Nulear Extracts of Crithidia fasciculata Contain a Factor(s) That Binds to the 5′-Untranslated Regions ofTOP2 and RPA1 mRNAs Containing Sequences Required for Their Cell Cycle Regulation*

The Crithidia fasciculata replication protein A gene, RPA1, and topoisomerase II gene,TOP2, encode proteins involved in the replication of nuclear and mitochondrial DNA, respectively. Transcripts of both genes accumulate periodically during the cell cycle and attain their maximum levels just before S phase. Octamer consensus sequences within the 5′-untranslated region (UTR) of both genes have been shown to be necessary for cycling of these transcripts. Using a gel retardation assay, we show here that nuclear extracts of C. fasciculatacontain a protein factor(s) that binds specifically to RNA from 5′-UTRs of TOP2 and RPA1 genes. In addition, mutations in the consensus octamer sequence abolish binding to the RNA in both cases. Ultraviolet cross-linking using a radiolabeled TOP25′-UTR probe identified proteins with apparent molecular masses of 74 and 37 kDa in the RNA-protein complex. Nuclear extracts prepared from synchronized cells show that the binding activity varies during the cell cycle in parallel with TOP2 and RPA1mRNA levels. These results suggest that the cell cycle regulation of the mRNA levels of trypanosomatid DNA replication genes may be mediated by binding of specific proteins to conserved sequences in the 5′-UTR of their transcripts.

The Crithidia fasciculata replication protein A gene, RPA1, and topoisomerase II gene, TOP2, encode proteins involved in the replication of nuclear and mitochondrial DNA, respectively. Transcripts of both genes accumulate periodically during the cell cycle and attain their maximum levels just before S phase. Octamer consensus sequences within the 5-untranslated region (UTR) of both genes have been shown to be necessary for cycling of these transcripts. Using a gel retardation assay, we show here that nuclear extracts of C. fasciculata contain a protein factor(s) that binds specifically to RNA from 5-UTRs of TOP2 and RPA1 genes. In addition, mutations in the consensus octamer sequence abolish binding to the RNA in both cases. Ultraviolet cross-linking using a radiolabeled TOP2 5-UTR probe identified proteins with apparent molecular masses of 74 and 37 kDa in the RNA-protein complex. Nuclear extracts prepared from synchronized cells show that the binding activity varies during the cell cycle in parallel with TOP2 and RPA1 mRNA levels. These results suggest that the cell cycle regulation of the mRNA levels of trypanosomatid DNA replication genes may be mediated by binding of specific proteins to conserved sequences in the 5-UTR of their transcripts.
The trypanosomatid Crithidia fasciculata is a protozoan parasite containing a single mitochondrion with an unusual form of DNA called kinetoplast DNA (kDNA) 1 (1,2). The kDNA consists of a single network of catenated minicircles and maxicircles. During replication of kDNA, the minicircles are released from the network and are reattached to the network periphery after replication, whereas maxicircles replicate while still attached to the network (3). An unusual feature of DNA replication in trypanosomes is that both kinetoplast and nuclear DNA replicate in approximate synchrony (4,5). In other eukaryotes, mitochondrial DNA replication occurs throughout the cell cycle (6,7). Since both the kinetoplast and nuclear DNA replication genes are encoded in the nucleus, their coordinated expression may play a role in synchronizing nuclear and kDNA replication.
Expression of protein-coding genes in trypanosomatids involves mechanisms that are different from those in most other eukaryotes. All mRNAs in trypanosomatids contain two exons, a 39-nucleotide miniexon at the 5Ј end of the mRNA and the main coding exon (8,9). Genes are usually grouped in polycistronic transcription units, and the polycistronic pre-mRNAs are processed by 5Ј trans-splicing and 3Ј polyadenylation to yield monocistronic mRNAs. The differential expression of steady state mRNAs from a single polycistronic transcript involves post-transcriptional controls by mechanisms that are still unclear. Pre-mRNA turnover in combination with differential rates of trans-splicing and polyadenylation and of mRNA turnover may all play a role in regulating mRNA levels. So far, only two RNA polymerase II promoters for protein-coding genes have been described in trypanosomes, the Trypanosoma congolense glutamic acid/alanine-rich protein promoter and the Trypanosoma brucei hsp70 promoter, and neither promoter appears to be transcriptionally regulated (10,11). The regulated expression of T. brucei hsp70 upon heat shock appears to be controlled entirely at a post-transcriptional level, since heat shock does not induce increased transcription of the hsp70 genes (11).
