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Department of Epidemiology and Public Health,1 Department of Internal Medicine, Yale University School of Medicine, New Haven, Connecticut2
Received 1 November 2004/ Returned for modification 6 December 2004/ Accepted 9 December 2004
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ABSTRACT |
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AmpAmpCmpm3Um) at the 5' end of all mRNAs. The cap 4 becomes part of the mRNA through trans-splicing of a 39-nucleotide-long sequence donated by the spliced leader RNA. Although the cap 4 modifications are required for trans-splicing to occur, the underlying mechanism remains to be determined. We now describe an unconventional nuclear cap binding complex (CBC) in Trypanosoma brucei with an apparent molecular mass of 300 kDa and consisting of five protein components: the known CBC subunits CBP20 and importin-
and three novel proteins that are only present in organisms featuring a cap 4 structure and trans-splicing. Competitive binding studies are consistent with a specific interaction between the CBC and the cap 4 structure. Downregulation of several individual components of the T. brucei CBC by RNA interference demonstrated an essential function at an early step in trans-splicing. Thus, our studies are consistent with the CBC providing a mechanistic link between cap 4 modifications and trans-splicing. |
INTRODUCTION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Studies performed mainly in yeast and human cells have shown that the m7GpppN cap structure of nascent Pol II transcripts has key functions in various aspects of RNA processing and that these effects are mediated by two distinct cap binding complexes (CBCs) (17, 22, 39). The predominantly nuclear CBC is comprised of two subunits, termed cap binding proteins 20 (CBP20) (16) and 80 (CBP80) (17, 31). CBP20, in contrast to CBP80, is highly conserved from yeast to humans and contains an RNA binding motif (the RNP domain). Of note is that binding to capped RNA requires an association of the two subunits, with CBP20 directly contacting the cap and CBP80 ensuring high-affinity cap binding (6, 17, 28). Although the CBC subunits are not essential for viability in Saccharomyces cerevisiae (8), they play an active role in both splicing and RNA export (17, 19). CBC augments pre-mRNA processing by increasing the splicing efficiency of cap-proximal 5' introns. Furthermore, CBC positively affects the efficiency of 3'-end formation (12). In metazoa, where U-snRNP assembly has a cytoplasmic phase, CBC, in cooperation with an adaptor protein named PHAX (for phosphorylated adaptor for RNA export), is required for the nuclear export of U-snRNAs (32). In contrast, export of mRNA does not appear to require CBC, although it accompanies mRNA to the cytoplasm (15, 22, 47), where it is exchanged for eukaryotic initiation factor 4E, the cytoplasmic cap binding protein of eukaryotic translation initiation factor 4F (13).
The mRNA cap in T. brucei has the unusual feature of containing, in addition to 7-methylguanosine, four modified nucleotides making it by definition a cap 4 structure (m7
AmpAmpCmpm3Um) (3). Whereas this highly modified cap appears to be conserved throughout the evolution of the family Trypanosomatidae (34), there is at present no evidence that a similar cap structure exists outside this group of organisms. Although permeable cell studies showed that the hypermodified cap is essential for utilization of the SL RNA in trans-splicing (46) and genetic analysis in Leptomonas collosoma established that each of the four nucleotides of the cap 4 is essential for SL RNA function in trans-splicing (27), a specific role for the modified nucleotides has not been elucidated. Nevertheless, these observations reinforce the notion that cap structures have key functions in RNA metabolism. In this paper we have characterized the T. brucei nuclear CBC, which consists of five subunits and is essential for cell viability.
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MATERIALS AND METHODS |
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Protein purifications. Whole-cell and S100 extracts were prepared as described previously (10). For gel filtration chromatography, 500 µl of an S100 extract (the equivalent of about 500 ml of cells at a density of 6 x 106 cells/ml) was fractionated on a Superdex-200 column (Amersham Biosciences) as described elsewhere (10).
Tandem affinity purification (TAP) was carried out according to the methods described in reference 36. Purified protein samples were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) for silver staining with the SilverQuest kit (Invitrogen). Individual protein bands (with apparent molecular masses of 30, 55, 75, and 110 kDa) were cut out and digested with trypsin, and the resulting peptides were then subjected to nano-liquid chromatography and tandem mass spectrometry (MS) analysis at the Protein Chemistry Core Facility at Columbia University. The output file from this procedure was then used to search a T. brucei database. The following matches were predicted: TbCBP30 with the 30-kDa protein MS data with an expect value of 3.1 x 10–9; T. brucei importin- with the 55-kDa protein MS data with an expect value of 0.10; TbCBP66 with the 75-kDa protein MS data with an expect value of 0.43; TbCBP110 with the 110-kDa protein MS data with an expect value of 8.1 x 10–5.
