From the Molecular Biology Institute and Department of Microbiology, Immunology and Molecular Genetics, University of California, Los Angeles, California 90095
Received for publication, April 24, 2003
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ABSTRACT |
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INTRODUCTION |
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Regulation of gene expression in trypanosomatids is predominantly post-transcriptional (reviewed in Ref. 5). Polycistronic messages are generated through constitutive transcription of protein-coding genes by RNA polymerase II, which then undergo 5'-trans-splicing and 3'-polyadenylation to produce the mature mRNA. Multiple points of regulation controlling expression of specific transcripts have been investigated. Cis-acting factors that affect mRNA stability have been identified within the 3'-untranslated region (UTR)1 of specific transcripts. AU-rich cis-regulatory elements present in the 3'-UTR have been shown to regulate the stability of transcripts of procyclic acidic repetitive proteins (EP and GPEET) or procyclins (6, 7) and the variable surface glycoprotein gene transcripts in Trypanosoma brucei (2, 8) and those of mucin (9), amastin (10), and H2A histone (11) genes in Trypanosoma cruzi. Trans-acting factor, a developmentally regulated U-rich RNA-binding protein involved in selective mRNA destabilization, has recently been identified in T. cruzi (12). The transacting factor 1 protein recognizes 44-nucleotide instability elements in the 3'-UTR region of mucin SMUG mRNA (13) as well as GU-rich sequences. Homologs of the poly(A)-binding protein 1 have been cloned from T. brucei (14) and Leishmania major (3). In higher eukaryotes, the poly(A)-binding protein 1 binding to the poly(A) tail of matured transcripts has been shown to enhance message stability.
Crithidia fasciculata, a member of the family trypanosomatidae that includes many human pathogens like T. brucei causing sleeping sickness and the Leishmania parasites causing a range of disease forms including the fatal visceral leishmaniasis, is infective to insect cells. Most biochemical studies of kinetoplast DNA replication have been carried out in Crithidia as it can be easily grown in large scale cultures that can also be synchronized by hydroxyurea treatment. In synchronized cultures, the transcript levels of the genes encoding the large and middle subunits of the nuclear protein RPA (RPA1 and RPA2, a homolog of the human replication protein A), dihydrofolate reductase-thymidylate synthase (DHFR-TS), the kinetoplast-specific topoisomerase II (TOP2), and the histone-like kinetoplast-associated protein 3 have been shown to cycle in parallel, reaching maximum levels during the late G1 and S phases and then declining rapidly during the G2 and M phases (15). Transcripts of these genes were found to possess one or more copies of a consensus octameric sequence (C/A)AUAGAA(G/A) with a highly conserved hexameric core in either their 5'- or 3'-UTR (16, 17). The sequence has been proposed to have a destabilizing role as mutations in the octamer sequence lead to accumulation of the messages to the highest level (16). Introduction of six copies of the octamer sequence (6x octamer RNA) in the 5'-UTR of a gene that does not cycle under normal conditions resulted in cycling of the message (18).
In our effort to understand the mechanism of this message cycling, we identified and purified a high molecular mass complex, the cycling sequence-binding protein (CSBP) that binds to the cycling sequence with high sequence specificity (1820). The binding of CSBP to the TOP2 5'-UTR RNA and to the 6x octamer RNA probe varied during the cell cycle in parallel with the levels of the putative target mRNAs (18). The putative target mRNAs are found to be most stable at times when the cycling sequence binding activity is high, and the message levels decline sharply in parallel with the decrease in binding activity. Based on these observations, we had proposed that the binding of the CSBP protein complex to the octamer sequence might confer a cell cycle-dependent stabilization of transcripts containing these sequence elements.
To study its functional role, the gene encoding CSBPA, one of the two subunits of the CSBP complex (20), has been disrupted by gene replacement. Although the knock-out of the gene resulted in a loss of both CSBPA and CSBPB proteins, the putative target message levels were found to still cycle in the CSBPA null mutant cells. This mutant cell line has allowed us to detect the presence of an alternate cycling sequence binding activity. This activity termed CSBP II is also a high molecular mass complex comparable to the earlier described CSBP complex. CSBP II also shows high sequence specificity in binding to RNA probes. We report here the identification, purification, and biochemical characterization of this CSBP II protein complex.
