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. 2009 Aug;15(8):1554–1564. doi: 10.1261/rna.1426609

Identification of the HIT-45 protein from Trypanosoma brucei as an FHIT protein/dinucleoside triphosphatase: Substrate specificity studies on the recombinant and endogenous proteins

Hiren Banerjee 1,2, Jennifer B Palenchar 3, Maciej Lukaszewicz 4, Elzbieta Bojarska 4, Janusz Stepinski 4, Jacek Jemielity 4, Andrzej Guranowski 5, Stephanie Ng 1, David A Wah 6, Edward Darzynkiewicz 4, Vivian Bellofatto 1
PMCID: PMC2714743  PMID: 19541768

Abstract

A new member of the FHIT protein family, designated HIT-45, has been identified in the African trypanosome Trypanosoma brucei. Recombinant HIT-45 proteins were purified from trypanosomal and bacterial protein expression systems and analyzed for substrate specificity using various dinucleoside polyphosphates, including those that contain the 5′-mRNA cap, i.e., m7GMP. This enzyme exhibited typical dinucleoside triphosphatase activity (EC 3.6.1.29), having its highest specificity for diadenosine triphosphate (ApppA). However, the trypanosome enzyme contains a unique amino-terminal extension, and hydrolysis of cap dinucleotides with monomethylated guanosine or dimethylated guanosine always yielded m7GMP (or m2,7GMP) as one of the reaction products. Interestingly, m7Gpppm3N6, N6, 2′OA was preferred among the methylated substrates. This hypermethylated dinucleotide is unique to trypanosomes and may be an intermediate in the decay of cap 4, i.e., m7Gpppm3N6, N6, 2′OApm2′OApm2′OCpm2N3, 2′OU, that occurs in these organisms.

Keywords: RNA processing, trypanosomes, FHIT proteins, cap-containing nucleotides, dinucleoside triphosphatase activity

INTRODUCTION

Gene expression in trypanosomes is controlled in part by cellular processes that regulate cytoplasmic steady-state mRNA levels (Clayton and Shapira 2007). Many of the regulatory proteins recognize mRNA-specific modifications. For example, mRNA decay pathways initiate at either the 5′ end m7G-cap or the 3′ end polyadenylated tail (Shatkin and Manley 2000; Shuman 2002). The most hypermethylated m7G- or cap-containing structure found in nature is the trypanosome cap 4 (m7Gpppm3N6, N6, 2′OApm2′OApm2′OC pm2N3, 2′OU). It contains base methylations on the first and fourth transcribed nucleotides and 2′-O-methylations on the ribose of all four transcribed nucleotides (Bangs et al. 1992). Little is known about how trypanosomes catabolize the hypermethylated m7G-cap within their mRNAs.

Extensive analyses of yeast and mammalian mRNA decay pathways reveal that m7G-capped mRNA first loses its 3′ polyadenylated tail through the action of specific deadenylases. One fate of this mRNA is 3′-5′ exonucleolytic decay mediated by the exosome, leaving a m7G-capped dinucleotide (Mitchell et al. 1997; Anderson and Parker 1998). This m7GpppX dinucleotide is hydrolyzed by a scavenger nuclease, DcpS, that catalyzes the cleavage of the γ-β pyrophosphate bond of the 5′,5′′′-P1,P3-triphosphate bridge to yield pm7G and ppX (Nuss et al. 1975; Wang and Kiledjian 2001; Liu et al. 2002). DcpS proteins constitute their own branch within the histidine triad (HIT) family of hydrolases and include human DcpS, Saccharomyces cerevisiae Dcs1p and Dcs2p, Caenorhabditis elegans dcs-1, and Schizosaccharomyces pombe Nhm1p (Fig. 1).

FIGURE 1.

FIGURE 1.

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Diagram of histidine triad superfamily of nucleotide pyrophosphatases. The triad is defined by HψHψHψψ (where ψ is a hydrophobic amino acid) (Seraphin 1992). Representative proteins within each group are limited to those for which detailed functional studies have been published. References: Human FHIT (Brenner 2002); S. pombe Aph1p (Ingram and Barnes 2000), A. thaliana FHIT (Guranowski et al. 2008), trypanosome HIT-45 (this paper), human DcpS (Gu et al. 2004), S. cerevisiae Dcs1p2p (Liu et al. 2002), C. elegans dcs-1 (Cohen et al. 2004), S. pombe Nhm1p (Salehi et al. 2002), human Hint1 (Brenner et al. 1997), S. cerevisiae Hnt1p and Gal7p (Tajima et al. 1985).

