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The Trypanosoma brucei CCCH zinc finger proteins ZC3H12 and ZC3H13
Molecular & Biochemical Parasitology 183 (2012) 184–188
Contents lists available at SciVerse ScienceDirect
Molecular & Biochemical Parasitology
Short communication
The Trypanosoma brucei CCCH zinc finger proteins ZC3H12 and ZC3H13 Benard Aswani Ouna, Mhairi Stewart 1 , Claudia Helbig, Christine Clayton ∗ Zentrum fur Molekular Biologie Heidelberg, ZMBH-DKFZ Alliance, Im Neuenheimerfeld 282, Heidelberg 69120, Germany
a r t i c l e
i n f o
Article history: Received 28 April 2011 Received in revised form 8 February 2012 Accepted 10 February 2012 Available online 17 February 2012 Keywords: Trypanosoma CCCH zinc finger Xrn1 TTP BRF1
a b s t r a c t CCCH-type zinc finger proteins have various roles in RNA metabolism. We here analysed the functional relevance of two such proteins from Trypanosoma brucei, TbZC3H12 and TbZC3H13. Each protein has a single CCCH motif very similar to those seen in metazoan proteins that regulate mRNA degradation. TbZC3H12 is expressed in bloodstream form parasites at low levels. It is phosphorylated, cytosolic and not required for normal growth of cultured bloodstream trypanosomes. RNA interference targeting TbZC3H13, on a TbZC3H12 null background, also had no effect on bloodstream trypanosome growth, but over-expression of tagged TbZC3H13 inhibited procyclic trypanosome growth. Tandem affinity purification of both proteins revealed various interesting potential interactions; specificity was assessed against a list of proteins that were found in 24 other pull-down experiments, which is provided. The conservation of TbZC3H12 in all kinetoplastids, and TbZC3H13 in Salivaria, suggests that the two proteins may be required for optimal growth at some stage of the parasite life-cycle. © 2012 Elsevier B.V. All rights reserved.
1. Introduction Trypanosoma brucei multiplies in mammalian blood and tissue fluids as the bloodstream form (BS) and in the midgut of Tsetse flies as the procyclic form (PC). Since most trypanosome genes are constitutively transcribed, rates of mRNA turnover and translation are important in controlling trypanosome gene expression [1]. The control of mRNA turnover in eukaryotes is often mediated by proteins which bind the 3 -untranslated region of a target mRNA. One such protein class, the Tis11 family, contains two finger domains of the type C-x-C-x-C-x3 -H [2], separated by a short linker and immediately preceded by a consensus sequence, R/K-Y-K-T-E-L. Well-studied examples of mammalian Tis11-family proteins include tristetraprolin (TTP) and Butyrate Response Factors BRF1 and BRF2; each of these binds to AU-rich elements (AREs) in the 3 -UTRs of mRNAs and induces their decay [2]. Forty-nine genes encoding CCCH zinc finger proteins are found in the T. brucei genome. A few have known functions in splicing and mRNA export [3], and so far, four have been implicated in post transcriptional gene regulation [4]. TbZFP1, TbZFP2 and TbZFP3 are all required for normal differentiation [5–7]; TbZFP3 is required for normal patterns of translation of the major surface proteins of
∗ Corresponding author. Tel.: +49 6221 546876. E-mail address: [email protected] (C. Clayton). 1 Current address: Wellcome Trust Centre for Molecular Parasitology, Institute of Infection and Immunity, MVLS, GBRC, 120 University Place, University of Glasgow, Glasgow G12 8TA, United Kingdom. 0166-6851/$ – see front matter © 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.molbiopara.2012.02.006
procyclic forms. Over-expression of TbZFP2 in the procyclic forms caused abnormal remodelling of the cytoskeleton [5]. Meanwhile TbZC3H20 is required for growth of procyclic forms, binding to and stabilizing at least two developmentally regulated mRNAs [8]. TbZC3H18 has been implicated in control of differentiation but the mechanism is unknown [9]. In this paper, we investigated the functions of two more T. brucei CCCH zinc finger proteins, TbZC3H12 (Tb927.5.1570) and TbZC3H13 (Tb927.5.1580) (henceforth written without the Tb prefix). Each has a single CCCH motif starting at residue 20 near the N-terminus. The proteins are 50% identical in the first 49 amino acid residues, and then diverge completely (Fig. 1A). ZC3H13 (60 kDa) is 400 residues longer than ZC3H12 (18.8 kDa). Within the core CCCH motif, the key aromatic residues that are involved in base stacking are conserved (Fig. 1A sequence, arrowheads). We were particularly interested in these two proteins because each has a sequence resembling the Tis11 consensus immediately preceding the CCCH motif: KYRTTL for ZC3H12 and KYKTSL for ZC3H13 (Fig. 1A, greyshaded sequence). The genes encoding ZC3H12 and ZC3H13 are located next to each other on chromosome 5 of T. brucei TREU927; the ZC3H13 gene is the first ORF in a polycistronic transcription unit as indicated by both deep sequencing [10] and histone modification patterns [11] (Fig. 1B). ZC3H12 is conserved in all Kinetoplastid parasite genomes sequenced to date, while ZC3H13 is present in all salivarian trypanosomes (T. brucei, T. congolense and T. vivax). Messenger RNAs encoding ZC3H12 and ZC3H13 are present at similar levels in both BS and PC trypanosomes (Fig. 1B) [12]; the half-lives (12–18 min) and abundances (1–2 mRNAs per cell) are similar to those for most trypanosome open reading frames (ORFs) [13]. The