Immunoprecipitations and affinity selection. The following antibodies were used: mouse monoclonal anti-BB2 antibodies were raised against 10 amino acids from the immunologically well-characterized major structural protein of the S. cerevisiae Ty1 virus-like particle in Keith Gull's laboratory (4); horseradish peroxidase-conjugated rat anti-HA antibodies (Roche); mouse monoclonal anti-FLAG antibodies (Sigma); mouse monoclonal anti-myc antibodies (a generous gift from Susan Baserga); and rabbit polyclonal anti-protein A antibodies (Sigma). Immunoprecipitations were performed as described elsewhere (41).
For m7GTP-Sepharose pull-down experiments, 15 µl of m7GTP-Sepharose beads (Amersham Biosciences) was washed three times in NET-2 buffer (150 mM NaCl, 50 mM Tris-HCl [pH 7.5], 0.05% NP-40) and then mixed with 150 µl of S100 extract, 150 µl of NET-2 buffer, and 33 µl of yeast tRNA (10 mg/ml). The mixture was rotated at 4°C for 30 min, then the beads were spun down and washed five times with NET-2 buffer, and the affinity-selected material was subjected to Western blot analysis.
EMSAs. Electrophoretic mobility shift assays (EMSAs) were carried out essentially as described previously (18). The binding reaction mixture contained 1.6 µg of yeast tRNA/µl, 1 U of RNasin (Roche)/µl, and 103 cpm of labeled SL RNA probe/µl (5.1 fmol/µl) in BB buffer (10 mM potassium phosphate [pH 8.0], 0.25 M KCl, 2 mM EDTA, and 5% glycerol). The total volume of the samples was 10 µl. Reaction mixtures were incubated at 28°C for 30 min and then on ice for 10 min. Next, each sample was mixed with 3 µl of sample buffer (5% Ficoll, 20% glycerol, and 0.05% bromophenol blue in BB buffer) and loaded onto a 5% polyacrylamide gel (40:1 acrylamide-bis-acrylamide). Gels were run at room temperature at 4 V/cm in 0.5x Tris-borate-EDTA for 6 h. Following drying, the gels were exposed to a phosphorimager screen, and data were acquired on a Cyclone PhosphorImager (Packard). Quantitation of bands was carried out using the Optiquant software (Packard).
TbCBC used in band shift assays (except for the results shown in Fig. 4, below) was purified from an S100 extract of cells expressing TAPmyc-CBP20 up to the TEV cleavage step. The concentration of TbCBC was determined by Western blot analysis, using as a standard a myc tag-containing protein (Positope; Invitrogen). The recombinant human CBC sample was a generous gift from Kristin Wilson and Richard Cerione, and its concentration was determined by SDS-PAGE followed by Coomassie blue staining, using bovine serum albumin as a standard.
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-32P]UTP (45). Labeled total RNA was separated on a 6% acrylamide-8 M urea gel, and the SL RNA was cut out from the gel and recovered by soaking the gel piece in water (25). Similarly, unlabeled competitor SL RNA was prepared using the permeable cell system without the addition of [-32P]UTP. The concentration of the unlabeled SL RNA was determined by Northern blotting using in vitro-transcribed SL RNA as a standard. For in vitro RNA synthesis, the T7 Ampliscribe kit (Epicentre) was used. To prepare unlabeled, uncapped RNA, XhoI-digested pBluescript II KS was used as a template to yield a 96-nt-long RNA. Unlabeled m7G-capped RNA was synthesized in the presence of 6 mM m7GpppG and 1.5 mM GTP.
To calculate the apparent disassociation constant (Kd), 5.1 fmol of labeled SL RNA/µl was used, and increasing amounts of CBCs were used in EMSAs. With Kaleidagraph software, the Kd was calculated by fitting the collected data as a function of protein concentration and using the equation 
= [protein]/([protein] + Kd), where is the fraction of bound RNA.
For competition assays, we used a concentration of CBCs that resulted in 10 to 15% of the labeled SL RNA forming an RNP. A total of 5.1 fmol of probe/µl was mixed with various amounts of cold competitor, and then 5.0 fmol of TbCBC/µl or 5.2 fmol of HsCBC/µl was added to assemble the EMSA reaction mixture. The mixtures were processed as described above. Using Kaleidagraph software, Kd1/2 was calculated by fitting the data to the equation Y = bottom + (top - bottom)/(1 + 10 exp(log [competitor] - log [Kd1/2]), where top and bottom are the Y values at the top and bottom plateau of the curve (http://www.curvefit.com).