Bioinformatic and biochemical analyses have uncovered deadenylase activities and an exosome complex that participate in mRNA decay in trypanosomes (Estevez et al. 2001; Milone et al. 2004; Clayton and Shapira 2007). However, the absence of a trypanosome DcpS ortholog motivated us to determine if a unique Trypanosoma brucei HIT protein catabolizes cap-containing (oligo)nucleotides. The T. brucei database revealed four HIT proteins. Interestingly, only one protein, HIT-45, remotely resembled DcpS in that it contained an N-terminal extension in addition to its C-terminal HIT motif. Detailed analysis allowed us to assign this protein to the Fhit branch of HIT proteins and to demonstrate that HIT-45 is active on NpnNs, including cap-containing molecules and m7Gpppm3N6, N6, 2′OA, which contains the hypermethylated adenosine only found within the trypanosome cap 4.

RESULTS

HIT-45 is present exclusively in the Tri-Tryp genomes and is a novel FHIT family member

There are no obvious homologs of the decapping enzyme DcpS in the Tri-Tryp database (genomic sequences of T. brucei, T. cruzi, and Leishmania major) (Berriman et al. 2005; El-Sayed et al. 2005; Ivens et al. 2005). Given that DcpS is a member of the HIT family of hydrolases, we searched in T. brucei for a candidate protein with a HIT motif that could function as a nucleotide-specific hydrolase (Lima et al. 1997; Brenner 2002). The T. brucei database has four HIT motif-containing proteins. Two proteins were not pursued as they contain significant homology with copper oxidase and kinesin (Tb927.3.2870 and Tb927.3.3400, respectively). One of the other two proteins, Tb927.7.4480, is a member of the Hint branch of HIT proteins that possess AMP-lysine hydrolase activity (Fig. 1; Krakowiak et al. 2004), making it an unlikely component of mRNA catabolism. Thus, we chose to investigate HIT-45 (Tb927.8.2980), as its primary amino acid sequence is arranged similarly to DcpS in that it contained an N-terminal extension followed by a C-terminal HIT motif.

An amino acid alignment of the HIT-45 orthologs present in T. brucei, T. cruzi, and L. major to members of the HIT protein family has revealed that it most closely resembles proteins within the Fhit branch. Two motifs characteristic for Fhit proteins (motif I: M/LVNxKPV/IxPxHL/VM/LV/IxPxR, and motif II: I/V/MQQ/DGxxAGQT/SVP/E/KHL/VHV/THV/I I/LP; embedded in this motif is the histidine triad) are well conserved within trypanosome proteins (Supplemental Fig. S1; Barnes et al. 1996; Pace et al. 1998; Brenner 2002; Ingram et al. 2003). These motifs occur within the C-terminus of HIT-45 and align with human FHIT, the founding member of the Fhit branch of HIT proteins (Klein et al. 1998; Brenner et al. 1999; Brenner 2002; Pekarsky et al. 2002). However, HIT-45 differs in four features from other proteins in the Fhit branch of the HIT family of hydrolases as follows.

First, HIT-45 has an N-terminal extension that is unique to the trypanosome family (Fig. 2). The only other N-terminal extension found on an Fhit protein is in worms and flies, where an N-terminal nitrilase is fused to a C-terminal Fhit protein (Pekarsky et al. 1998).

FIGURE 2.

FIGURE 2.

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Multiple sequence alignment of trypanosome HIT-45 and the human FHIT protein. The entire amino acid sequences of HIT-45 from T. brucei (Tb, Tb927.8.2980), T. cruzi (Tc, Tc00.1047053509857.20), and L. major (LmjF23.1055, with the correct N-terminal extension) N termini are shown. The human FHIT protein (gene: P49789, protein: EC 3.6.1.29) is included because this protein contains the canonical fHIT motif (striped boxes) that define FHIT orthologs. (Black shading) High conservation, (gray shading with white lettering) 75% conservation, (gray shading with black letters) 50% conservation. (Box) HIT motif. The C termini of the proteins are not included. (Arrow) Internal histidine that was mutated.

Second, the highly conserved motifs I and II in the C terminus of HIT-45 are separated by a stretch of amino acids that is longer than the region that normally separates these two motifs in the Fhit branch of HIT proteins. Interestingly, this sequence shares no significant homology among the T. brucei, T. cruzi, and L. major HIT-45 orthologs (although several glycines and two adjacent serines seem to be similarly arranged), and it is approximately two times longer in T. brucei than in the other trypanosomes (Supplemental Fig. S1).

Third, the highly conserved motif II in the C terminus of HIT-45 is within a longer sequence that is conserved among T. brucei, T. cruzi, and L. major. Specifically, motif II is flanked at its N terminus by four conserved amino acids (F/Y SIA) and at its C terminus by 25 conserved amino acids (FDPxG K/R LAGEPExDE E/A xQ R/Q R Q/R P P/C RT) (Supplemental Fig. S2). As a separate point, this C-terminal conserved region is markedly different from the corresponding region that flanks motif II in the Fhit proteins described to date (human FHIT, Arabidopsis thaliana FHIT, S. pombe Aph1, S. cerevisiae Hnt2). HIT-45 motif II's C-terminal extension begins with a phenylalanine that is followed by aspartic acid, then (separated by PxG) by either positively charged lysine or arginine, and finally by conserved regions (patches) of negatively and positively charged residues (Supplemental Fig. S2). In contrast to HIT-45, other Fhit proteins contain a motif II that is followed by two positively charged resides and then less conserved negatively charged amino acids.