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Fig. 1. ZC3H12 and ZC3H13 protein properties. (A) Above: Cartoon of T. brucei ZC3H12 and ZC3H13. The conserved motif with the CCCH domain is shown in black, and the remaining N-terminal conserved region in grey, with number of amino acid residues (aa) indicated. Below: The aligned N-terminal sequences, with double dots for identical residues and single dots for functional conservation: R/K (basic), F/Y (aromatic), S/T (aliphatic, hydroxyl group). The CCCH residues are white on black, and the conserved 6mer is underlain in grey. The arrows indicate the aromatic groups that are involved in base stacking in Tis11 proteins [22]. (B) Chromosomal context of ZC3H12 and ZC3H13. The gene map is shown at the top, with ORFs in black and the approximate extent of the mRNAs as open boxes. The arrows show the direction of transcription, and abbreviated gene numbers (e.g. Tb927.5.1570 is abbreviated to “1570”). Below are shown the map of histone H3 trimethylation at lysine 4, showing enrichment at the transcription start region [11], and the RNA sequencing read densities for PC and BS trypanosomes [12]. The figure is based on a screen shot from TritrypDB. (C) Localization of in situ V5-tagged TbZC3H12 in BS parasites. Nuclei and cytoplasm were separated by NP40 lysis and centrifugation, and the resulting fractions were analysed by Western blotting. Lysate equivalent to 2 × 107 cells was loaded for each fraction. T, total lysate; C, cytosol; N, Nuclei. The marker proteins are XRND (nucleus) and peroxiredoxin (cytosolic). All methods are described in the Supplementary Material. (D) Localization of inducibly expressed myc tagged TbZC3H13 in both BS and procyclic (PC) parasites. Experimental details as in (A). For each fraction, lysate equivalent to 5 × 106 cells was loaded on the gel. In PC cells, a cytoplasmic protein that migrates slightly slower than XRND cross-reacts with the anti-XRND antiserum. XRND is indicated by an arrowhead. (E) Phosphorylation of ZC3H12-myc. Lysates from myc- tagged TbZC3H12 bloodstream form cells were incubated at 30 ◦ C for 20 min with the indicated chemicals and enzymes. The phosphatase was -phosphatase. MG132 is a proteasome inhibitor and was added 30 min prior to extract preparation, as well as to the extraction buffer. EDTA and Vanadate are -phosphatase inhibitors. In situ V5-tagged protein gave similar results but the bands were fainter (not shown). (F) Dephosphorylation of ZC3H13-myc. Experimental details as in (E); a second gel from another experiment, with slightly better resolution, is shown beneath the first.
ZC3H12 mRNA had the expected size of 1.5 kb (Supplementary Fig. S1B); the ZC3H13 mRNA was usually not detectable (not shown). To examine the properties of ZC3H12, we inserted a sequence encoding a V5 tag at the 5 -end of the endogenous ORF: this should result in expression of approximately normal levels of mRNA [14] (see Supplementary Material for methods). BS cells were obtained. Using 1 × 107 parasites, we detected the expected band migrating
at 20 kDa, and an additional band migrating slightly slower (Fig. 1C and Supplementary Fig. S1B), but with smaller cell numbers, the protein could not be detected. In contrast, in situ V5-tagged UBP2 – estimated to be present at around 6 × 106 molecules per BS cell – was previously readily detected using 1 × 105 cells [15]. This comparison suggests that ZC3H12 is much less abundant than UBP2 in BS trypanosomes. Heat shock for 1 h at 41 ◦ C, and cell densities
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above 1.8 × 106 cells/ml did not alter V5-ZC3H12 protein levels (not shown). We did not make PC cells that stably expressed V5-ZC3H12. All attempts to integrate either tagging or knockout plasmids at the ZC3H13 locus yielded no live trypanosomes. This was not a consequence of the location of this ORF at the beginning of a transcription unit since integrating a V5-tagging plasmid into the neighbouring locus Tb927.5.1590 was unproblematic (not shown). We generated BS cells that inducibly expressed ZC3H12 or ZC3H13 with C-terminal myc tags, and also with C-terminal tandem affinity purification tags, with no effect of expression on growth. Sub-cellular fractionation of BS cells showed that tagged ZC3H12 and ZC3H13 were predominantly in the cytoplasm, together with the cytosolic control, peroxiredoxin [16] (Fig. 1C and D); XRND served as a nuclear marker [17]. To find out whether the two bands of ZC3H12 were caused by phosphorylation, we reacted lysates from cells expressing myc-tagged proteins with lambda phosphatase. The two bands (Fig. 1E, lanes 1–5) were not affected by incubation of the lysates with EDTA (lane 3). Addition of a proteasome inhibitor 30 min prior to lysis, and in the lysis buffer, caused only a marginal increase in the upper band, while the lower one remained (Fig. 1E, lane 1). This result, together with the fact that both N-terminally and C-terminally tagged ZC3H12 appeared as two bands of similar mobility, ruled out proteolysis in the lysates as the source of the lower band. In contrast, addition of phosphatase almost completely eliminated the upper band (Fig. 1E, lane 6); this was prevented if the phosphatase was inhibited by EDTA and Sodium Orthovanadate (Na3 VO4 ) (Fig. 1E, lanes 4 and 5). These results indicate that the upper ZC3H12 band is a phosphorylated form. Scans for possible phosphorylation sites using default parameters of NetPhos 2.0 [18] detected multiple potential phosphorylation sites. ZC3H13 was already known to be phosphorylated, since it was found in the T. brucei bloodstream-form phosphoproteome [19] and correspondingly, the myc-tagged protein showed two very closely spaced bands on the Western blot (Fig. 1F). The upper band was lost after digestion by lambda phosphatase (Fig. 1F, lane 6), confirming that the tagged protein is