Characterization of deadenylation in trypanosome extracts and its inhibition by poly(A)-binding protein Pab1p

Identification and characterization of deadenylation activities in two trypanosome species

We have previously demonstrated the presence of two decapping activities and an AU-rich element-regulated 3′→5′ exonuclease activity in L. seymouri extracts (Milone et al. 2002). The existence of these activities prompted us to determine whether our extracts also contained a deadenylase activity. We tested for this activity by using small artificial mRNAs. The RNA substrates used were labeled internally with [α-³²P]UTP and possess a 5′ m⁷G cap and a 60-nucleotide (nt) long poly(A) tail. Previous deadenylation assays in mammalian and yeast extracts required the addition of excess poly(A) homopolymer to the reaction. First, we tested whether the addition of poly(A) competitor could activate deadenylation in the L. seymouri cytoplasmic extracts. Figure 1A ▶ shows a titration of increasing amounts of either poly(A) or poly(C). The addition of poly(A) homopolymer specifically resulted in the change in substrate size from the polyadenylated form of the RNA (A60) to the deadenylated form of the RNA (A0). No significant change in size of the RNA substrate was observed by incubation of the RNA substrate in a reaction without poly(A) homopolymer or in the presence of poly(C) homopolymer. We next determined the biochemical nature of the nuclease activity. Figure 1B ▶ shows a titration from ~48 μg to 0 μg of protein extract in the presence of excess poly(A) homopolymer and internally radiolabeled polyadenylated RNA. Reducing the amount of extract, and thus the amount of nuclease, in the deadenylation reaction caused the accumulation of RNAs with differing poly(A) tail lengths. The increase in the amount of these decay intermediates under these conditions suggest an enzyme-limiting situation and are indicative of a distributive activity. The distributive nature of the nuclease is further supported by the additional observation that an excess amount of poly(A) homopolymer in the reaction shows a decay pattern similar to that observed in Figure 1B ▶.

In addition to L. seymouri, we prepared extracts from T. brucei and assayed them for deadenylation activities. Figure 1C ▶ shows titrations performed with poly(A) or poly(C) homopolymer using the T. brucei extracts. As observed for the L. seymouri extracts, the T. brucei extracts exhibited a poly(A)-activated nuclease that resulted in a size change from the polyadenylated form (A60) to the deadenylated form (A0). The deadenylase in the T. brucei extract is acting in a distributive fashion, similar to the L. seymouri extracts, as addition of excess amounts of poly(A) homopolymer (2 μg) to the reaction cause an accumulation of different-sized decay intermediates. The decay intermediates are similar to the ones observed in the L. seymouri reactions in Figure 1B ▶.

To determine the poly(A) specificity and directionality of the trypanosomal deadenylation activities, we used two additional substrates. Figure 2A ▶ shows L. seymouri extract incubated with an RNA substrate containing a 60-nt poly(A) tail and with an RNA substrate containing a 60-nt poly(A) tail blocked at its 3′ end by 20 additional nonadenylated nucleotides. The left panel shows both RNAs incubated for a 30-min time course with L. seymouri extracts. The unblocked RNA was completely deadenylated by 30 min, whereas the blocked RNA remained fully adenylated after 30 min. The right panel shows the same experiment using T. brucei extracts. Again, the unblocked RNA was deadenylated and the blocked RNA remained polyadenylated. These results demonstrate that the nuclease activity in both of the trypanosome extracts are occurring in a 3′→5′ direction. The direction of decay is supported by an additional experiment using cap-labeled polyadenylated RNA substrate in L. seymouri extracts (Fig. 4A ▶, below, right panel). The decay of cap-labeled RNA produces the same decay pattern as internally labeled RNA. Finally, a tract of nine cytosine residues was placed in the middle of the poly(A) tail to interrupt the poly(A) tract and examine specificity of the deadenylase-like activity. Figure 2 ▶, B and C, show time courses with the interrupted poly(A) tail in each trypanosome extract. Figure 2B ▶ shows the decay kinetics in L. seymouri extracts in a 60-min time course using either the noninterrupted or interrupted RNA substrates. A deadenylation pause is detected by 30–60 min in the reaction containing the substrate with the interrupted poly(A) tail. This pause appeared approximately halfway between the A60 and A0 markers and is consistent with the location of the nine-cytosine residue interruption in the poly(A) tail. A pause was not observed in the time course performed in parallel using an RNA with a noninterrupted poly(A) tail. A graphical representation of the data is shown below Figure 2B ▶. The data are represented as distance of migration of RNA products relative to the location of the RNA A0 marker. The presence of the poly(C) tract imbedded in the middle of the poly(A) tail caused a pause in deadenylation that is midway in distance (~12.5 mm) between the polyadenylated (~25 mm) and deadenylated forms (~0 mm) of the RNA. The T. brucei extracts functioned similarly to the L. seymouri extracts. Figure 2C ▶ shows the reaction products from the incubation of an uninterrupted and poly(C) tract-interrupted RNA substrate with T. brucei extracts. The graph below Figure 2C ▶ shows the data in a graphical format. As observed for the L. seymouri reactions, the unblocked RNA deadenylated unimpeded, while the interrupted RNA substrate paused to form a decay intermediate approximately half the distance (~15 mm) between the polyadenylated (~28 mm) and deadenylated (~0 mm) RNA. Therefore, we conclude that both T. brucei and L. seymouri extracts contain a 3′→5′ adenylate-specific nuclease that is activated by the addition of poly(A) homopolymer. This activity is a trypanosomal deadenylase.

