The exosome of Trypanosoma brucei - PubMed (original) (raw)

The exosome of Trypanosoma brucei

A M Estévez et al. EMBO J. 2001.

Abstract

The yeast exosome is a complex of at least 10 essential 3'-5' riboexonucleases which is involved in 3'-processing of many RNA species. An exosome-like complex has been found or predicted to exist in other eukaryotes but not in Escherichia coli. The unicellular parasite Trypanosoma brucei diverged very early in eukaryotic evolution. We show here that T.brucei contains at least eight exosome subunit homologs, but only a subset of these associate in a complex. Accordingly, the T.brucei exosome is smaller than that of yeast. Both free and complex-associated homologs are essential for cell viability and are involved in 5.8S rRNA maturation. We suggest that the exosome was present in primitive eukaryotes, and became increasingly complex during subsequent evolution.

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Figures

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Fig. 1. _Tb_RRP4 and _Tb_RRP45, but not _Tb_RRP44, are found in a complex. (A) Cytosolic and (B) nuclear extracts from procyclic T.brucei were fractionated through 10–30% glycerol gradients. Aliquots of each fraction and original extracts were subjected to SDS–PAGE and immunoblotting analysis using antibodies against _Tb_RRP4, _Tb_RRP45 or _Tb_RRP44 (A and B) or assayed for 3′–5′ exonuclease activity (C). The exonuclease activity is expressed as the percentage of substrate digested (see Materials and methods). The sedimentation coefficients of marker proteins processed in parallel are indicated.

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Fig. 2. Recombinant His6-_Tb_RRP4 and the purified T.brucei exosome show 3′–5′ exoribonuclease activity in vitro. Purified His6-_Tb_RRP4 was incubated in the presence of a 5′-labeled (A) or 3′-labeled RNA substrate (B) for the times indicated, and the reactions electrophoresed in PAGE–urea gels. In the mock lane the RNA substrate was incubated for 120 min using the same reaction conditions, without recombinant His6-_Tb_RRP4. Exonuclease activity was assayed for the purified exosome complex under the same conditions using the 5′- (C) or 3′-labeled (D) substrate. (E) Separation of the reaction products by TLC. The RNA substrate was incubated in the absence (mock) or in the presence of His6-_Tb_RRP4. The migration of 5′- and 3′-nucleoside monophosphates is also indicated. (F) Exonuclease activity of His6-_Tb_RRP4 measured in the presence of the RNA substrate without (open squares) or with (filled squares) a phosphate group at the 3′ end.

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Fig. 3. Purification of the T.brucei exosome. The expression of TAP-tagged _Tb_RRP4 was induced by the addition of tetracycline to the culture medium for 48 h, and the associated proteins purified using the TAP method. (A) Aliquots from each step (or the whole EGTA-eluate fractions) were analyzed by SDS–PAGE and silver staining. Lane 1, S100 extract; lane 2, IgG chromatography flow-through; lane 3, TEV eluate; lane 4, calmodulin chromatography flow-through; lane 5, EGTA eluate (5 fractions). (B and C) Immunoblotting analyses of the TAP fractions using _Tb_RRP45 (B) or _Tb_RRP44 (C) antisera. The protein marker is the 10 kDa protein ladder (Gibco BRL).

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Fig. 4. Depletion of the exosome protein homologs by inducible RNA interference and effects on growth. Trypanosome lines were created that expressed gene-specific dsRNA in a tetracycline-inducible fashion. Each trypanosome line was grown in the absence (open squares) or presence (filled squares) of 100 ng/ml tetracycline to induce RNAi. Cultures were followed for four to nine days and were diluted to 0.4 × 106 cells/ml every 2 days as required.

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Fig. 5. Effect of depletion of each exosome component homolog on 5.8S rRNA maturation in vivo. Total RNA was extracted from parasites grown in the absence (–) or in the presence (+) of tetracycline for 24 or 48 h, and separated in PAGE–urea gels. In the case of _Tb_CSL4, an additional sample taken at 96 h after induction was also included. After electrophoresis the gels were transferred and hybridized to detect (A) extended 5.8S rRNA species, (B) mature 5.8S rRNA or (C) the T.brucei signal recognition particle RNA (loading control). The arrow indicates full-length 7S rRNA and the vertical dashed line shows incompletely processed 7S rRNA species. 6S-like species are indicated with a bracket. The asterisk (A, left panel) indicates the mature 5.8S rRNA, which in this particular blot was not completely stripped from a previous hybridization. The size marker is ØX174 DNA digested with _Bsu_R I (MBI fermentas) that was dephosphorylated and labeled with [γ-32P]ATP and polynucleotide kinase.

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