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Eukaryotic Cell, May 2006, p. 876-879, Vol. 5, No. 5
1535-9778/06/$08.00+0 doi:10.1128/EC.5.5.876-879.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.
Sir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford OX1 3RE, United Kingdom,1 Department of Biological Sciences, University of Hull, Cottingham Road, Hull HU6 7RX, United Kingdom2
Received 20 December 2005/ Accepted 8 March 2006
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ABSTRACT |
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Trypanosoma brucei is unusual among eukaryotes as it harbors two separate RNA polymerase I (Pol I) compartments within its nucleus during certain stages of its life cycle. rRNAs are transcribed in the primary Pol I-containing organelle, the nucleolus. However, in bloodstream-form cells, genes encoding the variant surface glycoprotein are transcribed by Pol I in an extranucleolar expression site body (10). This presents trypanosomes with the unique challenge of linking the RNA polymerase II mRNA processing machinery to Pol I. Here, we describe two homologues of Nopp140 in trypanosomatids that are potentially involved in RNA processing and demonstrate an intriguing convergence in evolution.
Trypanosomatids encode two Nopp140-like proteins.
We generated a mouse monoclonal antibody (NUMAG) that detects the nucleolus and recognizes two proteins of 
180 kDa and 80 kDa by Western blotting in T. brucei (11). Screening a T. brucei cDNA expression library identified clones encoding a repeat protein with a predicated mass of 128 kDa (accession no. XP_825186
[GenBank]
). We identified this protein as the T. brucei orthologue of Nopp140 (TbNopp140) due to the presence of the SRP40 C-terminal domain. Searching the completed T. brucei genome sequence revealed a related predicted protein of 54 kDa (accession no. XP_827326
[GenBank]
) This predicted protein, herein named TbNoLP (T. brucei Nopp140-like protein), is 19.7% identical across the N-terminal region and 51.4% identical across the repetitive central region to TbNopp140 but shares no detectable homology in the C-terminal domain (Fig. 1A and B). NUMAG binds to both proteins by shared epitopes in the central repetitive region (data not shown).
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75-amino-acid region at the extreme C terminus (Fig. 1C). We generated a novel hidden Markov model for this domain and used it to identify several highly divergent homologues of this eukaryote-specific domain, including those of Entamoeba, Giardia, and Chlamydomonas, which have been truncated by up to 60% (Fig. 1C).
The central domains of Nopp140 homologues are poorly alignable. They do, however, share some traits. They are rich in amino acids A, D, E, K, P, and S (73% of all residues), which are arranged in repeats of alternating charge. Assuming all the available serines are phosphorylated (as has been shown for mammalian Nopp140) (8), the mean charge per residue for the repeat region is also well conserved (Fig. 1D). The central domains of four organisms from our analysis, Saccharomyces, Schizosaccharomyces, Dictyostelium, and Giardia, do not fit this pattern as their central domains are composed mainly of serine (61%, 50%, 32%, and 61%, respectively). The central domain of Saccharomyces Srp40p is phosphorylated to a much lesser extent than that of vertebrate Nopp140 (8), and this is likely to be true for other S-biased domains.
TbNoLP contains the N-terminal LisH and central repeats of a canonical Nopp140 protein. However it differs by replacement of the SRP40 domain with an RGG repeat containing a GAR domain similar to those in nucleolar proteins such as fibrillarin, nucleolin, and the kinetoplastid-specific Nopp44/46 (6). Intriguingly, a protein with an identical domain structure to TbNoLP is produced as a splice variant of DmNopp140 (15). Moreover, we have identified a hypothetical NoLP protein in Anopheles gambiae, suggestive of a more widespread distribution within insects. Additional Nopp140 variants can be found in other organisms, for example, the mammalian Treacher Collins syndrome protein, treacle (13). Like Nopp140, treacle contains an N-terminal LisH domain and a charged central domain, but lacks either an SRP40 domain or GAR domain at its C terminus.