To determine the mechanism of coordinate expression of nuclear and kinetoplast DNA replication genes, we have studied the cell cycle-regulated expression of RPA1 and TOP2 genes. The TOP2 gene encodes a kinetoplast-associated type II topoisomerase (12), and the RPA1 gene encodes the large subunit of replication protein A, the single-stranded DNA-binding protein that has been immunolocalized to the nucleus (13,14). The steady state levels of the mRNAs for these two genes and that of dihydrofolate reductase-thymidylate synthase gene are cell cycle-regulated in a similar manner (4). Sequence elements required for periodic accumulation of RPA1 and TOP2 mRNAs are present in the 5Ј-untranslated region (15,16). Examination of the 5Ј-UTR of these genes reveals the presence of a consensus octamer sequence with a conserved hexameric core. Mutation analysis showed that these octamer sequences are necessary for periodic accumulation of TOP2 and RPA1 transcripts. We show here that specific proteins present in nuclear extracts bind to the 5Ј-UTR RNA of TOP2 and RPA1 genes and that the octamer sequences are necessary for binding to occur.

EXPERIMENTAL PROCEDURES
Preparation of Nuclear and Cytosolic Extracts-C. fasciculata Cf-C1 cells were grown in brain heart infusion medium (Difco) supplemented with 20 g/ml hemin and 100 g/ml streptomycin sulfate at 28°C with shaking. The cells were grown to a density of 6 -7 ϫ 10 7 cells/ml and then harvested by centrifugation (Sorvall GS-3 rotor, 15 min, 5000 rpm at 4°C). All procedures for extract preparation were performed at * This work was supported by National Institutes of Health Grant GM53254. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18  0 -4°C. Buffers B, C, and D also contained 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, and 5 g/ml leupeptin. The cell pellet was washed twice with phosphate-buffered saline and then once with Buffer A (10 mM HEPES-KOH, pH 7.9, 1.5 mM MgCl 2 , 10 mM KCl). The washed cell pellet was resuspended in an equal volume of Buffer B (Buffer A containing 0.5% nonidet P-40) and incubated on ice for 10 min with vortexing every 2 min. The cell extract was then centrifuged for 15 min at 15,000 rpm (SS-34 rotor). The supernatant was removed and mixed with an equal volume of Buffer D (20 mM HEPES-KOH, pH 7.9, 50 mM KCl, 0.2 mM EDTA, 20% glycerol) and represents the cytosolic extract. The pellet was resuspended in an equal volume of Buffer C (20 mM HEPES-KOH, pH 7.9, 1.5 mM MgCl 2 , 420 mM NaCl, 0.2 mM EDTA, 25% glycerol) and passed five times through a 21-gauge needle to lyse the nuclei (17). The extract was centrifuged for 15 min at 15,000 rpm (SS-34 rotor). The supernatant was removed and mixed with an equal volume of Buffer D to give the nuclear extract. The cytosolic and nuclear extracts were stored at Ϫ70°C or in liquid nitrogen. Protein was assayed by the bicinchoninic acid method using a micro BCA kit (Pierce) according to the manufacturer's instructions. Bovine serum albumin was used as the standard.
The plasmids pRM9-pRM12 containing the Ϫ291 to Ϫ209 region of the TOP2 gene were constructed (the A residue of the initiating ATG codon is ϩ1). These plasmids had both mutant octamers (pRM9), left octamer mutant only (pRM10), right octamer mutant (pRM11), or both wild type octamers (pRM12). These plasmids were constructed by amplifying the Ϫ291 to Ϫ209 region of the TOP2 gene by PCR with Vent DNA polymerase using primers containing wild type or mutant octamers and plasmid pt2-1 as template and then cloning the various PCR products into the SmaI site of pUC19. Oligonucleotides used in PCR were C46 (AGCAGTGCGGCCGCGTGCGAGGATATTGCGGGTAACA-CGCCGC) and C47 (CGGGAGTCGGCCGATTCCTCGCAGGCTTTCG-ACACCTCTCTGT) for pRM9, C46 and B93 (CGGGAGTCGGCCGATC-TTCTATGGGCTTTCGAC) for pRM10, B92 (AGCAGTGCGGCCGCG-CATAGAAGTATTGCGGG) and C47 for RM11, and B92 and B93 for pRM12. Mutant octamer sequences are underlined, and wild type octamer sequences are shown in bold face. These plasmids were used as templates in PCR to give products that were in turn used as templates for in vitro synthesis of RNA probes (see Table I).