Fourth, the highly conserved motif II in HIT-45 contains a leucine located seven residues N-terminal to the first histidine in the HIT triad (Supplemental Fig. S2). In contrast, other Fhit proteins contain an aspartic acid or a glutamic acid. This is interesting because in the case of the DcpS branch of HIT proteins, which comprise the scavenger decapping enzymes, a conserved histidine (H268 of human DcpS) at this position relative to the HIT triad is essential for decapping activity (Gu et al. 2004). Thus, we conclude that HIT-45 is a unique HIT protein and a novel member of the FHIT branch of hydrolases. We predict that the specific amino acid sequence of HIT-45 may influence its enzymatic activities in the parasite.

HIT-45 requires its HIT motif to hydrolyze dinucleoside 5′, 5′′′-P1,P3-triphosphates

Recombinant HIT-45 protein was purified and tested for specific hydrolase activity using m7GpppG (Fig. 3). The enzyme hydrolyzed only the anhydride bond between the γ and β phosphates within the tripolyphosphate bridge to produce *pm7G from m7G*pppG (Fig. 3B,C; asterisk indicates 32P at the γ-phosphate) in a way characteristic of the DcpS branch of the HIT family of proteins. HIT motif-containing proteins rely on their central histidine for enzymatic activity. To determine whether the HIT motif within HIT-45 conferred enzymatic activity to the protein, a H355N mutant enzyme was tested for activity on m7G*pppG and was found to be inactive (Fig. 3A,D). Thus, HIT-45 was able to cleave a capped dinucleotide, recognizing it as a substrate using the central histidine within the HIT motif for catalytic activity. We conclude that HIT-45 is a bona fide HIT family member.

FIGURE 3.

FIGURE 3.

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HIT-45 requires its HIT motif to function as a nucleotide pyrophosphatase. (A) Analysis of the purity of recombinant wild-type and mutant T. brucei HIT-45 proteins, synthesized in E. coli, and containing N-terminal His6-tags. Proteins were separated by SDS-PAGE and stained with Coomassie brilliant blue. (Lanes 1,2) BSA (5 and 1 μg) to assess protein concentrations; (lanes 3,4) TbHIT-45 wild-type (WT) (10 and 5 μL); (lanes 5,6) H355N mutant protein (10 and 5 μL). (Arrow) Purified proteins. Protein size markers are the Benchmark ladder (Invitrogen). (B) Thin-layer chromatogram (TLC) of products from m7G*pppG hydrolysis by HIT-45. (Input lane) m7G*pppG. (C) Two-dimensional TLC analysis of HIT-45 hydrolysis of m7G*pppG. (D) TLC analysis of m7G*pppG substrate incubated with wild-type HIT-45 and HIT-45 H355N. (Control lane) Human DcpS that converts m7G*pppG to m7G*p.

To determine if endogenous HIT-45 has a similar activity to the recombinant protein, T. brucei was stably transfected with a tandem affinity purification (TAP) tagged HIT-45 under the inducible control of a transgenic T7 RNA polymerase-dependent promoter. The dual chromatography steps used for protein purification and the elution profiles are shown in Figure 4A. Enzyme activity purified with the HIT-45 protein, as expected (Fig. 4B). A highly enriched HIT-45 fraction was active on m7G*pppG, releasing *pm7G (Fig. 4B, fraction E-2). This enzyme fraction converted substrate to *pm7G in a time-dependent fashion (Fig. 4C).

FIGURE 4.

FIGURE 4.

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HIT-45 purified from trypanosomes hydrolyzes m7G*pppG. (A) (IgG lanes) Fractions applied to the IgG resin (ON), flowed through the column (FT), and eluted in the wash (W) or after TEV-protease cleavage (E). Fraction E was separated on Calmodulin resin (Calmodulin lanes) and flow-through, wash, and protein elutions before and after concentration (lanes E-1,E-2) are shown. Protein size markers (Invitrogen) and TEV protease are labeled on the silver-stained SDS-PAGE gel. (B) TLC analysis of m7G*pppG hydrolysis by HIT-45-containing fractions during the purification. Comparison of m7G*pppG hydrolysis by recombinant protein (50 ng; WT lane; see Fig. 3), with T. brucei S100 extract (25 μg; lysate), TEV-eluate (60 ng; E from the IgG resin), E-1 (80 ng), and E-2 (130 ng). E-3 eluate (50 ng) is from the terminal elution from the calmodulin resin. (Mock) Filtrate (10 μL) produced after E-1 concentration to generate E-2. (Input lanes) Substrate incubated in buffer. (C) A time course of E-2 activity.