phosphorylated. The phosphorylation of these two proteins is interesting because the activities of TTP, BRF-1 and BRF-2 are regulated by phosphorylation [2]. To find out whether either protein was important for trypanosome growth, we used RNA interference and gene knock-out. RNAi against ZC3H12 sometimes caused a slight and transient growth defect, but this occurred only in freshly isolated clones in which dsRNA was produced from opposing T7 promoters (Supplementary Fig. S1A and B). The same result was obtained in a recently published high-throughput RNAi screen [20]: a significant decrease in the competitiveness of cells with ZC3H12 RNAi was seen after 3 days of RNAi induction, but the cells had recovered within 6 days; and there was no effect in procyclics. Indeed, we were able to obtain bloodstream forms completely lacking ZC3H12: even after a single round of transfection, we accidentally obtained cells that had completely lost the gene (Fig. 2A). There was no growth defect (Fig. 2A and B, compare column 1 with columns 6–8). In the published high-throughput RNAi screen, RNAi against ZC3H13 caused a mild growth defect in BS trypanosomes but had no effect on competitiveness of differentiating or PC forms [20]. We saw transient effects in some BS clones when using dsRNA transcribed from opposing T7 promoters (Supplementary Fig. S1C). Using a stem-loop RNAi construct, three out of four clones showed very mild growth defects, with doubling-time increases of between 3% and 12% (Fig. 2B, columns 2–5). Upon further cultivation, the clones grew faster and the RNAi effect was lost (not shown). Finally, to check for functional redundancy, RNAi against ZC3H13 was done in the zc3h12 knockout cell line. There was ∼80% RNA knock down after 48 h (not shown), and the ZC3H13 RNAi resulted
in extremely mild growth inhibition (Fig. 2B, columns 7 and 8). A similar result was obtained by RNAi targeting both mRNAs simultaneously (Fig. 2B, columns 10 and 11). Overall the results indicated that ZC3H13 expression might give a slight growth advantage in BS forms. Such equivocal RNAi results are impossible to interpret, because a small amount of residual gene product may be sufficient for normal growth. Expression of tagged versions of either protein in PC trypanosomes was much more problematic than for BS forms. We obtained only one, slow-growing PC line expressing ZC3H13-myc and no PC line for ZC3H12-myc. We also tried expressing each protein with a tandem affinity purification (TAP) tag bearing IgG binding domains and a calmodulin binding peptide. BS lines could be made, but no PC cells expressing ZC3H12-TAP were obtained. One PC line expressing ZC3H13-TAP grew slowly in the absence of tetracycline; tetracycline addition reproducibly caused further growth inhibition (Fig. 2C and Supplementary Fig. 2C and D). We therefore suspect that expression of tagged versions of either protein in PC trypanosomes may be toxic. We attempted to identify RNA targets of ZC3H12 or ZC3H13 by pull-down of the TAP-tagged proteins [21], but the yields were too low for further analysis. Since a single BRF-2 (TIS11d) CCCH domain interacts with only 4 bases of an AU-rich element [22], ZC3H12 and ZC3H13 would probably need to homo- or hetero-dimerize in order to bind RNA specifically. To look for partner proteins of ZC3H12 and ZC3H13, we performed tandem affinity purification of both TAP-tagged proteins, followed by mass spectrometry of the entire preparation. To determine the specificity of the detected interactions, we compared the results with those obtained from 22 other pull-downs, from our own and other laboratories; these included a TAP-only control, precipitation with human serum, and anti-Myc-tag immunoprecipitation using cells with no myc tag. The list of “frequent potential contaminants” is supplied as Supplementary Table S1. ZC3H12 and ZC3H13 did not detectably co-purify, ruling out formation of a stable dimer. Various other partners were found for both proteins but none has so far been verified. ZC3H13-TAP copurified with a hypothetical SET-domain protein (Tb927.10.8060, 18 different peptides identified with >95% confidence) and various ribosomal proteins. The most interesting potential interaction partner of ZC3H12 was the exoribonuclease XRNA (Supplementary Fig. S3 and Table S2). XRNA is the trypanosome equivalent of yeast and human Xrn1, and is, like Xrn1, important in mRNA degradation [17]. Mammalian TTP is thought to be involved in recruitment of Xrn1 to unstable mRNAs in mammalian cells, and both TTP and BRF-1 co-localize with Xrn1 in processing bodies, which are likely loci of RNA degradation [2]. Unfortunately, the instability of tagged XRNA prevented us from confirming this interaction by reciprocal immunoprecipitation, so the validity of the interaction remains uncertain. Finally, we speculated that artificial “tethering” of ZC3H12 to an mRNA might result in recruitment of XRNA and mRNA destabilization. We therefore created a plasmid that encoded a ZC3H12 fusion protein bearing an N-terminal lambda-N peptide and a Cterminal myc tag (not shown). This was expressed in cells that were expressing a chloramphenicol acetyltransferase reporter mRNA that specifically binds to the lambda-N peptide via recognition sequences (B-boxes) in the 3 untranslated region [23]. Expression of lambda-N-ZC3H12-myc was easily detected; it caused a slight (less than 20%) increase in CAT activity (not shown), which is similar to the effects seen after expression of lambda-N-GFP [23]. Thus the tethered ZC3H12 had no significant effect on reporter expression. In conclusion, ZC3H12 and ZC3H13 are cytoplasmic, phosphorylated zinc finger proteins with similar N-termini but divergent C-termini. Their roles in trypanosomes remain unclear. Any roles in BS cells may be redundant with other proteins. It is difficult to tell whether the minor (and usually transient) effects that we saw