The trypanosomal deadenylase activities possess characteristics similar to other known deadenylase activities

It has been reported that PARN is inhibited by neomycin-B sulfate (Ren et al. 2002). To determine which of the known deadenylase activities might correspond to the trypanosomal activity identified above, we treated the trypanosomal extracts with an increasing concentration of neomycin B sulfate. Similarly treated HeLa cell extracts were used as a control. Figure 3A ▶ shows neomycin-B sulfate titrations for both L. seymouri and T. brucei cytoplasmic extracts (Fig. 3A ▶, left panel). Inhibition of the L. seymouri activity begins at 100 μM neomycin-B sulfate. The activity in the HeLa extract behaved in a similar pattern to the L. seymouri activity and was partially inhibited at a 100 μM concentration. However, the T. brucei extracts were not inhibited at a neomycin-B sulfate concentration of 100 μM. All three extracts were completely inhibited at a concentration of 1 mM neomycin-B sulfate. We conclude that the trypanosomal activities are inhibited at relatively similar levels of neomycin-B sulfate as other known deadenylases. Minor differences in inhibitory concentration may not be informative because of differences between extracts.

Because most deadenylases are Mg²⁺-dependent enzymes (Dlakic 2000; Daugeron et al. 2001; Zuo and Deutscher 2001), we tested whether the trypanosomal deadenylase also requires a divalent cation. A total of 10 mM EDTA was added to deadenylation reactions using either trypanosomal extract, and reaction products were analyzed after 30 min. In both cases, EDTA blocked deadenylation of the RNA substrate (Fig. 3B ▶). Reactions were also performed without the addition of magnesium to extracts. These reactions resulted in a greatly diminished deadenylation activity (data not shown). Therefore, we conclude that the trypanosomal deadenylase activity requires divalent cations, just as other known deadenylases.

The product of known deadenylase activities is 5′-AMP. We tested our activities to determine the product of the trypanosomal deadenylation reactions. Figure 3C ▶ shows one-dimensional TLC analysis of the deadenylation reaction products of the RNA substrate labeled at the poly(A) tail with [α-³²P]ATP (Martinez et al. 2001). The radioactive product of the deadenylation reaction comigrated with 5′-AMP in the assay. A potential 3′-AMP product would have migrated lower than 5′-AMP using the one-dimensional TLC assay. These data show that the trypanosome nuclease activity produces the same product as other known deadenylases. Ultimately, these collective data shown in Figures 1 ▶–3 ▶ provide evidence that the trypanosome nuclease activity possesses many of the properties of known deadenylases.

The trypanosomal deadenylase activity is cap independent and not stimulated by the presence of an AU-rich element

The mammalian deadenylase, PARN, interacts with the 5′ cap (Gao et al. 2000). Accordingly, processive deadenylation acting in mammalian extracts is cap dependent. We tested whether the 5′ m⁷G cap is a requirement for trypanosomal deadenylases to degrade mRNA. Figure 4A ▶ (left panel) shows a time course of uncapped (5′ triphosphate containing) internally labeled RNA incubated in L. seymouri extracts. The RNA was completely deadenylated by 30 min, exhibiting kinetics similar to m⁷G-capped RNAs (radiolabeled at the γ Phosphate of the cap structure; Fig. 4A ▶, right panel). The deadenylation activity in the T. brucei extracts was also found to be cap independent (Fig. 4B ▶). m⁷G-capped and uncapped RNAs were incubated in T. brucei extracts for the times indicated. In both cases, the RNAs were deadenylated by 30 min. These results suggest a similarity between the trypanosomal deadenylase activity and that of yeast, which is cap independent in vitro (Wilusz et al. 2001a).