Specificity of HIT-45 toward methylated and unmethylated dinucleoside 5′, 5′′′-P1, P3- triphosphates

Several dinucleotides modified within the oligophosphate bridge, ribose ring, and base moiety were used to study the substrate specificity of HIT-45. The kinetics of HIT-45 hydrolysis of various substrates and the resulting products identified by HPLC analysis are shown in Table 1. Most of the investigated compounds were cleaved by HIT-45 with formation of nucleoside monophosphates as one of the reaction products. Nonmethylated dinucleotides ApppA and GpppG were hydrolyzed, yielding pA+ppA and pG+ppG, respectively. Cleavage of the hybrid dinucleotide ApppG gave two product pairs, pA+ppG and pG+ppA, with a preference for the pA+ppG pair.

TABLE 1.

Summary of substrate specificity and kinetic parameters for HIT-45

graphic file with name 1554tbl1.jpg

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Cap analogs were also good substrates for HIT-45. The preference of cleavage of these compounds strongly depends on the position and number of methyl groups. Mono- and dimethylated dinucleoside triphosphates (m7GpppG and m2N2,7GpppG) were hydrolyzed, with formation of methylated monophosphates (pm7G or pm2N2,7G, respectively) and nonmethylated diphosphate (ppG). pG and ppm7G (or ppm2N2,7G) were not produced. In contrast, m7GpppA was cleaved, giving two product pairs, pm7G+ppA and ppm7G+pA, with a significantly higher yield of the pm7G+ppA pair. This indicates a preferential recognition of methylated bases by HIT-45. Ribose modification of the cap analog m7GpppG, produced by introducing a 2′-O-methyl substitution, changed the cleavage preference; hydrolysis products of m27,2′OGpppG were identified as pG and ppm27,2′OG. Trimethylated dinucleotides m3N2,N2,7GpppG(A) were degraded in a similar way, yielding nonmethylated monophosphates pG (pA) and ppm3N2,N2,7G. Interestingly, enzyme recognition occurred when both bases in a dinucleotide were modified by a methyl group (m7GpppmN6A, m7Gpppm7G); however, the properties of these substrates were significantly different. m7Gpppm7G was a poor substrate for HIT-45, with a very low rate of hydrolysis (<1%) in comparison with m7GpppmN6A and other tested cap-containing substrates.

A cap-containing analog with a methylene group instead of oxygen between the β and γ phosphorus atoms (m7GpCH2ppG) was revealed to be a nonhydrolyzable compound, while m7GppCH2pG underwent very slow hydrolysis (similar to that of m7Gpppm7G), with formation of pm7G and pCH2pG.

The hypermethylated dinucleotide m7Gpppm3N6,N6,2′OA, which is a truncated form of cap 4, uniquely found in trypanosomes, was also hydrolyzed by HIT-45 to yield two products, pm7G and ppm3N6,N6,2′OA. This hypermethylated dinucleotide was also a preferred substrate for HIT-45 among all tested methylated dinucleoside triphosphates (it had the lowest K0.5 value and the highest Vmax determined for methylated dinucleotides, as shown in Table 1).

HIT-45 only hydrolyzes dinucleoside polyphosphates

Different nucleotides were tested as HIT-45 substrates to determine if the enzyme could cleave nucleotides other than dinucleotides. Four different mononucleotides (pm7G, ppm7G, pppm7G, pppG) were not HIT-45 substrates (data not shown). Capped and uncapped oligonucleotides including cap 4, m7GpppAACU, and longer RNAs (m7GpppN15 and m7GpppN30) were also not HIT-45 substrates (Table 1). In contrast, a wide variety of unmodified and differently modified dinucleotides (dinucleoside polyphosphates) presented in Table 1 were substrates for the enzyme. Thus, it appears that HIT-45 exclusively hydrolyzes the latter compounds, behaving as a typical Fhit protein/dinucleoside triphosphatase (EC 3.6.1.29).

HIT-45 activity is inhibited by dinucleoside triphosphates independently of their methylation status

We tested the ability of a wide range of small RNA molecules to block the hydrolysis of m7G*pppG catalyzed by HIT-45. As a standard, conditions under which HIT-45 alone converted 50% of m7G*pppG to *pm7G were used (defined as 100% hydrolysis in all panels). Enzyme inhibitors were added prior to addition of m7G*pppG, and the decrease in *pm7G production was measured (Figs. 5, 6). Mononucleotides GTP, m7GTP, m7GDP, m7GMP, and benzyl7GTP were poor inhibitors of enzyme activity, as concentrations up to 200 μM of each compound inhibited <80% of enzyme activity (data not shown).

FIGURE 5.

FIGURE 5.