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MATERIALS AND METHODS |
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CSBPA Expression ConstructTo make the CSBPA expression construct, a 4.9-kb HindIII fragment of C. fasciculata genomic DNA containing CSBPA coding sequence and 1.6-kb 5'- and 2.0-kb 3'-flanking sequences was cloned at the HindIII site of the blasticidin resistance plasmid pGL437B (22) to give pMS38.10. A 235-bp fragment containing the N terminus of the CSBPB gene was deleted from the construct by PCR mutagenesis to yield the CSBPA expression plasmid pMS38.20. This plasmid was electroporated into C. fasciculata CSBPA null mutant cells as described previously (15) and plated on brain heart infusion medium (Invitrogen) plus hemin (20 µg/ml) and streptomycin (100 µg/ml) in the presence of blasticidin S HCl (Invitrogen) at 100 µg/ml.
Northern Blot AnalysisRNA was prepared from 2 x 107 Crithidia cells using an RNeasy kit (Qiagen). RNA samples (10 µg) were loaded onto 1.2% formaldehyde-agarose gels and electrophoresed for 17 h at 22 V with continuous circulation of buffer. The RNA was then transferred to Hybond-XL membrane (Amersham Biosciences), UV-cross-linked, and subsequently probed with different radioactive probes.
Western BlottingC. fasciculata cell lysate from 3 x 106 cells was fractionated by 10% SDS-PAGE and immunoblotted as described previously (15). Blots were probed with polyclonal anti-CSBPA and anti-CSBPB sera at 1:5000 dilution.
Preparation of RNA ProbesRadioactive 32P-labeled RNA probes were prepared by in vitro transcription reactions from the T7 polymerase promoter as described previously (18) using the Maxiscript kit (Ambion). Plasmids pRM16 and pRM23 (18) linearized with NotI were used as templates for preparation of the 6x wild-type (CAUAGAAG) and mutant (CAUAGcAG) octamer probes. TOP2 wild-type or mutant probes were prepared as described previously (19). The two octamer sequences present in the TOP2 5'-UTR were mutated singly or both from CATAGAAG to TGCGAGGA. Plasmids containing the wild-type 291 to 209 fragment of the TOP2 5'-UTR or its mutant forms were linearized with HindIII and used as templates for PCR reactions to introduce a T7 promoter upstream of the sequence to be transcribed. The PCR product was then used as template for synthesizing 32P-labeled RNA using the Ambion Maxiscript Kit. The RNA probes were gel-purified, heated at 65 °C for 15 min, and then allowed to cool to room temperature before being used in assays.
Gel Retardation AssaysCycling sequence binding activity was monitored by performing binding assays (19) using RNA probes in reactions containing 10 mg/ml heparin and RNase inhibitor (10 units/reaction). Formation of RNA-protein complex was observed by relative shift in mobility of the radiolabeled probe when electrophoresed in a polyacrylamide gel. Following incubation of the binding reactions at 28 °C for 30 min, the samples were analyzed by electrophoresis in a 6% (60:1 acrylamide:bisacrylamide) polyacrylamide gel (3 h at 4 °C, 150 V, in 0.5x Tris borate EDTA), which had been pre-electrophoresed for 45 min under the same running conditions. The gels were then dried and exposed to x-ray films at 70 °C with intensifying screens. To quantitate the extent of RNA-protein complex formation, dried gels were exposed to a PhosphorImager screen and radioactive bands were quantitated with a PhosphorImager (Amersham Biosciences).
UV-cross-linkingBinding reactions were performed as described previously with DE52 purified cell extracts or RNA affinity column-purified CSBP II activity using 32P-labeled 6x wild-type or mutant probes. Following incubation at 28 °C for 30 min, the reactions were chilled in ice for 5 min and then transferred onto a parafilm strip, which was again placed on ice. The binding reactions were then irradiated with UV light (Stratalinker, Stratagene) for 1 min at a distance of 9 cm from the light source. The reactions were transferred to fresh tubes to which RNase A and RNase T1 were added and further incubated at 37 °C for another 30 min. The samples were then combined with one-third volume of 4x Laemmli buffer (23) and heated at 100 °C for 3 min before being loaded onto a 0.5-mm thick, 15-cm long 10% SDS-polyacrylamide gel. Electrophoresis was carried out in TGS buffer (27 mM Tris, 187 mM glycine, 0.1% SDS) under a constant current of 20 mAmp for 3 h at room temperature. The gel was then dried and analyzed by phosphorimaging.