We have previously shown that a 34-nt ARE sequence from the 3′ UTR of TNF-α functions to destabilize RNAs lacking a poly(A) tail in L. seymouri cytoplasmic extracts (Milone et al. 2002). This sequence was previously reported to slightly enhance the rate of deadenylation of RNAs in mammalian extracts (Ford et al. 1999). Therefore, we determined the deadenylation rate in trypanosome extracts of RNAs that possess a TNF-α ARE sequence. Figure 4C ▶ shows time courses containing an RNA with the ARE sequence and an RNA possessing the reverse ARE sequence (ERA). To the right of Figure 4C ▶ is a graphical representation of the data. Similar deadenylation kinetics were observed between the two substrates. We conclude that removal of a 60-nt poly(A) tail is not regulated by this element in our trypanosomal extracts.

PAB1p is an inhibitor of deadenylation activity

Pab1p is a ubiquitous cytoplasmic RNA-binding protein in eukaryotes that may play a major role in the regulation of mRNA stability and translation initiation (Wilusz et al. 2001b). Studies on mammalian cells and yeast indicate that Pab1p may function as a deadenylase inhibitor (Wilusz et al. 2001a). As a first step in identifying the interplay among different proteins that regulate mRNA turnover in trypanosomes, we sought to determine whether Pab1p functions as a deadenylase inhibitor in trypanosome extracts. As L. seymouri and Leishmania major are closely related trypanosomes, we used an available anti-L. major Pab1p (LmPab1p) antibody for immunodepletion studies (Bates et al. 2000). Figure 5A ▶ shows Western analysis using the LmPab1p polyclonal antisera. The antisera detected the purified recombinant His-tagged LmPab1p, as well as a similar-sized band in L. seymouri cytoplasmic extracts. We conclude that the L. major antiserum was able to cross-react and detect Pab1p in L. seymouri extracts.

We next used the anti-LmPab1p antisera to specifically deplete a L. seymouri extract of Pab1p. Figure 5B ▶ shows Western analysis of the Pab1p antisera-depleted fractions of the L. seymouri extract. Four consecutive rounds of immunodepletion were required to remove Pab1p from L. seymouri extracts (Fig. 5B ▶, left panel). In control experiments, parallel immunodepletions using rabbit normal sera did not significantly affect Pab1p levels in the extract (Fig. 5B ▶, right panel). Figure 5C ▶ shows functional deadenylase assays utilizing the supernatants from the fourth-round immunodepletions. Surprisingly, deadenylation of polyadenylated RNA substrate still required the addition of poly(A) homopolymer following removal of Pab1p. Therefore, the deadenylation activity remained inhibited in the Pab1-depleted extracts. These results suggest that Pab1p may not be the sole inhibitor of deadenylase activity in trypanosomes.

To further explore the inhibitory nature of Pab1p, we depleted a L. seymouri extract of all deadenylase inhibitors by incubation with poly(A) Sepharose. Figure 6A ▶ shows that poly(A) Sepharose batch treatment of the extract specifically removed the requirement of poly(A) homopolymer added in trans to stimulate deadenylation. As a control, the Protein G Sepharose batch-treated extract maintained its requirement for the addition of poly(A) homopolymer. We next added purified His-tagged LmPab1p or BSA to the poly(A) Sepharose-depleted extracts to restore the inhibition of deadenylation (Fig. 6B ▶). Addition of LmPab1p, but not BSA, was sufficient to block the deadenylase activity. Therefore, we conclude that Pab1p can function independently as an inhibitor in vitro of deadenylation in trypanosomes.