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Dinucleotide inhibitors of HIT-45. Each panel shows a TLC analysis of substrate to product conversion in the presence of a titrated amount of a different (unlabeled) dinucleotide as indicated: (A) GpppG, (B) GpppA, (C) m7Gppp m3N6N6,2′OA, (D) m3N2,N2,7GpppG and m3N2,N2,7GpppA, (E) m7GpCH2ppG, (F) m7GppCH2pG. (G) A graphic comparison of inhibitor effects on m7G*pppG hydrolysis. In all cases, m7G*pppG was preincubated with inhibitor prior to HIT-45 addition. (Input lanes) m7G*pppG incubated with buffer and no enzyme. Percent hydrolysis was calculated as defined in the text and was the average of three independent experiments.

FIGURE 6.

FIGURE 6.

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Pentanucleotide inhibitors of HIT-45 activity. Each panel shows a TLC analysis of substrate to product conversion in the presence of a titrated amount of a different (unlabeled) inhibitor as indicated: (A) cap 4 (m7Gpppm3N6, N6, 2′OApm2′OApm2′OC pm2N3, 2′OU), (B) cap 4-unmodified (m7GpppAACU). (C) Graphic comparison of inhibitor effects on m7G*pppG hydrolysis. Percent hydrolysis was calculated as defined in the text and was the average of three independent experiments.

We then tested the inhibitory effect of seven dinucleotides on HIT-45 hydrolysis of m7G*pppG (Fig. 5). Whereas GpppG and GpppA were robust inhibitors of the enzyme (Fig. 5A,B,G), trimethylguanosine forms of these dinucleotides were 10-fold less inhibitory (Fig. 5D,G). A hypermethylated dinucleotide, m7Gpppm3N6,N6,2′OA, showed an inhibition profile similar to the m3N2,N2,7GpppA profile (Fig. 5C,G). These data suggest that both unmethylated and hypermethylated dinucleotides inhibit HIT-45 activity and thus may function as substrates for the enzyme in vivo. Moreover, as both GpppA and m3N2,N2,7pppA were better inhibitors than were GpppG and m3N2,N2,7GpppG, base composition may affect enzyme recognition of substrates. When we used dinucleotide analogs that contained a methylene group in place of either oxygen atom within the phosphoanhydride bond of m7GpppG, only a small decrease in m7G*pppG hydrolysis was observed (Fig. 5E–G). Thus, both phosphoanhydride bonds within a dinucleoside triphosphate are required for HIT-45 recognition of substrate.

HIT-45 activity is inhibited by cap 4

Since the 5′ end of trypanosome mRNAs is cap 4, we determined if cap 4 competed with m7G*pppG for HIT-45 binding (Fig. 6). Low amounts of cap 4 (4–10 μM) were sufficient to block HIT-45 hydrolysis of m7G*pppG; 50 μM cap 4 allowed only 7% conversion of m7G*pppG to *pm7G (Fig. 6A). In contrast, high amounts of m7GpppAACU (cap 4-unmodified) were insufficient to block that reaction; at 50 μM concentration it allowed 70% conversion of m7G*pppG to *pm7G (Fig. 6B). In no case did an inhibitor alter the enzyme specificity of the reaction (Fig. 3C; data not shown). A comparison of the inhibition profiles of cap 4 and m7GpppAACU indicated that cap 4 inhibited enzyme activity much more efficiently than its unmethylated counterpart (Fig. 6C). Thus, cap 4 appears to bind HIT-45, whereas m7GpppAACU, which lacks the base and sugar modifications of cap 4, does not appear to interact with the enzyme.

DISCUSSION

HIT-45 is the first enzyme identified in trypanosomes involved in the cleavage of pyrophosphate bonds in dinucleoside polyphosphates, including those that contain caps and can originate from the 5′ ends of mRNAs. The uniqueness of the HIT-45 protein in trypanosomes, coupled with the distinctiveness of the trypanosome cap structure, implicate HIT-45 as a scavenger decapping enzyme identified in trypanosome mRNA decay. Four observations support this hypothesis. First, HIT-45 is a member of the HIT family of hydrolases that scavenge cap-containing dinucleotides resulting from 3′–5′ decay of mRNAs. As expected for an enzyme that utilizes its histidine triad for phosphoanhydride bond hydrolysis, mutation of the central histidine in the triad inactivates HIT-45. Second, HIT-45 and all HIT family members specifically cleave 5′, 5′′′-P1,P3 dinucleotides, and not mononucleotides or 5′, 3′-linked oligonucleotides. Third, HIT-45 is likely active in trypanosomes, as suggested by the finding that HIT-45 purified from these organisms is active on cap-containing dinucleotides. Fourth, HIT-45 hydrolyzes m7Gpppm3N6,N6,2′OA, which is the dinucleotide that would be released after 3′–5′ decay of cap 4-containing mRNAs. As m7Gpppm3N6,N6,2′OA is unique to trypanosomes, it likely requires a trypanosome-specific decapping enzyme, such as HIT-45, for its hydrolysis. In summary, our data suggest that HIT-45 plays a role in RNA metabolism, possibly by hydrolyzing residual capped nucleotides generated after 3′ to 5′ exonucleolytic decay during mRNA turnover.