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Fig. 2. Effects of alterations in ZC3H12 and ZC3H13 expression. (A) BS cells lacking ZC3H12 are viable. Cumulative growth curves are shown for wild-type cells, cells with one drug-resistance marker (puromycin, “SKO”), and cells with two resistance markers (“DKO”). The Southern blots to the right (grey background) are of restriction digested genomic DNA, with detection of bands of the expected sizes including puromycin (PURO) and blasticidin (BLA) resistance cassettes. The ethidium bromide-stained gel below shows bands from PCR using genomic DNA as template, and primers for the ZC3H12 open reading frame (ORF) (lanes 1–9) or, as positive control, the ZC3H13 5’-UTR (lanes 10–12). The results for cells with only the puromycin resistance cassette (so called “SKO”) show that these cells already had no copy of ZC3H12. The BLA resistance cassette in “DKO” cells presumably merely replaced a copy of “PURO”. (B) Division times of cells with various alterations in ZC3H12 and ZC3H13 expression. Each line was measured once but independent replicate lines were present in all cases. The grey bar in lane 6 is the “SKO” line. For details see Supplementary Fig. S1. (C) Expression of TAP-tagged ZC3H13 inhibits trypanosome growth. Growth of cells expressing tet repressor alone, or tetracycline-inducible ZC3H13-TAP, in the presence (filled symbols) or absence (open symbols) of tetracycline. The division times are indicated next to the lines. Additional growth curves for cells inducibly expressing tagged ZC3H13 are in Supplementary Fig. S2. The lower panel illustrates expression of ZC3H13-TAP during the experiment. The TAP tag was detected using an antibody that binds to the IgG-binding domain. Some “leaky” expression was detected in the absence of tetracycline, which may explain the slow growth rate without tetracycline.
after RNAi or knock-out are biologically significant or due to clonal variation. It is however important to note that even mild growth defects can lead to inviability in the wild: for example, if ZC3H12 wild-type and knock-out bloodstream-form cultures were equally mixed, then allowed to grow, the knock-out would comprise less than 10% of the cell population after only 2 weeks. The conservation of ZC3H12 throughout Kinetoplastids, and of ZC3H13 in all Salivaria, does suggest that both proteins might confer a competitive advantage at some stage of the life cycle. It is possible, though, that their main role is in natural hosts or non-culturable life-cycle stages. Acknowledgements We thank Mark Carrington for plasmids and Luise KrauthSiegel for antibodies. BO was supported by a grant from the Deutsches Akademisches Austauschdienst. MS did the initial RNAi
experiments; CH did the tethering and made numerous attempts at the ZC3H13 knock-out. BO did all the other experiments and also assisted CC in writing the paper. We thank Dr Pegine Walrad (University of Edinburgh) and Dr Richard Burchmore (University of Glasgow) for sharing mass spectrometry data for the frequent contaminant Table. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.molbiopara.2012.02.006. References [1] Fernández-Moya S, Estévez A. Posttranscriptional control and the role of RNAbinding proteins in gene regulation in trypanosomatid protozoan parasites. WIREs RNA 2010;1:34–46.
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[2] Sanduja S, Blanco F, Dixon D. The roles of TTP and BRF proteins in regulated mRNA decay. WIREs RNA 2010;2:42–57. [3] Kramer S, Kimblin N, Carrington M. Genome-wide in silico screen for CCCHtype zinc finger proteins of Trypanosoma brucei, Trypanosoma cruzi and Leishmania major. BMC Genomics 2010;11:283. [4] Kramer S, Carrington M. Trans-acting proteins regulating mRNA maturation, stability and translation in trypanosomatids. Trends Parasitol 2011;27:23–30. [5] Hendriks EF, Robinson DR, Hinkins M, Matthews KR. A novel CCCH protein which modulates differentiation of Trypanosoma brucei to its procyclic form. EMBO J 2001;20:6700–11. [6] Hendriks EF, Matthews KR. Disruption of the developmental programme of Trypanosoma brucei by genetic ablation of TbZFP1, a differentiation-enriched CCCH protein. Mol Microbiol 2005;57:706–16. [7] Walrad P, Paterou A, Acosta-Serrano A, Matthews K. Differential trypanosome surface coat regulation by a CCCH protein that co-associates with procyclin mRNA cis-elements. PLoS Pathog 2009;5:e1000317. [8] Ling A, Trotter J, Hendriks E. A zinc finger protein, TbZC3H20, stabilises two developmentally regulated mRNAs in trypanosomes. J Biol Chem 2011;286:20152–62. [9] Benz C, Mulindwa J, Ouna B, Clayton C. The Trypanosoma brucei zinc finger protein ZC3H18 is involved in differentiation. Mol Biochem Parasitol 2011;177:148–51. [10] Kolev N, Franklin J, Carmi S, Shi H, Michaeli S, Tschudi C. The transcriptome of the human pathogen Trypanosoma brucei at single-nucleotide resolution. PLoS Pathog 2010;6:e1001090. [11] Siegel T, Hekstra D, Kemp L, Figueiredo L, Lowell J, Fenyo D, et al. Four histone variants mark the boundaries of polycistronic transcription units in Trypanosoma brucei. Genes Dev 2009;23:1063–76. [12] Siegel T, Hekstra D, Wang X, Dewell S, Cross G. Genome-wide analysis of mRNA abundance in two life-cycle stages of Trypanosoma brucei and identification of splicing and polyadenylation sites. Nucleic Acids Res 2010;38: 4946–57.