RNA substrate preparation

All RNA substrates were prepared as previously described (Ford and Wilusz 1999; Milone et al. 2002). Briefly, the DNA templates for SL-MCS A60, SL-MCS ARE A60, SL-MCS ERA A60, and Gem A60 RNAs were generated by digestion of the parent plasmids pSL-MCS, pSL-MCS ARE, pSL-MCS ERA, and pGem (Promega) with HindIII, followed by ligation to a DNA A₆₀-T₆₀ homopolymer with HindIII compatible ends. Recombinant PCR was performed using the ligated DNA as a template with two primers, M13 forward and JM37, that amplified the region containing the SP6 RNA polymerase promoter, plasmid multiple cloning site, and 60-bp A-T region. Following amplification, the PCR product was digested with SspI, phenol-chloroform extracted, and ethanol precipitated. MCS RNAs were produced using an initial recombinant PCR step to remove the L. seymouri SL–RNA sequence as previously described (Milone et al. 2002). SL-MCS ARE A60+20 and Gem A60+20 RNAs were prepared as described above, except that an SspI digestion was omitted. MCS A30-C9-A30 and Gem A30-C9-A30 RNAs were prepared using a DNA template generated by substitution of the A₆₀-T₆₀ homopolymer described above with a homopolymer interrupted after the first 30 A-T residues by a stretch of nine cytosine residues followed by the remaining 30 A-T residues. These recombinant PCR-amplified A30-C9-A30 DNAs were digested with NsiI to generate poly(A) ends.

DNA templates were in vitro transcribed using SP6 RNA polymerase (New England Biolabs) with [α-³²P]UTP for internal labeling or [α-³²P]GTP for cap labeling. All RNAs are capped by inclusion of cap analog (Amersham) in the transcription reaction, unless noted. Uncapped RNAs were prepared by transcribing RNA in the absence of cap analog. Cap-labeled RNA was prepared as previously described (Milone et al. 2002). Following transcription, all RNAs were phenol-chloroform extracted, ethanol precipitated, and purified following separation using a 7 M urea, 5% polyacrylamide gel. Gel-purified RNA was phenol-chloroform extracted twice, chloroform extracted once, and ethanol precipitated. Finally, the RNAs were resuspended in ddH₂O and adjusted to 100,000 cpm/μL for use in assays. A0 makers were prepared as above by transcription of the pertinent linearized plasmid following HindIII digestion.

Cytoplasmic extracts

Leptomonas seymouri extracts were prepared as previously described (Milone et al. 2002). Trypanosoma brucei cytoplasmic extracts were similarly prepared. A total of 1 L of T. brucei strain 427 wild-type trypanosomes were grown to a density of 1–3 × 10⁷ cells/mL in two 1-L flasks, gently shaking at 26°C. Parasites were washed three times with PBS and resuspended in TbCE (25 mM HEPES at pH 7.2, 100 mM NaCl, 10% glycerol, 0.1% triton X-100, 1 mM DTT, 1 μM Pepstatin A, 1 μM Leupeptin, and 1 mM PMSF). Parasites were disrupted using a 21-guage needle and freeze-thaw lysed for several cycles (typically four to six cycles) in a dry-ice ethanol bath for 2 min, and thawed at 42°C for 1 min. Parasite lysis was ascertained through light-microscopy analysis of the suspension and the appearance of ghost cells. After lysis, the extract was precleared of debris by centrifugation (11,500g, 10 min, 4°C), and then centrifuged (100,000g, 1 h, 4°C) to generate the S100 fraction. The extract was aliquoted, quick-frozen, and stored at −80°C.

Deadenylation assays

Assay conditions were the same as used for decapping analysis (Milone et al. 2002). Briefly, an 11-μL reaction volume contained 50 mM Tris-HCl (pH 7.9), 2.4 mM MgCl₂, 20 mM KCl, 30 mM (NH₄)₂SO₄, 4% glycerol, and 4 μL of cytoplasmic extract (typically 40–80 μg of total protein), and 10–50 fmole of RNA. Similarly, 4 μL of poly(A)/Protein G Sepharose-depleted or Pab1p-immunodepleted extracts were also used per reaction. To activate deadenylation, 500 ng of poly(A) homopolymer (Amersham) was added to the reaction unless otherwise stated. Poly(C) homopolymer (Amersham) was used as a alternative competitor to poly(A) for the indicated reactions. All reactions were preformed at 26°C for the indicated times, and reactions were terminated by addition of 300 μL of HSCB (400 mM NaCl, 25 mM Tris-HCl at pH 7.6, 1% SDS, and 1 mM EDTA). The terminated reaction products were phenol extracted, ethanol precipitated, and separated using 7 M urea 5% polyacrylamide denaturing gels. Radioactive gels were exposed to PhosphorImager screens.