The HIT family of nucleotide hydrolases shares an essential Histidine triad motif, which is the sequence HψHψHψψ (where ψ is a hydrophobic amino acid) that explains their name. The HIT motif is directly involved in the nucleotide binding properties of these proteins. There are several branches within the HIT family, each defined by additional amino acid motifs as well as evolutionary relationships. HIT-45 most closely resembles proteins within the FHIT branch, as it is specific for 5′, 5′′′ dinucleotides and shares with human FHIT and S. pombe Aph1p the signature motifs conserved in some diadenosine polyphosphate hydrolases (Fig. 2; Lima et al. 1997; Ingram et al. 2003). Monomers of α2 human FHIT and S. pombe Aph1p are relatively small proteins, ∼15 kDa in size. In contrast, HIT-45 contains a large N-terminal extension. There is a precedent for N-terminal extended Fhit proteins in worms and flies. These organisms contain an fHIT protein fusion, designated NitfHIT, in which a nitrilase enzyme is fused to a fHIT protein. The trypanosome HIT-45 resembles this fusion protein in that it contains a well-conserved Fhit region and a large N-terminal domain. However, the N-terminal domain of the trypanosome enzyme is distinctly different from that of NitfHIT and is unique to trypanosomes by bioinformatic analysis. As the amino acid sequence of the HIT-45 “extension” is unique to trypanosomes, it may be responsible for interactions between HIT-45 and the trypanosome-specific cap 4. This could explain why cap 4, but not m7GpppAACU, blocks enzyme activity. In summary, we propose that this region may be important in either cap 4 binding or m7Gpppm3N6,N6,2′OA binding and hydrolysis, both of which are likely to be trypanosome-specific requirements.

The Fhit branch of the HIT family, which contains proteins found exclusively in Eukarya, likely plays a fundamental role in eukaryotic cell metabolism. It is known that FHIT in humans has at least two distinct functions. First, FHIT regulates signaling pathways triggered by specific substrate–enzyme complexes (Trapasso et al. 2003). This activity is independent of the enzyme's hydrolase activity. FHIT suppresses tumor formation in humans in the absence of its hydrolysis activity, suggesting that FHIT-dependent signaling cascades, triggered by specific substrate–enzyme complexes, function during cancer surveillance in mammalian cells (Brenner 2002). Similarly, it is possible that cap 4–HIT-45 complexes signal biological activities integral to the metabolic circuitry in trypanosomes. Second, FHIT hydrolyzes diadenosine polyphosphates, which are generated as side products of some aminoacyl tRNA synthetases activity under conditions of cell stress (McLennan 2000; Murphy et al. 2000; Sillero and Sillero 2000). It has been shown that Fhits recognize as substrates several other naturally occurring nucleotides, such as adenosine 5′ phosphosulfate and adenosine 5′ phosphoramidate (Guranowski et al. 2008). Thus, we speculated that HIT-45 functions to turn over ApnX or related molecules that are produced during intracellular anabolic and catabolic metabolism in the parasite cytoplasm. Trypanosomes require purine acquisition for growth and are avid scavengers of oligonucleotides, nucleosides, and nucleobases. They are also replete with nitrogenous base and nucleoside interconverting enzymes. Thus, it is possible that the hydrolase function of HIT-45 is important for the acquisition and recycling of nucleotides. The high affinity of the enzyme for an array of unmethylated dinucleotides supports this contention.

We envision that during trypanosome mRNA decay, deadenylation of the polyadenylate tail releases an RNA that is readily destroyed by 3′ to 5′ exonucleases within the exosome complex. The exosome complex has been well characterized in trypanosomes (Estevez et al. 2001). In our model, the exosome releases the cap 4 pentanucleotide that is catabolized by phosphodiesterase cleavage between A1 and A2 and removal of the 2′-O-methylribose modification on A1 to yield m7Gpppm3N6,N6,2′OA. This hypermethylated dinucleotide is then hydrolyzed to pm7G and ppm3N6,N6,2′OA by HIT-45 (Fig. 7). Our discovery of an enzyme that recognizes a partially catabolized cap 4 is the impetus for searches of the predicted phosphodiesterase and a demethylase activity that would generate m7Gpppm3N6,N6,2′OA after exosome activity. Enzymatic pathways that lead to cap 4 metabolism are beginning to be explored (Arhin et al. 2006; Takagi et al. 2007; Zamudio et al. 2007).

FIGURE 7.

FIGURE 7.