The RPA1 gene sequences from Ϫ341 to Ϫ231 containing wild type or mutant hexamers were cloned into p⌬10Not to give plasmids pDS17 and pDS19, respectively (16). The inserts in all plasmids were sequenced to confirm the DNA sequence.
In Vitro Transcription-RNA probes were generated by in vitro transcription of PCR products as described (18). PCR was performed using one primer that had a T7 promoter sequence added at the 5Ј end. Amplification of the target DNA yields a PCR product with the T7 promoter upstream of the sequence of interest. The PCR product then served as a template for run off in vitro transcription by purified T7 RNA polymerase. The templates and primers used in PCR to generate various RNA probes are given in Table I.
Labeled RNAs were synthesized by in vitro transcription with T7 RNA polymerase using 5 l of PCR product as template in the presence of [␣-32 P]ATP (800 Ci/mmol, NEN Life Science Products) using the Maxiscript kit from Ambion. After electrophoresis on 5% polyacrylamide gels containing 8 M urea, full-length transcripts were eluted from gel slices by overnight incubation at 37°C in 0.4 ml of 0.5 M ammonium acetate, 0.1% SDS, 1 mM EDTA buffer. The RNAs were phenol-chloroform extracted, precipitated with ethanol, and resuspended in diethyl pyrocarbonate-treated water. The probes were heated to 70°C for 15 min and slowly cooled to room temperature before use in gel retardation assays. Some purified RNA probes gave two bands on nondenaturing gels. These are probably due to formation of stable secondary structures, since electrophoresis of these RNAs in denaturing gels gave single bands.
Partial Purification of the Binding Activity-Nuclear extracts were thawed on ice and centrifuged for 60 min at 40,000 rpm in a Ti45 rotor at 2°C. Solid ammonium sulfate was added to the supernatant to give 40% saturation, and the mixture was stirred on ice for 60 min. The solution was centrifuged at 15,000 rpm for 20 min, and the pellet was dissolved in Buffer A (20 mM Tris-HCl, pH 8.0, 1 mM dithiothreitol, 5 mM MgCl 2 , 10% glycerol) and dialyzed against the same buffer for 60 min at 4°C. After a 30-min centrifugation at 40,000 rpm in a Ti45 rotor, the supernatant was applied on a 40 ml DEAE-cellulose column equilibrated in Buffer A containing 50 mM KCl. The column was washed with 120 ml of Buffer A containing 50 mM KCl, and the binding activity was eluted with Buffer A containing 100 mM KCl. The fractions containing binding activity were pooled, and the proteins were concentrated by precipitation with 40% ammonium sulfate as above and dissolved in buffer containing 20 mM HEPES-KOH, pH 7.9, 1 mM dithiothreitol, 5 mM MgCl 2 , and 20% glycerol. The partially purified proteins were also electrophoresed on 10% polyacrylamide gels containing 0.1% SDS (19), and the polypeptides present were visualized by staining the gels with Coomassie Blue.
UV Cross-linking and SDS-PAGE-The TOP2 Ϫ291 to Ϫ209 RNA probe was incubated with 10 g of protein partially purified by ammonium sulfate precipitation and DEAE-cellulose chromatography. After 15 min at 28°C, the RNA-protein complexes were resolved by nondenaturing polyacrylamide electrophoresis as above. The wet gel was exposed to x-ray film, and the gel slices containing the shifted complexes were excised, covered with Saran Wrap, and exposed to UV light for 1 min at a distance of 9 cm from the light source (Stratalinker, Stratagene). The gel slices were equilibrated with 180 l of buffer (12.5 mM Tris-HCl, pH 6.8, 0.1% SDS, 2% glycerol) for 30 min at 37°C and then heated to 95°C for 5 min. The solution was transferred to a fresh tube, and the RNA was digested by adding a 10-l solution containing a mixture of RNase A (5 g) and RNase T1 (100 units) and incubating for 60 min at 37°C. The sample was then mixed with 1 l of ␤-mercaptoethanol, heated at 95°C for 2 min, and applied on an 8.5% polyacrylamide gel containing 0.1% SDS (19). The gel was dried and autoradiographed. Prestained molecular weight markers (Bio-Rad) were used as standards in the electrophoresis.