Preparation of Crithidia Cell ExtractsWild type or CSBPA null mutant C. fasciculata cells were grown in brain heart infusion medium (Invitrogen) supplemented with hemin (20 µg/ml) and streptomycin (100 µg/ml) at 28 °C with shaking. Cells were harvested at a concentration of 57 x 107 cells/ml from 4 liters of culture. Cell extracts were prepared from the cells essentially as described previously (19). The harvested cells were washed once with phosphate-buffered saline and then with buffer A (10 mM HEPES-KOH, pH 7.9, 1.5 mM MgCl2, 10 mM KCl, 1 mM DTT, 1 mM phenylmethylsulfonyl fluoride). The cells were resuspended in 25 ml of buffer B (buffer A containing 0.5% Nonidet P-40), incubated on ice for 10 min, and then centrifuged at 12,000 rpm for 15 min in a Sorvall SS34 rotor. The cell pellet was further resuspended in buffer C (20 mM HEPES-KOH, pH 7.9, 420 mM NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 1 mM DTT, 1 mM phenylmethylsulfonyl fluoride), passed through a 20-G needle five times, and further incubated on ice for 15 min before being centrifuged (SS34 rotor, 12,000 rpm, 15 min, 4 °C). An equal volume of buffer D (20 mM HEPES-KOH, pH 7.9, 50 mM KCl, 0.2 mM EDTA, 1.5 mM MgCl2, 20% glycerol, 1 mM DTT, 1 mM phenylmethylsulfonyl fluoride) was then added to the supernatant. The pellet extraction step with buffers C and D was repeated twice. The total extracts were pooled together and used for assays or were stored at 70 °C until further use.
Purification of CSBP II Activity and SDS-PAGE AnalysisAll of the steps in the purification of CSBP II activity were performed at 4 °C or below. For further purification of the CSBP II activity, the cell extracts were first subjected to 040% (of saturation at 0 °C) ammonium sulfate precipitation. The precipitated protein was dissolved in buffer E (20 mM Tris-HCl, pH 7.9, 5 mM MgCl2, 10% glycerol, 1 mM DTT) and centrifuged in a Beckman Ti45 rotor for 30 min at 40,000 rpm at 4 °C. The supernatant was collected and dialyzed against buffer E lacking glycerol for 1 h at 4 °C. Following dialysis, glycerol was added to the dialysate to 20% final concentration and the dialysate was centrifuged at 12,000 rpm for 15 min in a Sorvall SS34 rotor. The cleared supernatant was loaded onto a 25-ml DE52 column pre-equilibrated with buffer E containing 50 mM KCl. The column was first washed with 5 column volumes of equilibration buffer, and the CSBP II activity was eluted with buffer E containing 125 mM KCl. The active DE52 fractions were pooled and further concentrated with a 040% ammonium sulfate cut. The precipitated proteins were dissolved in buffer F (20 mM Tris-HCl, pH 7.9, 20% glycerol, 1 mM EDTA, 1 mM DTT) and divided into three aliquots. Each aliquot was filtered though 0.22 µM SpinX filters (Costar) and subsequently loaded onto a UNO Q6 anion exchange column (Bio-Rad) pre-equilibrated with buffer F. The column was washed with 5 column volumes of equilibration buffer, and the bound proteins were eluted with a gradient of 00.35 M KCl in buffer F. The UNO Q-purified active fractions were diluted with an equal volume of buffer F and then purified further on a 6x octamer RNA affinity column. The column was prepared as described earlier (14) by attaching a RNA with six copies of the wild-type octamer cycling sequence (6x CAUAGAAG) followed by a (A)25 tail onto a oligo(dT) matrix. The column was successively eluted with buffer F containing 0.05, 0.15, 0.3, 0.6, and 1 M KCl. The CSBP II activity eluted with buffer containing 1 M salt. The purified CSBP II protein fraction was analyzed by electrophoresis in a 10% polyacrylamide gel containing 0.1% SDS, and the protein bands were visualized by Sypro Ruby staining (Molecular Probes).