Thin-layer chromatography analysis

TLC analysis was largely performed as previously described (Martinez et al. 2001). Deadenylation reactions were performed as above and terminated by the addition of 25 mM EDTA. Reaction aliquots were increased to 20 μL, phenol-cholorform extracted, and 10 μL of the aqueous phase spotted onto a BakerFlex PEI-F cellulose TLC plate (Baker). Nonradioactive 5′-AMP and 3′-AMP (Sigma) were added into the decapping reaction’s aqueous phase prior to spotting as a marker. Plates were then washed for 20 min at room temperature in methanol, and air-dried. TLC plates were developed in 0.75 M KH₂PO₄ adjusted to pH 3.5 with H₃PO₄. Following development, the plate was air dried and UV shadowed to visualize the 5′ and 3′-AMP markers. Deadenylation reaction products were visualized by exposure to a PhosphorImager screen.

Pab1p analysis

L. seymouri cytoplasmic extracts were treated with either poly(A) Sepharose (Amersham) or Protein G Sepharose (Amersham) in parallel to remove deadenylation inhibitory proteins. A total of 500 μL of dry volume of Poly(A) or Protein G Sepharose resins were prepared by addition of 10 mL of PBS with protease inhibitors (1 μM Pepstatin A, 1 μM Leupeptin, and 1 mM PMSF) and allowed to swell. The swelled resin was then washed three times with 10 mL volumes of PBS with protease inhibitors, and resuspended in 1 mL of PBS with protease inhibitors. A total of 100 μL of resin slurry was washed with 1 mL of LsCE and used to deplete extract of deadenylase inhibitors. EDTA was added to 500 μL of extract to a final concentration of 5 mM to chelate any residual divalent cations. The extract volume was then equally split, and 250 μL of extract incubated with 100 μL of resin prepared above (30 min, rotating, 4°C). Following incubation, the extract was cleared of the resin by centrifugation (2000g, 2 min, 4°C) and the supernatant removed. A small aliquot of the depleted extract was saved (~50 μL) and the remaining amount incubated with resin for a second round of depletion performed as above. The poly(A)/Protein G Sepharose batch-purified fractions were quick-frozen and stored at −80°C.

A Protein A Sepharose suspension was prepared as described above for Protein G Sepharose. A total of 50 μL of resin slurry was added to 25 μL of rabbit polyclonal anti-LmPab1p antisera or rabbit normal sera and incubated with Protein A for 40 min while rotating at 4°C (Bates et al. 2000). These antibody/Protein A/resin complexes were washed three times with 500 μL of LsCE. A total of 300 μL of L. seymouri cytoplasmic extracts were precleared by incubation (40 min, rotating, 4°C) with purified rabbit IgG and 50 μL of washed protein A resin slurry. Following incubation, the extract was cleared by a short spin (2000g, 2 min, 4°C) and the supernatant removed. A total of 40 μL of precleared extract was removed for analysis, and the remaining extract was divided into equal portions. Each 130-μL portion was added to the anti Pab1p sera or normal sera-bound beads prepared above and incubated for 30 min. The extract was cleared of the beads as above, and 20 μL of depleted extract saved. Four separate rounds of depletion were done, leaving ~50–60 μL of depleted extracts. Depleted extracts were quick frozen and stored at −80°C.

Purified recombinant L. major Pab1p was prepared using the constructs and procedure as described (Bates et al. 2000). Purified His₆-tagged LmPab1p was quantified by Bradford (Pierce) or by visual inspection on an 8% SDS–polyacrylamide gel and compared with BSA standards.

Western analysis was performed as described (Harlow and Lane 1988). A total of 4 μL of L seymouri total cytoplasmic extracts or depleted extracts were separated using an 8% SDS–polyacrylamide gel. Protein was transferred to PVDF membrane and blocked in blocking solution (5% nonfat dry milk in PBS) for 1 h at room temperature. The membranes were blotted with a 1:1000 dilution of rabbit anti-LmPab1p antisera in blocking solution for 1 h at room temperature, followed by three washes in PBS 0.1% Tween for 10 min each. Pab1p antibody complexes were detected using an anti-rabbit IgG alkaline phosphatase conjugated antibody and detection reagents (Amersham). Briefly, a 1:10,000 dilution was made of secondary antibody in blocking solution and allowed to incubate with blot for 1 h at room temperature. The blot was washed as above, and development solution was added. Blots were visualized by exposure to film.