Hydroxyurea Synchronization-The synchronization of C. fasciculata cultures by hydroxyurea was performed as described previously (4,15). Nuclear and cytosolic extracts were prepared from cultures 90 and 180 min after release from hydroxyurea arrest and assayed for binding activity.

RESULTS
The TOP2 5Ј-UTR RNA Binds to a Factor(s) in Nuclear Extracts-An 83-base pair DNA fragment from Ϫ291 to Ϫ209 of the TOP2 5ЈUTR has been shown to confer periodic expression to a reporter gene (15). Since gene expression in trypanosomes is regulated mainly at the post-transcriptional level, we wanted to determine whether C. fasciculata cell extracts contained a factor(s) that would bind to RNA from this region. An electrophoretic method for detecting RNA-protein interactions was used for this purpose (20). Nuclear and cytosolic extracts were incubated with 32 P-labeled RNA, and the protein-RNA complexes were resolved by electrophoresis on nondenaturing polyacrylamide gels. Heparin, a polyanion, was used to displace proteins bound nonspecifically to the labeled RNA. Gel shift results show that C. fasciculata nuclear extracts contain a factor(s) that binds to the Ϫ291 to Ϫ209 sense RNA of the TOP2 gene (Fig. 1, lanes 3 and 4). A smaller amount of binding activity was present in cytosolic extract (lane 2) that could be due to some breakage of nuclei during cell lysis. Two gel-shifted bands were seen on the gel with nuclear extracts. At present the basis for the different mobilities of these complexes is unknown. The relative intensity of these two bands varies in different preparations of nuclear extracts.
To investigate the specificity of RNA binding activity in nuclear extracts, we examined binding to an antisense transcript of the Ϫ291 to Ϫ209 region and to a lacZ transcript. No

5Ј-UTR-binding Factor(s)
binding was observed when the antisense RNA was incubated with cytosolic or nuclear extracts (Fig. 1, lanes 6 and 7). The lacZ sense RNA also showed no binding with cytosolic or nuclear extracts (lanes 9 and 10). The specificity of binding is also indicated by gel retardation assays carried out in the presence of unlabeled competitor RNA (Fig. 2B). A 10-fold molar excess of unlabeled TOP2 Ϫ291 to Ϫ209 sense RNA greatly reduced binding to the labeled RNA (Fig. 2B, lanes 2 and 3). However unlabeled lacZ RNA did not compete for binding at a similar concentration (lane 4). Further experiments were done to characterize the nature and specificity of binding seen to Ϫ291 to Ϫ209 TOP2 RNA. No complex formation took place when the nuclear extract was pretreated with proteinase K, confirming the involvement of a protein factor(s) in the binding reaction ( Fig. 2A, lane 3). However, pretreatment of the nuclear extract with DNase I or inclusion of poly(dI-dC) had no effect on binding ( Fig. 2A, lanes 4 and 5), ruling out a role for DNA in the binding reaction. These results show that C. fasciculata nuclear extracts contain a protein factor(s) that binds specifically to an 83-nucleotide RNA from the TOP2 5Ј-UTR.