[13] Manful T, Fadda A, Clayton C. The role of the 5’-3’ exoribonuclease XRNA in transcriptome-wide mRNA degradation. RNA 2011;17:2039–47. [14] Shen S, Arhin GK, Ullu E, Tschudi C. In vivo epitope tagging of Trypanosoma brucei genes using a one step PCR-based strategy. Mol Biochem Parasitol 2001;113:171–3. [15] Hartmann C, Benz C, Brems S, Ellis L, Luu V-D, Stewart M, et al. The small trypanosome RNA-binding proteins TbUBP1 and TbUBP2 influence expression of F box protein mRNAs in bloodstream trypanosomes. Eukaryotic Cell 2007;6:1964–78. [16] Ceylan S, Seidel V, Ziebart N, Berndt C, Dirdjaja N, Krauth-Siegel R. The dithiol glutaredoxins of African trypanosomes have distinct roles and are closely linked to the unique trypanothione metabolism. J Biol Chem 2010;285:35224–37. [17] Li C-H, Irmer H, Gudjonsdottir-Planck D, Freese S, Salm H, Haile S, et al. Roles of a Trypanosoma brucei 5’–3’ exoribonuclease homologue in mRNA degradation. RNA 2006;12:2171–86. [18] Blom N, Gammeltoft S, Brunak S. Sequence- and structure-based prediction of eukaryotic protein phosphorylation sites. J Mol Biol 1999;294:1351–62. [19] Nett I, Martin D, Miranda-Saavedra D, Lamont D, Barber J, Mehlert A, et al. The phosphoproteome of bloodstream form Trypanosoma brucei, causative agent of African sleeping sickness. Mol Cell Proteomics 2009;8:1527–38. [20] Alsford S, Turner D, Obado S, Sanchez-Flores A, Glover L, Berriman M, et al. High throughput phenotyping using parallel sequencing of RNA interference targets in the African trypanosome. Genome Res 2011;21:915–24. [21] Archer SK, van Luu D, de Queiroz R, Brems S, Clayton CE. Trypanosoma brucei PUF9 regulates mRNAs for proteins involved in replicative processes over the cell cycle. PLoS Pathog 2009;5:e1000565. [22] Hudson B, Martinez-Yamout M, Dyson H, Wright P. Recognition of the mRNA AU-rich element by the zinc finger domain of TIS11d. Nat Struct Mol Biol 2004;11:257–64. [23] Delhi P, Queiroz R, Inchaustegui D, Carrington M, Clayton C. Is there a classical nonsense-mediated decay pathway in trypanosomes? PLoS One 2011;6:e25112.
Contents lists available at SciVerse ScienceDirect
Molecular & Biochemical Parasitology
Short communication
The Trypanosoma brucei CCCH zinc finger proteins ZC3H12 and ZC3H13 Benard Aswani Ouna, Mhairi Stewart 1 , Claudia Helbig, Christine Clayton ∗ Zentrum fur Molekular Biologie Heidelberg, ZMBH-DKFZ Alliance, Im Neuenheimerfeld 282, Heidelberg 69120, Germany
a r t i c l e
i n f o
Article history: Received 28 April 2011 Received in revised form 8 February 2012 Accepted 10 February 2012 Available online 17 February 2012 Keywords: Trypanosoma CCCH zinc finger Xrn1 TTP BRF1
a b s t r a c t CCCH-type zinc finger proteins have various roles in RNA metabolism. We here analysed the functional relevance of two such proteins from Trypanosoma brucei, TbZC3H12 and TbZC3H13. Each protein has a single CCCH motif very similar to those seen in metazoan proteins that regulate mRNA degradation. TbZC3H12 is expressed in bloodstream form parasites at low levels. It is phosphorylated, cytosolic and not required for normal growth of cultured bloodstream trypanosomes. RNA interference targeting TbZC3H13, on a TbZC3H12 null background, also had no effect on bloodstream trypanosome growth, but over-expression of tagged TbZC3H13 inhibited procyclic trypanosome growth. Tandem affinity purification of both proteins revealed various interesting potential interactions; specificity was assessed against a list of proteins that were found in 24 other pull-down experiments, which is provided. The conservation of TbZC3H12 in all kinetoplastids, and TbZC3H13 in Salivaria, suggests that the two proteins may be required for optimal growth at some stage of the parasite life-cycle. © 2012 Elsevier B.V. All rights reserved.