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Hypothetical model for degradation of cap 4. (A) Schematic degradation of cap 4, (B) HIT-45 catalyzed hydrolysis of m7Gpppm3N6,N6,2′OA to m7Gp and ppm3 N6,N6,2′OA as determined by HPLC analysis.

Our data illuminate a third function for FHIT proteins, namely that of coordinating mRNA decay and the intracellular signaling by dinucleoside polyphosphates. mRNA decay in trypanosomes probably produces cap 4 and m7Gpppm3N6,N6,2′OA. In humans, dinucleoside polyphosphates in pancreatic B-cells function in glucose sensing, and in bacteria, they function in the heat-shock response (Lee et al. 1983; Brenner 2002; Rubio-Texeira et al. 2002). Therefore, the control of dinucleoside polyphosphate levels in cells is important for their activities. HIT-45 may modulate m7Gpppm3N6,N6,2′OA levels in trypanosomes, linking mRNA decay products with cellular metabolic needs.

MATERIALS AND METHODS

Trypanosome cell lines

The genetically engineered strain 29-13 of T. brucei, which is derived from procyclic (tsetse midgut form) wild-type T. brucei Lister 427 parasites and constitutively expresses T7 RNA polymerase and tetracycline repressor coupled to drug resistance markers, was cultured as previously described (Wirtz et al. 1999). Transfectants containing tetracycline-inducible pHB45 integrated into the parasite genome were selected in 2.5 μg/mL phleomycin 24 h after electroporation. Stable, clonal cell lines were generated by limiting dilution.

Preparation of Trypanosoma brucei cytoplasmic extracts

T. brucei procyclic (tsetse midgut form) wild-type Lister strain 427 was cultured as described previously (Wirtz et al. 1999). Extracts (S100 fractions) were prepared as described (Milone et al. 2002, 2004) and dialyzed into Buffer D (20 mM HEPES pH 7.9, 50 mM KCl, 0.2 mM EDTA, 20% glycerol, 1 mM DTT, 1 μM each of pepstatin A, leupeptin, and PMSF) before storing at −80°C.

Preparation of cap analogs

All modified pentanucleotide, trinucleotide, dinucleotide, monucleotide, and methylene-modified cap analogs were synthesized by the methods referenced in Table 1.

Preparation of radiolabeled m7G*pppG

Plasmid DNA (pGEM-4, Promega) was digested with KpnI and transcribed in vitro (Milone et al. 2002). RNAs were uniquely labeled at the γ phosphate of the cap structure using α-32P-GTP and recombinant vaccinia virus capping enzymes. The dinucleotide m7G*pppG (* indicates position of 32P) was produced by P1 nuclease digestion of RNA followed by purification using thin-layer chromatography (van Dijk et al. 2003).

Plasmid constructs and recombinant protein purification

The T. brucei HIT-45 open reading frame (ORF) from GeneDB locus Tb927.8.2980 was PCR amplified from genomic DNA and cloned into pET15b (Novagen) to produce pET15b-HIT-45. The pET15b-HIT-45-H355N, in which His 355 was replaced with Asn, was generated using the QuickChange mutagenesis system (Stratagene). Plasmid pHB45, which contains a C-terminal tandem affinity protein (TAP) tagged HIT-45, was constructed by introducing the HIT-45-TAP fusion into NruI/BamHI-digested pLEW111 (Wirtz et al. 1999). All plasmid constructs were confirmed by sequencing.

Escherichia coli BL21 cells transfected with pET15b-HIT-45 or variant were used for recombinant protein production (Cohen et al. 2004), and His6-tagged HIT-45 was purified by affinity chromatography using Ni-NTA agarose (Qiagen). HIT-45-containing fractions were dialyzed into 20 mM HEPES pH 7.9, 100 mM KCl, 1 mM DTT, 0.2 mM EDTA, 10% glycerol and stored at −80°C. Protein concentrations were estimated by comparing Coomassie staining of the protein with titrated amounts of BSA in adjacent lanes of the gel. The human DcpS ORF in pET-28, a generous gift from Dr. Michael Kiledjian (Liu et al. 2002), was expressed and purified as described above.

Purification of TAP-tagged T. brucei HIT-45

Two liters of transgenic T. brucei containing the HIT-45-TAP construct were induced with 200 ng/mL tetracycline, and an S100 lysate was prepared and protein was purified using protocols adapted from Rigaut et al. (1999) and Das et al. (2006). Protein was stored in 10 mM Tris pH 8.0, 150 mM NaCl, 0.1% NP-40, 1 mM DTT, 10% glycerol, and 1 μM each of leupeptin, pepstatin, and PMSF.