Octamer Sequences Present in the TOP2 5ЈUTR RNA Are Involved in Binding-The Ϫ291 to Ϫ209 region of TOP2 gene contains two copies of the consensus octamer sequence found in the 5Ј or 3Ј-UTRs of several C. fasciculata mRNAs that vary as cells progress through the cell cycle (Table I). Deletion of either of the two octamer sequences resulted in reduced cycling of the mRNA of a reporter gene, whereas deletion of both octamers completely abolished cycling of the mRNA (15). To investigate the requirement for the consensus octamer sequence in binding to the RNA, we performed gel retardation assays using labeled RNAs in which one or both octamers were mutated to a different sequence (CAUAGAAG to UGCGAGGC). Mutation of either the left or right octamer sequence greatly reduced binding as compared with wild type RNA (Fig. 3). Interestingly, muta-tion of the right octamer had a greater effect on binding activity than mutation of the left octamer. We do not know the significance of this observation, although deletion of either octamer sequence reduced reporter mRNA cycling to the same extent

TABLE I RNA probes used in gel retardation assays
Plasmids linearized with HindIII were used as templates in PCR to introduce a T7 promoter upstream of the sequence to be transcribed by T7 RNA polymerase in the presence of ␣-32 P to generate the given 32 P-labeled RNA probes. Open and filled squares represent wild type and mutant octamer sequences, respectively (Probe 5 is the Ϫ291 to Ϫ209 antisense RNA). The sequences of the primers used are: C52, T7 promoter-CGCATAGAAGTATTGCGGGT; C53, T7 promoter-TCTTC-TATGGGCTTTCGACA; C54, T7 promoter-CGTGCGAGGATATTGC-GGGT; C95, T7 promoter-ATCACCAACGCTCACAGAAA; C69, ATAA-GAATGCGGCCGCTGAAATAGGTTACGTGGGAAGG. The T7 promoter sequence in these primers is GGATCCTAATACGACTCACTAT-AGGGAGG. The sequences of primers B92, B93, and C47 are given under "Experimental Procedures." 5Ј-UTR-binding Factor(s) (15). No binding was seen to an RNA in which both octamers had been mutated. These results suggest that the consensus octamer sequence is required for binding of RNA to a factor(s) in nuclear extracts. Moreover, the results from these binding assays complement those measuring cycling of mRNA levels in synchronized cells. Mutations that reduce or abolish mRNA cycling also affect the RNA binding in a similar manner.
Octamer Sequences Are Required for Binding of a Factor(s) to 5ЈUTR RNA of the RPA1 Gene-A 349-base pair fragment, from Ϫ523 to Ϫ174, of the RPA1 5Ј-UTR is required for periodic accumulation of the RPA1 transcript (16). This region contains two copies of the consensus octamer sequence. Subcloning into a reporter plasmid has shown that a 113-base pair DNA fragment from Ϫ343 to Ϫ231 containing the two octamer sequences can confer periodic accumulation on a heterologous transcript. Mutation of both octamer sequences abolished cycling of the reporter gene transcript, suggesting that these sequences function in a manner similar to that seen with the TOP2 gene.
We therefore wanted to determine whether C. fasciculata nuclear extracts contained a factor(s) that would also bind to the Ϫ343 to Ϫ231 RPA1 RNA. Fig. 4 shows that the wild type RPA1 RNA binds to a factor(s) in the nuclear extracts. Two retarded bands are present, as seen earlier with TOP2 RNA. No binding activity is present in the cytosolic extracts. Again, as seen with TOP2 RNA, no binding was observed when an RNA in which both octamers had been mutated to a different sequence was used in the gel retardation assay. These results show that nuclear extracts have a factor(s) that binds to RNA from the 5Ј-UTR of RPA1, and that octamer sequences present in the RNA are required for binding to occur.
TOP2 and RPA1 RNAs Compete for Binding to a Factor(s) in Nuclear Extracts-The above results from gel retardation assays show that octamer sequences are required for binding of TOP2 or RPA1 5Ј-UTR RNA to a factor(s) present in nuclear extracts. To determine whether the same factor(s) is involved in binding to both TOP2 and RPA1 RNA probes, a competition experiment was done in which nuclear extracts were incubated with wild type RPA1-labeled RNA in the presence of unlabeled TOP2 RNA. The presence of unlabeled TOP2 RNA prevents binding of a factor(s) in nuclear extracts to the RPA1 RNA probe (Fig. 5). This suggests that both RNAs bind to the same nuclear factor(s). The elution profile of the TOP2 and RPA1 RNA binding activity was also determined after column chromatography. The binding activity was partially purified by ammonium sulfate precipitation of proteins in the nuclear extract followed by successive chromatography over DEAE-cellulose, phosphocellulose, and heparin-Sepharose columns. A similar elution profile was observed when the heparin-Sepharose column fractions were assayed with either TOP2 or RPA1 RNA probes (data not shown). Results of the competition experiment and the column chromatographic profiles of the factor(s) that binds to RPA1 and TOP2 RNA strongly suggest that the same factor(s) is involved in binding to both RNA probes.