For supershift analysis, 100 ng of antibody was added to the EMSA reaction mixture after the 10-min ice incubation step, and then the samples were kept on ice for another 30 min, which was followed by gel electrophoresis and data collection as described for the EMSA.
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RESULTS |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, and three novel proteins.
To get a handle on the protein component(s) associated with TbCBP20, we used the TAP method (see Materials and Methods). A modified TAP tag consisting of two protein A binding peptides, a TEV protease cleavage site, a triple myc tag, and a calmodulin binding peptide was inserted at the N terminus of TbCBP20 in a strain where the second allele was replaced by the BSR resistance marker. Since TbCBP20 is encoded by an essential gene (see below), this strategy ensured that the TAP-tagged protein was functional. We monitored TAP-CBP20 during the purification by Western blotting with anti-myc antibodies and verified complex integrity and its associated cap binding activity by gel shift assays (data not shown). In parallel, an equivalent amount of S100 extract from wild-type cells was processed to control for specificity. Purified samples were separated alongside on an SDS-PAGE gel and silver stained (Fig. 2C). Protein bands that were present in both the control and sample lanes were considered contaminants and were not pursued further. In addition to the band corresponding to CBP20, four proteins with approximate molecular masses of 35, 55, 75, and 110 kDa were specifically and consistently present. The four protein bands were excised from the gel and subjected to MS analysis, which led to the identification of the corresponding genes. The 55-kDa protein was tentatively identified as importin-, or karyopherin-, a bona fide component of the nuclear CBC (15), and was not pursued further in this study. The most interesting result from the MS data was the identification of three novel proteins associated with the TbCBC, namely, TbCBP30, TbCBP66, and TbCBP110 (see the supplemental material for further information). We named the proteins migrating with approximate molecular masses of 35 and 75 kDa in SDS-PAGE gels (Fig. 2C) TbCBP30 and TbCBP66, respectively, to reflect their predicted molecular masses. Most intriguingly, BLAST searches of all three proteins revealed that these proteins appeared to be restricted to members of the Trypanosomatidae family, i.e., homologs were only found in Leishmania major, Trypanosoma cruzi, Trypanosoma congolense, and Trypanosoma vivax, identifying proteins with unknown functions. Additional searches of domain and motif databases were surprisingly negative for TbCBP30 and TbCBP110 and thus failed to provide us with any clues to their function. In contrast, TbCBP66 was found to contain an unusual zinc finger motif (CCCH) present in a diverse range of RNA binding proteins and an RNA recognition motif (RRM) close to the N terminus (see the supplemental material for further information).
TbCBP30, TbCBP66, and TbCBP110 are components of the TbCBC. To verify that TbCBP30, TbCBP66, and TbCBP110 were indeed subunits of the 300-kDa CBC, we generated a series of cell lines expressing epitope-tagged versions of the three proteins and used a combination of m7G pull-down experiments, gel filtration chromatography, coimmunoprecipitations, EMSAs, and supershifts (see Materials and Methods). In a first set of experiments, S100 extracts from cells expressing either epitope-tagged CBP30, CBP66, or CBP110 were fractionated on a Superdex-200 gel filtration column in parallel with an S100 extract from cells expressing BB2-tagged CBP20 (Fig. 3A). Western blotting with the appropriate antibodies showed a similar fractionation profile for all four proteins, underscoring that TbCBP20, TbCBP30, TbCBP66, and TbCBP110 are part of an approximately 300-kDa complex. We further showed that fractions containing the epitope-tagged protein bound the SL RNA by using an EMSA (Fig. 3B). This result was corroborated by affinity selections with m7G-Sepharose, which revealed that CBP30, CBP66, and CBP110 were pulled down by the affinity matrix (Fig. 4B and data not shown). Next, we examined whether TbCBP30, TbCBP66, and TbCBP110 could be coimmunoprecipitated with TbCBP20. For this, we inserted an epitope tag at the N terminus of either CBP30, CBP66, or CBP110 in the background of cells expressing BB2-tagged CBP20. Whole-cell extracts of each cell line were then immunoprecipitated with anti-BB2 antibodies (CBP20) and subjected to Western blot assays (Fig. 4A). This showed that CBP30, CBP66, and CBP110 were coimmunoprecipitated with CBP20, whereas a control protein (CE1 [reference 42]) remained in the supernatant, indicating that each protein was in a complex with CBP20.