Decapping assays

Reaction conditions were as described previously (Zhang et al. 1999). Briefly, a 20 μL reaction containing either fraction E-2 (100 nM HIT-45-TAP) or purified pET15b-HIT-45 or variant (200 nM) and m7G*pppG (5–25 nM) was incubated in CE buffer (50 mM Tris pH 7.9, 20 mM KCl, 30 mM [NH4]2SO4, 2.5 mM MgCl2) for 30 min at 27°C and terminated with 20 mM EDTA. Competition experiments were performed under identical conditions. Titrated amounts of unlabeled inhibitors (1–500 μM) were incubated with enzyme for 10 min before addition of 25 nM m7G*pppG substrate. Inhibitor stocks were 5 mM in deionized water. Product analysis was performed by thin-layer chromatography (TLC). After phenol/chloroform extraction, decapping products were separated on Bakerflex PEI-F-cellulose, pre-run in deionized water, and developed in 750 mM LiCl (Milone et al. 2002). Quantification was performed using a PhosphorImager and ImageQuant software (Molecular Dynamics), and hydrolysis was quantified by comparing the fraction of m7G*p released from m7G*pppG. For two-dimensional TLC, products were resolved using 1 M acetic acid at pH 3.5 in the first dimension and saturated (NH4)2SO4 in the second dimension (Bergman et al. 2004).

Enzymatic digestion of cap analogs with HIT-45

To determine the kinetic parameters for dinucleotide cap analog hydrolysis, the in vitro decapping assay was used as described above except that five to seven substrate concentrations of unlabeled material (5–120 μM) were present in the reaction. HPLC analysis was used to identify reaction products by matching their retention time with the appropriate peak of a reference compound, as well as by estimating the initial hydrolysis rate of nonmethylated dinucleotide GpppG and m7GpppG (as reference substrates). A Spectra-Physics SP 8800 with a Spectra 100 UV monitor, SP 4600 automatic integrator, and a Supelco LC-18-T reversed phase column were utilized in HPLC analysis (Spectra-Physics). The solvent was 0.1 M KH2PO4 or a linear gradient of methanol in 0.1 M KH2PO4 (0%–25% v/v within 15 min), and monitoring was performed at 260 nm.

Hydrolysis of other cap analogs was monitored using a fluorimetric method, in which the increase of fluorescence intensity as a result of enzymatic cleavage of pyrophosphate bond in dinucleotides was recorded as previously described (Kalek et al. 2006). The initial rate of hydrolysis was calculated by linear regression of substrate concentration versus time. Fluorescence measurements were carried out on a Perkin-Elmer LS 50B spectrofluorometer, and cuvettes had 4 mm and 10 mm path lengths for absorption and emission, respectively. HPLC data directly correlated with fluorometric analysis, as determined by comparing data obtained for the initial hydrolysis rate of m7GpppG using both methods.

Estimation of kinetic parameters of fHit-45 using the Hill equation

The Hill equation v = V * ah / (K0.5h + ah) (where v is the initial velocity; V is the maximal velocity; a is the substrate concentration; K0,5 is the substrate concentration that gives a velocity equal to half V; and h is the Hill coefficient) was rearranged to the logarithmic form: log(v/V − v) = h log a − h log K0,5. A plot of log(v/V − v) against log a is predicted to give a straight line.

To calculate kinetic parameters from the Hill equation we first estimated a maximal velocity V. An approximate V (Vapp) value was obtained from a Lineweaver–Burk plot for data obtained for higher concentrations of substrate. This was used to draw a Hill plot and estimate a Hill coefficient. Subsequently, the Lineweaver–Burk plot was redrawn, substrate concentration values (a) were replaced with ah, and Vmax was recalulated. This information was used to redraw the Hill plots and determine the K0,5 values.

To compare Vo data obtained for HIT-45 from different protein purifications, they were normalized against data for m7GpppG. For kinetic parameters, calculations were used to normalize data. Therefore Vmax values, presented in Table 1, are expressed relative to m7GpppG (Vmax = 1).

Protein alignments

Alignments of HIT-45 from T. brucei, T .cruzi, and Leishmania major were performed using CLUSTALW (Thompson et al. 1994). Alignments were generated using the ClustalW program and are represented using GeneDoc (http://www.nrbsc.org/gfx/genedoc/).

The L. major HIT-45 gene was assembled from Lm23_BIN_contig795 and LmjF23.1055 sequences.

SUPPLEMENTAL MATERIAL

Supplemental material can be found at http://www.rnajournal.org.

ACKNOWLEDGMENTS

We thank Karl Drlica for critical reading of the manuscript. We gratefully appreciate the excellent drawings and figure constructions done by Timothy Linteau. This work was supported by National Institutes of Health–NIAID grant AI535835 to V.B., by grants from the Howard Hughes Medical Institute (No. 55005604) and the Polish Ministry of Sciences and Higher Education (No.2 PO4A 006 28) to E.D., and by a grant from the Polish Ministry of Sciences and Higher Education (PBZ-MNiSW-07/1/2007) to E.D and A.G.

Footnotes

Article published online ahead of print. Article and publication date are at http://www.rnajournal.org/cgi/doi/10.1261/rna.1426609.

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