The subcellular localisation of trypanosome RRP6 and its association with the exosome

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Abstract

The exosome, a complex of 3′-exoribonucleases and associated proteins, is involved in the degradation of eukaryotic mRNAs in the cytoplasm, and has RNA processing and quality control functions in the nucleus. In yeast, the nuclear exosome differs from the cytoplasmic one in that it contains an additional non-essential component, Rrp6p. In contrast, a small proportion of human RRP6 has been shown to localise to the cytoplasm as well. When we purified the Trypanosoma brucei exosome from cytosolic extracts we found RRP6, apparently in stoichiometric amounts. We here confirm that RRP6 is in the trypanosome cytoplasm and nucleus. The level of RRP6 was unaffected by depletion of core exosome subunits by RNA interference and over-expression of tagged RRP6 was possible, indicating that RRP6 can be present independent of exosome association.

Introduction

The exosome is a protein complex which is involved in 3′  5′ degradation of diverse RNAs. It is composed of a “core” of six subunits related to Escherichia coli RNase PH, several of which have been shown to have 3′  5′ exonuclease activity, and three subunits with S1 domains, and is present in yeast, trypanosomes, animals and plants [1], [2], [3], [4], [5].

The exosome has multiple functions in the nucleus, including processing of rRNAs and small nucleolar (sno) RNAs, and quality control of mRNAs (reviewed in [6] and see [7], [8], [9], [10], [11]). In the cytoplasm, it is involved in the degradation of both normal and defective mRNAs [12], [13], [14], [15], [16], [17], [18], [19], [20], [21]. By analogy with the structure of prokaryotic polynucleotide phosphorylase, Aloy et al. [22] suggested that the six RNase PH subunits of the exosome form a ring, with the three S1 domain subunits balanced on top. This concept has been supported by analyses using yeast and mammalian two-hybrid systems [23], [24] and using mass spectrometry [25]. The archaeon Sulfolobus solfataricus has an exosome-like complex [26] in which three copies each of two RNase PH subunits form a hexameric core structure [27]. In yeast, all of the RNase PH and S1 domain subunits are essential for viability [2].

Various other proteins are associated with the yeast and human exosomes, often in sub-stoichiometric amounts. One of these sub-stoichiometric components is RRP6, an RNase D enzyme. S. cerevisiae Rrp6p is found only in the nucleus [2], where it is involved in multiple processes, including linkage of transcription with RNA processing [28]. Deletion of the gene encoding Rrp6p causes lethality at 37 °C, but not at 30 °C [2]. In Drosophila tissue culture cells, the Rrp6p homologue is predominantly, but not exclusively, nuclear [29]. The human Rrp6p is called PM/Scl-100, a target of auto-antibodies in patients with polymyositis-scleroderma overlap syndrome. Pm/Scl-100 was initially found to be undetectable in a cytoplasmic extract using the human autoimmune sera [2], but a later report showed that a small quantity was cytoplasmic [3]. In support of a partially cytoplasmic location for PM/Scl-100, depletion of the subunit using siRNA inhibited decay of an unstable mRNA containing a nonsense codon [14].

We have previously shown that the Trypanosoma brucei exosome is similar to those of other eukaryotes [30]. There are six RNase PH subunits (EAP1, EAP2, EAP4, RRP41A, RRP41B, RRP45), and three S1 domain subunits (RRP40, CSL4, RRP4). In addition, however, the RRP6 RNase D, and an additional small subunit called EAP3 were present in apparently stoichiometric amounts. RNA-interference (RNAi)-mediated depletion of each of the individual exosome components, including RRP6, in procyclic trypanosomes (the life-cycle stage which multiplies in the Tsetse fly) inhibited growth and 5.8 S rRNA processing [30], [31]. Depletion of the core subunit RRP45 also inhibited growth, and degradation of unstable mRNAs, in bloodstream forms [32]. Analyses using the yeast two-hybrid system gave structural predictions consistent with those from yeast and mammals; in addition, an interaction was found between trypanosome RRP6 and EAP3. The effects of genetic manipulation yielded more insights into exosome structure. First, over-expression of tagged version of either RRP4 (and S1 domain subunit) or RRP45 (a core subunit) resulted in a decrease in abundance of the endogenous, untagged protein, suggesting that both RRP4 and RRP45 are unstable when not associated with the exosome complex. Second, depletion of the core subunits RRP45, RRP41B, EAP2 and EAP4 caused co-depletion of RRP4 and RRP45, suggesting that the absence of any of these subunits destabilises the entire complex. More surprisingly, depletion of RRP6 and EAP3 also caused decreases in RRP4 and RRP45 [31]. This result suggested that – in dramatic contrast with the situation in yeast – RRP6 might either be a central, part of the exosome structure, or be involved in exosome assembly. This paper describes the results of further experiments to define the location and functional role of RRP6.

Section snippets

Trypanosomes

Procyclic forms of T. brucei expressing the tet repressor were cultured as described [33]. Cell lines harboring RNAi plasmids targeting RRP44 or each exosome components, or expressing TAP-tagged RRP4 or RRP45, were grown and induced as described [30], [31].

Expression of TbRRP6 in E. coli and generation of antiserum

Several attempts to produce full-length RRP6 in E. coli failed, so instead, a fragment of the TbRRP6 open reading frame corresponding to amino acids 468–588 was amplified using primers CZ1667 (CTC GGA TCC ATG TCT GCG GTT AAG) and CZ1668 (CAC

TbRRP6 is stable in the absence of other exosome subunits

We had previously shown that depletion of RRP6 results in a decrease in the amounts of both RRP45 and RRP4. We had, not, however, been able to determine the fate of RRP6 after depletion of other exosome subunits. To find out of exosome depletion affected RRP6, we made lysates of procyclic trypanosomes in which expression of individual exosome subunits had been reduced by RNAi, and measured RRP6 levels using a new specific antiserum (see Section 2 and Fig. 3A for details). The results from this

Discussion

We have previously shown that in trypanosomes, RRP6 is essential for maintaining the structural integrity of the exosome. Results presented here show that in contrast, a reduction in the amount of exosome does not affect the abundance of RRP6. The core exosome components RRP45, RRP41B, EAP2 and EAP4 are essential for exosome integrity [31] but RRP6 levels were not reduced by depletion of any of these subunits (Fig. 1) even though the migration of the protein in glycerol gradients was clearly

Acknowledgements

This work was supported by the Deutsche Forschungsgemeinshaft, CL112/7 and Cl112/9, and Graduiertenkolleg 300. We thank Noreen Williams and Marilyn Parsons for antibodies. The experiments shown in Fig. 1, Fig. 2 were done by SH under the supervision of AE, and those in Fig. 3 by SH, AE, and MC. MC also did the TAP-tagging and mutagenesis supervised by CC. CC wrote the grant applications and all authors contributed to writing the manuscript.

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    Present address: Centre de Recherche en Infectiologie, CHUQ, Pavillon Chul, 2705 Boul. Laurier, Que. G1V 4G2, Canada.

    2

    Instituto de Parasitologia y Biomedicina “Lopez-Neyra”, CSIC, Avda. del Conocimiento s/n, 18100-Armilla, Granada, Spain.

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