Glycerol Gradient and Gel Filtration AnalysesUNO Q-purified CSBP II activity was diluted by adding an equal volume of buffer containing 20 mM Tris-HCl, pH 7.9, and 1 mM DTT to reduce the glycerol and salt concentrations. BSA was added to the diluted protein for stabilization followed by concentration on a Centricon-10 column. The final buffer contained 20 mM Tris-HCl, pH 7.9, 100 mM KCl, 10% glycerol, and 1 mM DTT. Fifty microliters of this concentrated sample were analyzed by 1030% glycerol gradient sedimentation as described previously (20). Another 50-µl aliquot of CSBP II was subjected to gel-sieving chromatography on a Superose 12 column (Amersham Biosciences) as detailed earlier (20).
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RESULTS |
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Target Message Levels Continue to Cycle in the Absence of CSBPA and CSBPB ProteinsWe further investigated the role of CSBPA and CSBPB proteins in cycling of the putative target messages using CSBPA null mutant cells depleted of both CSBPA and CSBPB proteins. Northern blot analyses were performed with total RNA isolated at 30-min intervals from hydroxyurea-synchronized cells. The relative levels of TOP2, RPA1, and DHFR-TS mRNAs, which have been shown previously to cycle during the cell cycle, were determined (Fig. 3). The levels of these messages were found to still cycle even in the absence of CSBPA and CSBPB. The message levels were lowest between 90 and 150 min, the period in which the percentage of dividing cells is highest. The message levels then increased during the 180240-min time period corresponding to the highest levels of DNA synthesis (15). The level of CaBP message detected as the loading control was constant throughout the cell cycle. These data indicate that the cycling sequence-binding proteins CSBPA and CSBPB are not essential for cycling of the RPA1, TOP2, and DHFR-TS mRNA levels.
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Presence of a Cycling Sequence Binding Activity in CSBPA Null Mutant CellsEvidence of cycling of putative target messages in the CSBPA null mutant cells necessitated further search for any alternate cycling sequence binding activity other than CSBP. Gel-shifts assays were performed with whole-cell extracts of CSBPA null mutant cells using a 291 to 209 5'-UTR transcript of the TOP2 gene as probe (Fig. 4). This 83-nucleotide RNA includes two octamer cycling sequences, and binding to this RNA probe by CSBP was shown previously to depend on these cycling elements (19). The RNA probe (lane 1) containing two wild-type octameric sequences (represented by open squares) is efficiently bound by factor(s) in the CSBPA mutant cell extract (lane 2). However, when RNA probes with mutations in either one or both of the octamer sequences (represented by solid squares) were used in assays, binding was reduced (lanes 4 and 6) or completely abolished (lane 8). Mutation of the 3'-octamer (lanes 5 and 6) had a greater effect on binding than a mutation in the 5'-octamer (lanes 3 and 4). A similar observation was made previously with wild-type Crithidia nuclear extracts (19). No binding was observed when both the octamer sequences were mutated (lanes 7 and 8). A small shift of each of the radioactive probes relative to the bulk of the free probe is seen even in the absence of extract and is somewhat greater in the presence of extract. The nature of these species has not been investigated further. These results indicate the presence of additional cycling sequence binding protein(s) in CSBPA mutant cells other than CSBPA or CSBPB.
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Cycling Sequence Binding Activity in CSBPA Null Mutant Cells Is Sequence-specificTo determine the specificity of binding of the cycling sequence binding protein(s) in CSBPA null mutant cells, hereafter referred to as CSBP II, radiolabeled RNA containing six copies of the wild-type octamer sequence (separated by 2-nucleotide spacers) or its mutant forms with single nucleotide changes in each copy of the octamer were used as probes in gel-shift assays. CSBP II binding activity, which was partially purified on a DE52 column, bound to the wild-type sequence CAUAGAAG but not to mutant probes containing single nucleotide substitutions within the central hexamer (Fig. 5A). A minor and faster migrating species observed with the probe CAUAuAAG was not investigated further.
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Similar gel-shift experiments with extracts from wild-type Crithidia showed that the cycling sequence binding activity from wild-type Crithidia bound to the wild-type probe and also to a lesser extent to the mutant probes RM23 and RM24 that contain six copies of the mutated sequence CAUAGcAG and CAUAGAcG, respectively (Fig. 5B). These results indicate that CSBP II has a higher specificity for binding to the cycling sequence than does CSBP.