1. Introduction Trypanosoma brucei multiplies in mammalian blood and tissue fluids as the bloodstream form (BS) and in the midgut of Tsetse flies as the procyclic form (PC). Since most trypanosome genes are constitutively transcribed, rates of mRNA turnover and translation are important in controlling trypanosome gene expression [1]. The control of mRNA turnover in eukaryotes is often mediated by proteins which bind the 3 -untranslated region of a target mRNA. One such protein class, the Tis11 family, contains two finger domains of the type C-x-C-x-C-x3 -H [2], separated by a short linker and immediately preceded by a consensus sequence, R/K-Y-K-T-E-L. Well-studied examples of mammalian Tis11-family proteins include tristetraprolin (TTP) and Butyrate Response Factors BRF1 and BRF2; each of these binds to AU-rich elements (AREs) in the 3 -UTRs of mRNAs and induces their decay [2]. Forty-nine genes encoding CCCH zinc finger proteins are found in the T. brucei genome. A few have known functions in splicing and mRNA export [3], and so far, four have been implicated in post transcriptional gene regulation [4]. TbZFP1, TbZFP2 and TbZFP3 are all required for normal differentiation [5–7]; TbZFP3 is required for normal patterns of translation of the major surface proteins of
∗ Corresponding author. Tel.: +49 6221 546876. E-mail address: [email protected] (C. Clayton). 1 Current address: Wellcome Trust Centre for Molecular Parasitology, Institute of Infection and Immunity, MVLS, GBRC, 120 University Place, University of Glasgow, Glasgow G12 8TA, United Kingdom. 0166-6851/$ – see front matter © 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.molbiopara.2012.02.006
procyclic forms. Over-expression of TbZFP2 in the procyclic forms caused abnormal remodelling of the cytoskeleton [5]. Meanwhile TbZC3H20 is required for growth of procyclic forms, binding to and stabilizing at least two developmentally regulated mRNAs [8]. TbZC3H18 has been implicated in control of differentiation but the mechanism is unknown [9]. In this paper, we investigated the functions of two more T. brucei CCCH zinc finger proteins, TbZC3H12 (Tb927.5.1570) and TbZC3H13 (Tb927.5.1580) (henceforth written without the Tb prefix). Each has a single CCCH motif starting at residue 20 near the N-terminus. The proteins are 50% identical in the first 49 amino acid residues, and then diverge completely (Fig. 1A). ZC3H13 (60 kDa) is 400 residues longer than ZC3H12 (18.8 kDa). Within the core CCCH motif, the key aromatic residues that are involved in base stacking are conserved (Fig. 1A sequence, arrowheads). We were particularly interested in these two proteins because each has a sequence resembling the Tis11 consensus immediately preceding the CCCH motif: KYRTTL for ZC3H12 and KYKTSL for ZC3H13 (Fig. 1A, greyshaded sequence). The genes encoding ZC3H12 and ZC3H13 are located next to each other on chromosome 5 of T. brucei TREU927; the ZC3H13 gene is the first ORF in a polycistronic transcription unit as indicated by both deep sequencing [10] and histone modification patterns [11] (Fig. 1B). ZC3H12 is conserved in all Kinetoplastid parasite genomes sequenced to date, while ZC3H13 is present in all salivarian trypanosomes (T. brucei, T. congolense and T. vivax). Messenger RNAs encoding ZC3H12 and ZC3H13 are present at similar levels in both BS and PC trypanosomes (Fig. 1B) [12]; the half-lives (12–18 min) and abundances (1–2 mRNAs per cell) are similar to those for most trypanosome open reading frames (ORFs) [13]. The
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Fig. 1. ZC3H12 and ZC3H13 protein properties. (A) Above: Cartoon of T. brucei ZC3H12 and ZC3H13. The conserved motif with the CCCH domain is shown in black, and the remaining N-terminal conserved region in grey, with number of amino acid residues (aa) indicated. Below: The aligned N-terminal sequences, with double dots for identical residues and single dots for functional conservation: R/K (basic), F/Y (aromatic), S/T (aliphatic, hydroxyl group). The CCCH residues are white on black, and the conserved 6mer is underlain in grey. The arrows indicate the aromatic groups that are involved in base stacking in Tis11 proteins [22]. (B) Chromosomal context of ZC3H12 and ZC3H13. The gene map is shown at the top, with ORFs in black and the approximate extent of the mRNAs as open boxes. The arrows show the direction of transcription, and abbreviated gene numbers (e.g. Tb927.5.1570 is abbreviated to “1570”). Below are shown the map of histone H3 trimethylation at lysine 4, showing enrichment at the transcription start region [11], and the RNA sequencing read densities for PC and BS trypanosomes [12]. The figure is based on a screen shot from TritrypDB. (C) Localization of in situ V5-tagged TbZC3H12 in BS parasites. Nuclei and cytoplasm were separated by NP40 lysis and centrifugation, and the resulting fractions were analysed by Western blotting. Lysate equivalent to 2 × 107 cells was loaded for each fraction. T, total lysate; C, cytosol; N, Nuclei. The marker proteins are XRND (nucleus) and peroxiredoxin (cytosolic). All methods are described in the Supplementary Material. (D) Localization of inducibly expressed myc tagged TbZC3H13 in both BS and procyclic (PC) parasites. Experimental details as in (A). For each fraction, lysate equivalent to 5 × 106 cells was loaded on the gel. In PC cells, a cytoplasmic protein that migrates slightly slower than XRND cross-reacts with the anti-XRND antiserum. XRND is indicated by an arrowhead. (E) Phosphorylation of ZC3H12-myc. Lysates from myc- tagged TbZC3H12 bloodstream form cells were incubated at 30 ◦ C for 20 min with the indicated chemicals and enzymes. The phosphatase was -phosphatase. MG132 is a proteasome inhibitor and was added 30 min prior to extract preparation, as well as to the extraction buffer. EDTA and Vanadate are -phosphatase inhibitors. In situ V5-tagged protein gave similar results but the bands were fainter (not shown). (F) Dephosphorylation of ZC3H13-myc. Experimental details as in (E); a second gel from another experiment, with slightly better resolution, is shown beneath the first.
