is the first study to report on the localization of mRNA decay proteins within P-body-like structures in E. histolytica. Our findings should open up opportunities for deciphering the mechanisms of mRNA degradation and RNA-based gene silencing in this deep-branching eukaryote.

Dataset Information

Inhibition of mRNA maturation in trypanosomes causes the formation of novel foci at the nuclear periphery containing cytoplasmic regulators of mRNA fate.

Publications

Inhibition of mRNA maturation in trypanosomes causes the formation of novel foci at the nuclear periphery containing cytoplasmic regulators of mRNA fate.

Similar Datasets

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