Presence of Multiple Cycling Sequence Binding Activities in CrithidiaIdentification of cycling sequence binding activity in CSBPA null mutant cells indicates the presence of multiple cycling sequence-binding proteins in Crithidia. CSBP II activity identified in CSBPA null mutant cells could be present in addition to the previously identified CSBP complex in wild-type cells or alternatively may be induced in CSBP(A/) mutant cells. Wild-type RM16 probe and the mutant RM23 probe were used to address this question. Binding activity from wild-type cells bound to both RM16 and RM23 probes (Fig. 6A). Additional incubation with anti-CSBPA antibody supershifted bound complexes into the well for both the probes. However, anti-CSBPA antibodies only partially shifted the complex formed with the wild-type RM16 probe even when higher amounts of antibody were used. In contrast, the complex formed with the mutant RM23 probe, which binds CSBP but not CSBP II, was efficiently supershifted by anti-CSBPA antibodies. The inability of anti-CSBPA antibodies to supershift all of the wild-type probe (RM16) in wild-type extracts indicates the presence of another binding activity other than the CSBP complex in wild-type cells. Western blot analyses of the immunodepleted extract confirmed total removal of both CSBPA and CSBPB (Fig. 6B). The immunodepleted extract, when used in RNA gel-shift assays, bound the wild-type probe only and not the mutant probe (Fig. 6A). Also, the complex of the wild-type probe and CSBPA-immunodepleted extract could not be efficiently supershifted with anti-CSBPA antibody. The gel shift by the CSBPA-immunodepleted extract is similar to that of the CSBP II activity from CSBP(A/) cells described earlier. This observation suggests that the residual cycling sequence binding activity in the CSBPA- and CSBPB-depleted wild-type extract is probably attributed to CSBP II.
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UV-cross-linking experiments were performed using radiolabeled RM16 and RM23 RNA probes to identify polypeptides bound to the wild-type and mutant probes (Fig. 6C). Five polypeptides in the wild-type Crithidia extract corresponding to approximate molecular masses of 68, 52, 50, 38, and 35 kDa were cross-linked to the wild-type probe (lane 1), and only the 38- and 50-kDa polypeptides were cross-linked to the mutant probe (lane 2). The CSBPA-immunodepleted extract contained three polypeptides, 68, 52, and 35 kDa in size, that were cross-linked to the wild-type probe (lane 3). No radiolabeled bands were observed with the mutant RNA probe in the CSBPA-depleted wild-type extract (lane 4). These results suggest that the 38- and 50-kDa polypeptides that are missing in CSBPA-immunodepleted extracts are the previously identified CSBPA and CSBPB proteins, which were estimated from SDS gels earlier (18) to have molecular masses of 38 and 48 kDa, respectively. The difference in the estimate of the molecular mass of CSBPB is within the experimental error of such measurements. The 68-, 52-, and 35-kDa polypeptides appear to derive from the CSBP II protein. Since the CSBP complex has been shown to bind to the mutant RM23 probe in gel-shift experiments, UV-cross-linking experiments were performed in wild-type extracts with the addition of 30-fold excess of cold mutant RM 23 RNA as competitor to the radiolabeled RM16 probe (lane 5). The cold mutant RNA efficiently competed out binding of the 38- and 50-kDa polypeptides to the wild-type probe, consistent with their identification as CSBPA and CSBPB.
Cycling of CSBP II Activity in CSBPA Mutant CellsThe previously reported cycling sequence binding activity in Crithidia cells was shown to cycle in parallel with the levels of the putative target mRNAs that accumulate periodically during the cell cycle (18, 20). To determine whether the CSBP II activity also varies during the cell cycle, we prepared whole cell extracts from cell samples removed at 30-min intervals from a hydroxyurea-synchronized culture of CSBPA mutant cells. These extracts were assayed for their ability to bind RM16 RNA in gel-shift assays, which were quantitated by phosphorimaging analysis. The relative levels of CSBP II binding activity (Fig. 7A) and of the putative target mRNA (DHFR-TS) in the Northern blot (Fig. 7B) cycled synchronously. Relative levels of CSBP II binding activity are shown graphically in Fig. 7C. The percentage of dividing cells is shown as an indication of synchrony in the culture. Following release from hydroxyurea arrest, the CSBP II binding activity reaches its peak within 3060 min after cell division, attaining a maximum level around 180 min. The DHFR-TS mRNA levels follow the same trend and increase to a maximum at approximately the same time. CSBP II binding activity and DHFR-TS message levels decline rapidly between 180 and 300 min as the cell enters the G2/M phase. The CSBP II activity and DHFR-TS mRNA levels increase once more following another cell division. These results indicate that the binding activity of CSBP II, similar to that of CSBP, varies during the cell cycle in parallel with the mRNA levels of transcripts containing cycling sequence elements.