ZC3H12 mRNA had the expected size of 1.5 kb (Supplementary Fig. S1B); the ZC3H13 mRNA was usually not detectable (not shown). To examine the properties of ZC3H12, we inserted a sequence encoding a V5 tag at the 5 -end of the endogenous ORF: this should result in expression of approximately normal levels of mRNA [14] (see Supplementary Material for methods). BS cells were obtained. Using 1 × 107 parasites, we detected the expected band migrating
at 20 kDa, and an additional band migrating slightly slower (Fig. 1C and Supplementary Fig. S1B), but with smaller cell numbers, the protein could not be detected. In contrast, in situ V5-tagged UBP2 – estimated to be present at around 6 × 106 molecules per BS cell – was previously readily detected using 1 × 105 cells [15]. This comparison suggests that ZC3H12 is much less abundant than UBP2 in BS trypanosomes. Heat shock for 1 h at 41 ◦ C, and cell densities
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above 1.8 × 106 cells/ml did not alter V5-ZC3H12 protein levels (not shown). We did not make PC cells that stably expressed V5-ZC3H12. All attempts to integrate either tagging or knockout plasmids at the ZC3H13 locus yielded no live trypanosomes. This was not a consequence of the location of this ORF at the beginning of a transcription unit since integrating a V5-tagging plasmid into the neighbouring locus Tb927.5.1590 was unproblematic (not shown). We generated BS cells that inducibly expressed ZC3H12 or ZC3H13 with C-terminal myc tags, and also with C-terminal tandem affinity purification tags, with no effect of expression on growth. Sub-cellular fractionation of BS cells showed that tagged ZC3H12 and ZC3H13 were predominantly in the cytoplasm, together with the cytosolic control, peroxiredoxin [16] (Fig. 1C and D); XRND served as a nuclear marker [17]. To find out whether the two bands of ZC3H12 were caused by phosphorylation, we reacted lysates from cells expressing myc-tagged proteins with lambda phosphatase. The two bands (Fig. 1E, lanes 1–5) were not affected by incubation of the lysates with EDTA (lane 3). Addition of a proteasome inhibitor 30 min prior to lysis, and in the lysis buffer, caused only a marginal increase in the upper band, while the lower one remained (Fig. 1E, lane 1). This result, together with the fact that both N-terminally and C-terminally tagged ZC3H12 appeared as two bands of similar mobility, ruled out proteolysis in the lysates as the source of the lower band. In contrast, addition of phosphatase almost completely eliminated the upper band (Fig. 1E, lane 6); this was prevented if the phosphatase was inhibited by EDTA and Sodium Orthovanadate (Na3 VO4 ) (Fig. 1E, lanes 4 and 5). These results indicate that the upper ZC3H12 band is a phosphorylated form. Scans for possible phosphorylation sites using default parameters of NetPhos 2.0 [18] detected multiple potential phosphorylation sites. ZC3H13 was already known to be phosphorylated, since it was found in the T. brucei bloodstream-form phosphoproteome [19] and correspondingly, the myc-tagged protein showed two very closely spaced bands on the Western blot (Fig. 1F). The upper band was lost after digestion by lambda phosphatase (Fig. 1F, lane 6), confirming that the tagged protein is phosphorylated. The phosphorylation of these two proteins is interesting because the activities of TTP, BRF-1 and BRF-2 are regulated by phosphorylation [2]. To find out whether either protein was important for trypanosome growth, we used RNA interference and gene knock-out. RNAi against ZC3H12 sometimes caused a slight and transient growth defect, but this occurred only in freshly isolated clones in which dsRNA was produced from opposing T7 promoters (Supplementary Fig. S1A and B). The same result was obtained in a recently published high-throughput RNAi screen [20]: a significant decrease in the competitiveness of cells with ZC3H12 RNAi was seen after 3 days of RNAi induction, but the cells had recovered within 6 days; and there was no effect in procyclics. Indeed, we were able to obtain bloodstream forms completely lacking ZC3H12: even after a single round of transfection, we accidentally obtained cells that had completely lost the gene (Fig. 2A). There was no growth defect (Fig. 2A and B, compare column 1 with columns 6–8). In the published high-throughput RNAi screen, RNAi against ZC3H13 caused a mild growth defect in BS trypanosomes but had no effect on competitiveness of differentiating or PC forms [20]. We saw transient effects in some BS clones when using dsRNA transcribed from opposing T7 promoters (Supplementary Fig. S1C). Using a stem-loop RNAi construct, three out of four clones showed very mild growth defects, with doubling-time increases of between 3% and 12% (Fig. 2B, columns 2–5). Upon further cultivation, the clones grew faster and the RNAi effect was lost (not shown). Finally, to check for functional redundancy, RNAi against ZC3H13 was done in the zc3h12 knockout cell line. There was ∼80% RNA knock down after 48 h (not shown), and the ZC3H13 RNAi resulted
in extremely mild growth inhibition (Fig. 2B, columns 7 and 8). A similar result was obtained by RNAi targeting both mRNAs simultaneously (Fig. 2B, columns 10 and 11). Overall the results indicated that ZC3H13 expression might give a slight growth advantage in BS forms. Such equivocal RNAi results are impossible to interpret, because a small amount of residual gene product may be sufficient for normal growth. Expression of tagged versions of either protein in PC trypanosomes was much more problematic than for BS forms. We obtained only one, slow-growing PC line expressing ZC3H13-myc and no PC line for ZC3H12-myc. We also tried expressing each protein with a tandem affinity purification (TAP) tag bearing IgG binding domains and a calmodulin binding peptide. BS lines could be made, but no PC cells expressing ZC3H12-TAP were obtained. One PC line expressing ZC3H13-TAP grew slowly in the absence of tetracycline; tetracycline addition reproducibly caused further growth inhibition (Fig. 2C and Supplementary Fig. 2C and D). We therefore suspect that expression of tagged versions of either protein in PC trypanosomes may be toxic. We attempted to identify RNA targets of ZC3H12 or ZC3H13 by pull-down of the TAP-tagged