Since the MS data for CBP66 were not as convincing as those for CBP30 and CBP110 (see Materials and Methods), we felt it critical to add one additional experiment to confirm that CBP66 was indeed part of the complex binding the SL RNA. Thus, we performed supershift assays (Fig. 4C), which revealed a supershift with antibodies against epitopes present on CBP66 (lanes 3 and 4) but not with control antibodies (lane 5). Taken together with the TAP-tag purification results, our data provide convincing evidence that TbCBP30, TbCBP66, and TbCBP110 are components of the T. brucei nuclear CBC.
Binding specificity of the T. brucei CBC. The presence of novel proteins in the T. brucei CBC might be a reflection of a critical interaction with the hypermodified cap 4 structure. To begin to address this issue, we used binding titration and competition experiments coupled with an EMSA. We examined two parameters of the SL RNA complex formation, namely, binding affinity and specificity, and compared them with those of the human CBC under identical assay conditions. The rationale for using the human CBC was that it will bind the cap 4 through recognition of the 5'-terminal m7G, but that the binding specificity will not be affected by the additional modifications. In contrast, we speculated that in the case of the T. brucei CBC the interaction with the SL RNA will not only be through the m7G, but also that the cap 4 structure will play a role.
First, equilibrium binding titration assays were performed to determine the apparent disassociation constants (Kd). For this, increasing amounts of the T. brucei or human CBC were added to a constant amount of 32P-labeled SL RNA, which carries the cap 4 structure at the 5' end (Fig. 5A and B). Whereas only one RNP complex was formed for the T. brucei CBC, two distinct complexes were detected for the human CBC, indicating that complexes of different oligomerization states were formed. Indeed, due to the presence of a glutathione S-transferase tag at the N terminus of CBP20, the human CBC has a tendency to dimerize (Kristin Wilson, personal communication). For the results reported here, both shifted bands were quantitated with a phosphorimager. In the experiment shown, the trypanosome CBC bound the SL RNA substrate with a Kd of 26 ± 5 nM (mean ± error), while the human CBC bound the SL RNA substrate with a Kd of 31 ± 4 nM.
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To extend the above observation, we next performed competition experiments to examine the T. brucei and human CBCs for their ability to discriminate between m7G-capped and cap 4-containing RNA (Fig. 6). In particular, we determined the amount of competitor needed to dissociate 50% of the RNP complex (Kd1/2). In the case of human CBC, the Kd1/2 values were 4.4 and 29 nM for m7G and cap 4 RNA, respectively. On the other hand, for the trypanosome CBC, the Kd1/2 values were 410 and 27 nM for m7G and cap 4 RNA, respectively. Thus, for the trypanosome CBC, significantly more m7G competitor was needed than cap 4 competitor, while for the human CBC more cap 4 competitor was needed than m7G competitor. Finally, we calculated the relative binding affinities, using the equation Krel = [Kd1/2(m7G)]/[Kd1/2(cap 4)]. For the human CBC the ratio was 0.15, while the value for the T. brucei CBC was 15, which translates into an approximately 100-fold increase in specificity. Thus, it appeared from our experiments that the trypanosome CBC can distinguish between a cap 4 and m7G structure and that it has a much higher preference for a cap 4 substrate.
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- and ß-tubulin genes and have been the subject of previous studies (44). As shown in Fig. 7B, downregulation of TbCBP20, TbCBP30, or TbCBP110 over a period of 3 days revealed an increase of polycistronic RNA spanning two tubulin gene units. Concomitantly, the levels of mature -tubulin mRNA decreased in CBP20 and CBP30 RNAi cell lines, but this was less obvious in CBP110 RNAi cells. No change of these RNA levels was seen in control cells, where an essential gene not involved in RNA metabolism (ornithine decarboxylate cyclase [ODC] [48]) was downregulated, and RNA samples were subjected to the same analysis. To corroborate the involvement of the T. brucei CBC in pre-mRNA processing, the SL RNA and the Y-structure intermediate were assayed by primer extension analysis (Fig. 7C). Targeting either TbCBP20, TbCBP30, or TbCBP110 with RNAi increased the amount of the SL RNA over the 3-day induction period, with a concomitant decrease of the Y-structure intermediate. This effect was specific for the CBC-silenced cells, since the levels of both RNAs did not significantly change in ODC-silenced cells. Of note is that downregulation of CBC components did not affect trimethylguanosine-capped RNAs, like the U1 and U2 snRNAs (data not shown). Taken together, our data showed that downregulation of TbCBC components results in an accumulation of trans-splicing substrates (tubulin polycistronic RNA and the SL RNA) and in a decrease of a trans-splicing intermediate (Y-structure intermediate), as well as of the final product (tubulin mRNA), which is consistent with the TbCBC being required before the first step of the trans-splicing reaction.