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Purification of CSBP II ActivityCSBP II was purified from
nuclear extracts of CSBPA mutant cells based on its ability to bind
RM16 RNA. For purifying CSBP II, we followed the basic chromatographic steps
used to purify CSBP (18).
Initial purification was achieved upon DE52 and UNO Q fast protein liquid
chromatography. CSBP II activity eluted from the UNO Q column with
180210 mM KCl in buffer. CSBP II was finally purified on an
RNA affinity column to which RM16 RNA containing an A25 tail was
bound to an oligo(dT) matrix. As the CSBP II binding to the octamer sequence
was sensitive to the presence of magnesium ion, the UNO Q-purified CSBP II
activity was loaded onto the RNA affinity column in the absence of magnesium
ion in the buffer, which instead contained 1 mM EDTA. This buffer
condition allows most efficient CSBP II binding to the RNA affinity column.
Following subsequent washes with buffers containing up to 0.6 M
KCl, the binding activity was finally eluted from the column with buffer
containing 1 M KCl. SDS-PAGE analysis of the active fractions
(Fig. 8) eluted with 1
M KCl shows a prominent polypeptide at 68 kDa and a doublet of
very closely migrating polypeptides
5052 kDa in molecular mass.
Two additional polypeptides with molecular masses of
35 and 73 kDa are
also present in the affinity-purified fraction.
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Three Polypeptides in RNA Affinity-purified CSBP II Bind 6x Octamer RNAUV-crosslinking was performed in solution to identify the octamer-specific RNA-binding polypeptides in the purified CSBP II following binding reactions using the radiolabeled RM16 or RM23 mutant probes and RNA affinity-purified CSBP II complex from CSBP(A/) cells. The UV-cross-linked RNA-protein complexes were digested with RNase A and RNase T1, after which the samples were analyzed by SDS-PAGE followed by autoradiography (Fig. 9). Three polypeptides of approximate molecular masses 35, 52, and 68 kDa were labeled when the wild-type RM16 RNA was used as the probe. No labeled protein band was visible in experiments using the mutant probe. These results are in agreement with our observations with CSBPA-depleted wild-type extract where three polypeptides of 35, 52 and 68 kDa were also cross-linked specifically to the wild-type RM16 probe. Polypeptides of comparable size to the cross-linked ones were also observed in the purified CSBP II fractions upon SDS-PAGE analysis (Fig. 8). The 73-kDa protein in the affinity-purified CSBP II was not cross-linked to the labeled probe.
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CSBP II Has a High Molecular MassPrevious analysis of CSBP
indicated that the protein complex has a molecular mass in excess of 200 kDa.
Since gel shifts obtained with CSBP and CSBP II are of similar magnitude, CSBP
II is likely to have a similar molecular mass. To estimate the molecular mass
of CSBP II, the sedimentation coefficient
(S20,w) and the Stoke's radius of the
binding activity of CSBP II was determined by glycerol gradient sedimentation
and gel-sieving chromatography, respectively. UNO Q-purified CSBP II activity
was subjected to velocity gradient sedimentation on 1030% glycerol
gradient containing 80 µg/ml BSA. The position of CSBP II activity was
determined by RNA gel-shift analysis of the collected fractions following
centrifugation. From the relative sedimentation of marker proteins of known
S20,w, the
S20,w for the CSBP II activity was
estimated to be 5.2 S (Fig.
10A).
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The Stoke's radius for CSBP II was determined by gel-sieving chromatography
on a Superose 12 column (Amersham Biosciences). The CSBP II binding activity
eluted from the column consistent with a Stoke's radius of 61 Å
(Fig. 10B). These
data predict a native molecular mass of the protein of 160 kDa with a
frictional ratio (f/f0) of 1.41
(24). This predicted molecular
mass of CSBP II is consistent with the presence of the three RNA-binding
proteins in the complex but appears to be too small to include the 73-kDa
protein as well. We cannot exclude the possibility that the 73-kDa protein has
a labile association with the other three proteins. Further characterization
of the molecular architecture of CSBP II is required to more precisely define
its structure.