UV Cross-linking of Proteins in Nuclear Extracts to TOP2 5Ј-UTR RNA-We sought to identify proteins bound to the 5Ј-UTR of TOP2 RNA in gel-shifted complexes by cross-linking to a radiolabeled probe. The binding activity was partially purified and then incubated with the TOP2 Ϫ291 to Ϫ209 RNA probe. The RNA-protein complexes were separated from other proteins and free probe by nondenaturing polyacrylamide gel electrophoresis. Gel slices containing the shifted bands were irradiated with UV light, and the complexes were eluted from the slices and treated with RNases A and T1. SDS-PAGE analysis of the RNase-treated complexes showed two bands with apparent molecular masses of 74 and 37 kDa (Fig. 6A). These bands were absent when the eluted complexes were treated with protease K (data not shown). At present we do not know if these polypeptides represent two different subunits of the binding protein or if the 74-kDa polypeptide might represent a dimer of the 37-kDa protein held together by a short oligonucleotide resistant to nuclease digestion. The partially purified proteins used in the RNA gel shift were analyzed by FIG. 3. Effect of mutations in octamer sequences in TOP2 5-UTR RNA on binding. The Ϫ291 to Ϫ209 TOP2 sense RNA probes containing wild type or mutant octamer sequences (see Table I) were incubated in the absence (Ϫ) or presence (ϩ) of 20 g of nuclear extracts (NE) and analyzed by nondenaturing polyacrylamide gel electrophoresis.

5Ј-UTR-binding Factor(s)
SDS-PAGE. The Coomassie-stained gel showed that the 74and 37-kDa polypeptides were not major proteins in the partially purified nuclear extract (Fig. 6B). This result indicates that the RNA probe was not simply cross-linked to two abundant nuclear proteins.
The Binding Activity Varies during the Cell Cycle-We have examined the relative level of binding activity in cells synchronized by the hydroxyurea arrest. Northern blot analysis of RNA isolated at various time intervals after release from hydroxyurea arrest has shown that the steady state transcript levels of the genes encoding RPA1, RPA2 (the gene encoding the RPA middle subunit), TOP2, and dihydrofolate reductasethymidylate synthase gene accumulate periodically during the cell cycle (4), with maximum transcript levels present at G 1 /S phase. To determine whether the TOP2 and RPA1 5Ј-UTR RNA binding activities also vary during the cell cycle, cultures of C. fasciculata were synchronized by treatment with hydroxyurea, and nuclear extracts were prepared from cells 90 and 180 min after release from the hydroxyurea block. These times represent minimum and maximum levels of the TOP2 and RPA1 mRNAs in the synchronously growing cells (4,16). Nuclear extracts were assayed for binding activity using the wild type Ϫ291 to Ϫ209 TOP2 RNA as probe. Fig. 7 shows that nuclear extracts prepared from cells 180 min after release from hydroxyurea arrest had severalfold higher binding activity than extracts from cells 90 min after hydroxyurea arrest. Similar results were obtained when nuclear extracts were assayed with wild type RPA1 RNA as probe (data not shown). These results show that the binding activity varies during the cell cycle and parallels the mRNA levels of at least four DNA replication genes.  6. UV cross-linking of protein(s) in nuclear extracts to TOP2 5-UTR RNA. A, the binding activity was partially purified as described under "Experimental Procedures," and 10 g of protein was incubated with TOP2 Ϫ291 to Ϫ209 RNA probe. After nondenaturing polyacrylamide gel electrophoresis, the gel slices containing the shifted complexes were exposed to UV light. The cross-linked RNA-protein complexes were eluted from the gel slices, digested with RNases A and T1, and separated by SDS-PAGE. The autoradiogram of the dried gel is shown above. The position and sizes (in kDa) of prestained protein standards are indicated at the side of the gel. B, the partially purified proteins (10 g) used in UV cross-linking were subjected to SDS-PAGE. The Coomassie Blue-stained gel showing the various polypeptides present in these partially purified proteins is shown above.