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DISCUSSION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The conservation of the amino acid sequence of CBP20 proteins contrasts with a relatively low overall conservation of CBP80s. Indeed, it has been argued that CBP80 has diverged through coevolution with its other protein binding partners and that one of its main functions is to provide a platform for binding to proteins participating in different steps of the biogenesis and transport of capped RNAs (14, 28, 29). Our efforts to identify subunits of the T. brucei nuclear CBC, either biochemically or through database mining, have so far not revealed a credible CBP80 homolog. In addition to the well-characterized CBP20 and CBP80, there are reports of several other proteins specifically associated with capped RNA. Using different cross-linking techniques, proteins of 89, 115, and 120 kDa have been identified (33, 38), but so far no function has been ascribed to these proteins and they remain poorly characterized. Furthermore, a fraction of importin- was found in a nuclear complex with S. cerevisiae or Xenopus laevis CBC (15). Thus, we were not too surprised to recover importin- as a subunit of the T. brucei CBC. One corollary of our finding is that the T. brucei complex most likely shuttles between the nucleus and the cytoplasm and is imported into the nucleus through importin-, similar to what has been described for the yeast and human CBCs (15). However, at present we do not have evidence to support this view.
In addition to CBP20 and importin-, the T. brucei CBC has subunits of 30, 66, and 110 kDa. As mentioned above, none of these proteins appears to be a homolog of CBP80, but further studies on the architecture and subunit interactions of TbCBC will be required to clarify this point. Nevertheless, our studies exposed a striking difference in the composition of the T. brucei CBC compared to other CBCs: three subunits are only found in trypanosomatid protozoa that feature cap 4 structures and trans-splicing. Our experiments further demonstrated that TbCBP30, TbCBP66, and TbCBP110 (Fig. 7A and data not shown), as well as TbCBP20, are essential genes and that RNAi-induced silencing results in trans-splicing inhibition. The fact that trans-splicing substrates accumulated and that at least one intermediate decreased suggested to us that these subunits, and most likely TbCBC, are essential at an early step of trans-splicing. Previous studies have shown that yeast and human CBCs are not absolutely essential for cis-splicing but that they enhance the assembly of an early splicing complex, the commitment complex (21, 23). Although the precise role of the T. brucei CBC in trans-splicing needs to be elucidated, it can nevertheless be speculated that TbCBC plays an essential role in assembling an early trans-spliceosome. One possibility could be that TbCBC is bringing the SL RNA to the spliceosome. In this scenario, TbCBP30, TbCBP66, and/or TbCBP110 could provide a platform to interact with components of the trans-spliceosome, or they could even be bona fide trans-splicing factors. Regrettably, the rarity of recognizable domains in their amino acid sequences makes it at present impossible to shed light on these possibilities.
One goal of the studies presented here was to extend our previous investigations on the requirement of the cap 4 structure in trans-splicing. We now have characterized a novel T. brucei CBC that binds with a 15-fold-higher affinity to cap 4-containing RNA than to m7G-capped RNA. We would predict that initial cap 4 binding involves recognition of the 5'-terminal m7G residue by CBP20 (see above). The increased specificity is then brought about by an interaction of one or more of the additional subunits with the hypermodified cap structure. Taken together with a requirement for TbCBC in trans-splicing, this complex offers an attractive link between the cap 4 structure and trans-splicing. Our results provide a starting point for functional and structural studies aimed at understanding how TbCBC interacts with the cap 4 structure, as well as with the trans-splicing machinery.
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ACKNOWLEDGMENTS |
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We are grateful to Kristin Wilson and Richard Cerione for providing us with recombinant human CBC. We thank Susan Baserga, Keith Gull, and Paul Englund for their generous supply of reagents and Susan Baserga, Tony Koleske, Elisabetta Ullu, Kristin Wilson, and Sandy Wolin for critical comments on the manuscript.
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FOOTNOTES |
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Supplemental material for this article may be found at http://mcb.asm.org/.
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