TbNopp140 and TbNoLP have different subnuclear localizations. To determine the cellular localization of both TbNopp140 and TbNoLP, we used the NUMAG monoclonal antibody, which recognizes both proteins by Western blotting, and specific anti-TbNopp140 and anti-TbNoLP polyclonal antibodies. These antibodies behave differently following different fixation procedures. Following methanol fixation, NUMAG stains a subcompartment of the nucleolus (Fig. 2A) (11). Consistent with this, the affinity-purified anti-Nopp140 antiserum stains the same subcompartment (Fig. 2A and B). Anti-TbNoLP antiserum also stains the nucleolus after methanol fixation (Fig. 2C). The localization of TbNopp140 does not change following paraformaldehyde fixation (Fig. 2B). However, in paraformaldehyde-fixed cells, NUMAG and the anti-TbNoLP antiserum recognize both the nucleolus and the nucleoplasm (Fig. 2D). These patterns are maintained throughout the cell cycle, during which time the nucleolus remains intact and is segregated along the mitotic spindle (Fig. 2B). These contrasting fixation procedures reveal the dual localization of TbNoLP both within the nucleolus and the nucleoplasm.
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The behavior of the two Nopp140 homologues in immunofluorescence experiments is mirrored by their differential solubilization. The majority of TbNoLP, but not TbNopp140, is readily released from cells by treatment with nonionic detergent (see data in the supplemental material). Nucleolar TbNopp140 and a small pool of TbNoLP remain insoluble and are not released by treatment with RNase A or DNase I, suggesting that both are being held in the nucleolus by protein-protein interactions.
Both TbNopp140 and TbNoLP are phosphorylated and interact with Pol I. Mammalian Nopp140 proteins are phosphorylated (9) and interact with Pol I (1). We asked whether this was true for both trypanosomatid Nopp140 homologues. In vivo labeling with [32P]phosphate followed by immunoprecipitation with the NUMAG antibody revealed that both TbNopp140 and TbNoLP are phosphorylated (see data in the supplemental material). We also performed coimmunoprecipitations using an anti-T. brucei Pol I monoclonal antibody (10) demonstrating an interaction with Pol I (Fig. 1E).
RNA interference (RNAi)-induced knockdown of TbNopp140 and TbNoLP. To address the function of TbNopp140 and TbNoLP, we used an inducible RNAi system to specifically downregulate expression of each, both independently and simultaneously. Inducible knockdown of either TbNopp140 or TbNoLP resulted in minor slow-growth phenotypes detectable 4 days postinduction with simultaneous knockdown of both producing a more pronounced, earlier onset, phenotype (see data in the supplemental material). These growth defects phenocopy the yeast SRP40 knockout (18). Depletion of TbNopp140 resulted in no observable defects in nuclear or nucleolar morphology and had no impact on localization of TbNoLP. However, depletion of TbNoLP caused nucleolar enlargement, with Nopp140 foci becoming more dispersed (Fig. 2). Given the almost ubiquitous distribution of Nopp140 throughout Eukarya, it is surprising that neither deletion of the yeast protein, nor RNAi-mediated depletion of the trypanosomatid protein, produce more severe phenotypes.
NoLP proteins may function in snoRNP shuttling. Trypanosomatids are unusual among eukaryotes as pseudouridylation is not limited to rRNA or sn(o)RNAs but is also found on every mRNA. This modification arises through trans-splicing of a pseudouridylated spliced leader sequence onto every pre-mRNA (7, 14). Drosophila also has an unusual capacity for RNA modification, exhibiting a large diversity of box H/ACA snoRNAs and the highest degree of pseudouridylation found in eukaryotes (3, 4). It is very suggestive that two organisms with such a dependence on RNA modification should have evolved NoLP proteins with a similar domain structure.
In trypanosomatids, pseudouridylation of spliced leader RNA is mediated by a canonical snoRNA-guided eukaryotic pathway (7), yet spliced leader RNA cannot be detected in the nucleolus where the modifying enzymes reside. We hypothesize that TbNoLP may recruit snoRNPs from the nucleolus so they can modify spliced leader RNA in the nucleoplasm. The enlargement of the nucleolus caused by TbNoLP knockdown may thus be indicative of accumulation of material within the nucleolus due to a breakdown in a TbNoLP-mediated snoRNP export process.
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ACKNOWLEDGMENTS |
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We acknowledge the technical support of Val Tilston and Lemy Tsikna.
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FOOTNOTES |
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Supplemental material for this article may be found at http://ec.asm.org/.
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