proteins [21], but the yields were too low for further analysis. Since a single BRF-2 (TIS11d) CCCH domain interacts with only 4 bases of an AU-rich element [22], ZC3H12 and ZC3H13 would probably need to homo- or hetero-dimerize in order to bind RNA specifically. To look for partner proteins of ZC3H12 and ZC3H13, we performed tandem affinity purification of both TAP-tagged proteins, followed by mass spectrometry of the entire preparation. To determine the specificity of the detected interactions, we compared the results with those obtained from 22 other pull-downs, from our own and other laboratories; these included a TAP-only control, precipitation with human serum, and anti-Myc-tag immunoprecipitation using cells with no myc tag. The list of “frequent potential contaminants” is supplied as Supplementary Table S1. ZC3H12 and ZC3H13 did not detectably co-purify, ruling out formation of a stable dimer. Various other partners were found for both proteins but none has so far been verified. ZC3H13-TAP copurified with a hypothetical SET-domain protein (Tb927.10.8060, 18 different peptides identified with >95% confidence) and various ribosomal proteins. The most interesting potential interaction partner of ZC3H12 was the exoribonuclease XRNA (Supplementary Fig. S3 and Table S2). XRNA is the trypanosome equivalent of yeast and human Xrn1, and is, like Xrn1, important in mRNA degradation [17]. Mammalian TTP is thought to be involved in recruitment of Xrn1 to unstable mRNAs in mammalian cells, and both TTP and BRF-1 co-localize with Xrn1 in processing bodies, which are likely loci of RNA degradation [2]. Unfortunately, the instability of tagged XRNA prevented us from confirming this interaction by reciprocal immunoprecipitation, so the validity of the interaction remains uncertain. Finally, we speculated that artificial “tethering” of ZC3H12 to an mRNA might result in recruitment of XRNA and mRNA destabilization. We therefore created a plasmid that encoded a ZC3H12 fusion protein bearing an N-terminal lambda-N peptide and a Cterminal myc tag (not shown). This was expressed in cells that were expressing a chloramphenicol acetyltransferase reporter mRNA that specifically binds to the lambda-N peptide via recognition sequences (B-boxes) in the 3 untranslated region [23]. Expression of lambda-N-ZC3H12-myc was easily detected; it caused a slight (less than 20%) increase in CAT activity (not shown), which is similar to the effects seen after expression of lambda-N-GFP [23]. Thus the tethered ZC3H12 had no significant effect on reporter expression. In conclusion, ZC3H12 and ZC3H13 are cytoplasmic, phosphorylated zinc finger proteins with similar N-termini but divergent C-termini. Their roles in trypanosomes remain unclear. Any roles in BS cells may be redundant with other proteins. It is difficult to tell whether the minor (and usually transient) effects that we saw
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Fig. 2. Effects of alterations in ZC3H12 and ZC3H13 expression. (A) BS cells lacking ZC3H12 are viable. Cumulative growth curves are shown for wild-type cells, cells with one drug-resistance marker (puromycin, “SKO”), and cells with two resistance markers (“DKO”). The Southern blots to the right (grey background) are of restriction digested genomic DNA, with detection of bands of the expected sizes including puromycin (PURO) and blasticidin (BLA) resistance cassettes. The ethidium bromide-stained gel below shows bands from PCR using genomic DNA as template, and primers for the ZC3H12 open reading frame (ORF) (lanes 1–9) or, as positive control, the ZC3H13 5’-UTR (lanes 10–12). The results for cells with only the puromycin resistance cassette (so called “SKO”) show that these cells already had no copy of ZC3H12. The BLA resistance cassette in “DKO” cells presumably merely replaced a copy of “PURO”. (B) Division times of cells with various alterations in ZC3H12 and ZC3H13 expression. Each line was measured once but independent replicate lines were present in all cases. The grey bar in lane 6 is the “SKO” line. For details see Supplementary Fig. S1. (C) Expression of TAP-tagged ZC3H13 inhibits trypanosome growth. Growth of cells expressing tet repressor alone, or tetracycline-inducible ZC3H13-TAP, in the presence (filled symbols) or absence (open symbols) of tetracycline. The division times are indicated next to the lines. Additional growth curves for cells inducibly expressing tagged ZC3H13 are in Supplementary Fig. S2. The lower panel illustrates expression of ZC3H13-TAP during the experiment. The TAP tag was detected using an antibody that binds to the IgG-binding domain. Some “leaky” expression was detected in the absence of tetracycline, which may explain the slow growth rate without tetracycline.
after RNAi or knock-out are biologically significant or due to clonal variation. It is however important to note that even mild growth defects can lead to inviability in the wild: for example, if ZC3H12 wild-type and knock-out bloodstream-form cultures were equally mixed, then allowed to grow, the knock-out would comprise less than 10% of the cell population after only 2 weeks. The conservation of ZC3H12 throughout Kinetoplastids, and of ZC3H13 in all Salivaria, does suggest that both proteins might confer a competitive advantage at some stage of the life cycle. It is possible, though, that their main role is in natural hosts or non-culturable life-cycle stages. Acknowledgements We thank Mark Carrington for plasmids and Luise KrauthSiegel for antibodies. BO was supported by a grant from the Deutsches Akademisches Austauschdienst. MS did the initial RNAi
experiments; CH did the tethering and made numerous attempts at the ZC3H13 knock-out. BO did all the other experiments and also assisted CC in writing the paper. We thank Dr Pegine Walrad (University of Edinburgh) and Dr Richard Burchmore (University of Glasgow) for sharing mass spectrometry data for the frequent contaminant Table. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.molbiopara.2012.02.006. References [1] Fernández-Moya S, Estévez A. Posttranscriptional control and the role of RNAbinding proteins in gene regulation in trypanosomatid protozoan parasites. WIREs RNA 2010;1:34–46.
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