DISCUSSION
We have used a reporter plasmid previously to analyze deletion mutants of 5Ј-UTRs of TOP2 and RPA1 genes to identify cis elements involved in periodic expression of these genes (15,16). Both TOP2 and RPA1 genes have 5Ј-UTR elements that can confer periodic expression on the mRNA of a reporter gene. Comparison of the TOP2, RPA1, RPA2, and dihydrofolate reductase-thymidylate synthase gene 5Ј-UTR sequences showed the presence of a consensus octamer sequence CATAGAAG that was also shown to be required for cycling of the reporter gene transcript. Furthermore, deletion analysis involving TOP2 5Ј-UTR showed that the essential sequence elements had to be present on the mature mRNA and not just within the flanking DNA sequence. These results indicated that the regulation of gene expression is primarily at the post-transcriptional level and involves sequences within the 5Ј-UTR of the TOP2 mRNA. This is in agreement with the general observation that in trypanosomes, the expression of protein-coding genes transcribed by RNA polymerase II is regulated mainly at the post-transcriptional level (21,22).
In the present work we have attempted to identify transacting factor(s) that interact with RNA encoded by the 5Ј-UTR elements shown to be required for periodic expression of TOP2 and RPA1 genes. Our results provide evidence that proteins present in nuclear extracts of C. fasciculata interact specifically with 5Ј-UTR of TOP2 and RPA1 mRNA. The interaction was shown to be specific by three independent criteria. First, excess unlabeled 5Ј-UTR RNA of the TOP2 gene prevents formation of complexes, whereas no reduction in binding is seen with an unlabeled nonspecific RNA as competitor. Second, mutation of both octamer sequences present in either TOP2 or RPA1 RNA probes abolished binding to the probes. Since deletion of these octamer sequences or mutation to a different sequence abolishes cycling of the reporter gene transcript, it appears that these elements function by virtue of being recognized by nuclear proteins at the RNA level. Third, ultraviolet cross-linking of the gel-shifted complexes identified two specific proteins cross-linked to the probe RNA.
The proteins that bind to the 5Ј-UTR RNA of TOP2 and RPA1 genes appear to be the same for several reasons. First, binding activities to both RNAs are present in the nuclear extracts and not in cytosolic extracts. Second, the same sequence elements are required for binding of both RNAs; mutations in octamer sequences abolish binding to both RNA molecules. Third, both activities vary during the cell cycle in a similar manner. Fourth, the activities that bind to both RNA molecules show identical chromatographic elution profiles. Finally, the addition of unlabeled TOP2 RNA eliminates binding to labeled RPA1 RNA probe. Thus we suggest that the cell cycle regulation of genes involved in replication of nuclear DNA and kDNA is mediated at a posttranscriptional level by a common protein factor(s). It remains to be determined whether the coordinate regulation of DNA replication genes plays an important role in synchronizing nuclear DNA and kDNA replication in C. fasciculata.
There are several examples of 5Ј-and 3Ј-UTR elements affecting gene expression in trypanosomes. In Leishmania the intergenic region of the tubulin gene was shown to be essential when placed on the 5Ј side of the reporter chloramphenicol acetyltransferase gene. The region contained signals that did not affect transcription rate and probably modulated RNA stability (23). The developmentally regulated Leishmania donovani A2 genes are regulated by differential RNA stability that involves the 3Ј-UTR of A2 mRNA (24). In T. brucei, the expression of procyclin mRNA is regulated at the post-transcriptional level by cis elements in the 3Ј-UTR (25). In this case regulation occurs at the level of both mRNA stability and translation. Our results also indicate that the 5Ј-UTR of TOP2 and RPA1 regulates gene expression at a post-transcriptional level.
An interesting observation in these studies is that the level of binding activity varies in nuclear extracts prepared from synchronized cells at different stages of the cell cycle. The binding activity is severalfold higher in nuclear extracts prepared from cells 180 min after release from a hydroxyurea block, the time at which DNA replication gene transcript levels are maximal, as compared with extracts from cells 90 min after release from hydroxyurea, the time at which these transcript levels are at a minimum (4,15). As a working model we propose that the presence of the conserved hexamer sequence in an mRNA targets the RNA for destruction and that a binding activity expressed periodically during the cell cycle protects the RNA from destruction when bound to the RNA. The target mRNAs would therefore vary during the cell cycle in parallel with the level of the binding activity. Another feature of this model is the protection from degradation of mRNAs in which the hexamer sequence is mutated. In this case, the mRNA level would no longer cycle regardless of the variation of the level of the binding factor. Further studies are aimed at testing this model and elucidating the biochemical mechanism of the cell cycle regulation of DNA replication genes in trypanosomatids.