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DISCUSSION |
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To further examine the role of CSBPA and CSBPB proteins and their possible involvement in mRNA cycling, we performed a knock-out of the CSBPA gene by gene replacement. Knock-out of CSBPA resulted in the loss of expression of both CSBPA and CSBPB in CSBPA null mutant cells. However, the cycling of the target mRNA levels remained unaffected even in the absence of both the CSBP proteins. Thus, although CSBPA and CSBPB proteins have a high specificity for binding to the octameric cycling sequence, they are not essential for the cell cycle regulation of the levels of mRNAs containing the octamer sequences.
We have identified an alternate cycling sequence-specific binding activity
called CSBP II in CSBPA null mutant cells. CSBP II RNA binding
activity is found to be sensitive to point mutations and very specific for the
hexameric core of the cycling sequence. Three polypeptides in the RNA
affinity-purified CSBP II activity, 68, 52, and 35 kDa in molecular mass,
are radiolabeled specifically when the wild-type 6x CAUAGAAG RNA is used
as probe in "in-solution" UV-cross-linking experiments. Similar
cross-linking experiments with the partially purified wild-type
Crithidia extract show five radiolabeled polypeptides approximately
68, 52, 50, 38, and 35 kDa in molecular mass. This finding differs from our
earlier observation where polypeptides of
74 and 38 kDa were labeled
(18,
19) when cross-linking was
performed within the polyacrylamide gel followed by elution of the
cross-linked complex from the gel. Our recent UV-cross-linking experiments
done in-solution maximized the yield of the RNA-binding proteins. The 74-kDa
species observed earlier probably corresponds to the 68-kDa protein observed
here considering the experimental error in such experiments. The correlation
between the protein species cross-linked to the 6x octamer probe in cell
extracts and the polypeptides present in CSBP and CSBP II indicates that both
CSBP and CSBP II activities are expressed in the wild-type Crithidia
cells.
Binding of CSBP II to the wild-type probe in extracts of the mutant cells varies during the cell cycle, reaching maximum levels as the cells transit S phase and declining sharply prior to cell division. Levels of the cycling target mRNAs also follow a similar pattern. These results are similar to those observed in wild-type cells (20). We had earlier hypothesized that the periodic binding of trans-regulatory factor(s) to cycling sequence elements in target mRNAs could stabilize the messages in a cell cycle-dependent manner. Parallel cycling of cycling sequence binding activity and target mRNA levels in the absence of CSBPA and CSBPB proteins in CSBPA null mutant cells is consistent with but does not prove a role for the CSBP II complex in the cycling of target mRNAs. Identification of proteins directly involved in the degradation of target mRNAs will be essential for further assessing the possible role of CSBP II in mRNA cycling.
It will also be important to determine the molecular composition of the
CSBP II complex. Three of the five polypeptides in the RNA affinity-purified
activity are specific RNA-binding proteins. The molecular masses of these
three proteins taken together add up to 160 kDa, which is comparable to
the molecular mass calculated for the CSBP II complex with data available from
size exclusion chromatography and glycerol gradient experiments. Whereas the
presence of multiple specific RNA-binding proteins in a single complex is
unusual, we note that previous studies of CSBP indicated that both CSBPA and
CSBPB subunits bind to the cycling sequence element and also
co-immunoprecipitate (15).
SDS-PAGE shows the presence of an additional protein of
73 kDa in the
purified fraction apart from the three cycling sequence-binding proteins.
Cloning of the genes encoding the proteins in the CSBP II complex will allow
us to develop the immunological reagents necessary for addressing these
questions. Also, it will enable us to examine the intracellular localization
of CSBP II and its association with specific mRNA sequences in the cell. A
more complete picture of the proteins that interact with the cycling mRNAs
will be needed to understand the mechanism of cycling of these mRNA
levels.
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FOOTNOTES |
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To whom correspondence should be addressed: 301A Paul D. Boyer Hall, 611
Charles Young Dr. E., UCLA, Los Angeles, CA 90095-1570. Tel.: 310-825-4178;
Fax: 310-206-7286; E-mail:
danray{at}ucla.edu.
1 The abbreviations used are: UTR, untranslated region; RPA, replication
protein A; DHFR-TS, dihydrofolate reductase-thymidylate synthase; TOP,
topoisomerase; CSBP, cycling sequence-binding protein; DTT, dithiothreitol;
BSA, bovine serum albumin.
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REFERENCES |
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