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• W2015646332 abstract "A consensus sequence present in the 5′- or 3′-untranslated regions of several Crithidia fasciculata messenger RNAs encoding proteins involved in DNA metabolism has been shown to be necessary for the periodic accumulation of these mRNAs during the cell cycle. A protein complex termed cycling sequence-binding protein (CSBP) has two subunits, CSBPA and CSBPB, and binds the consensus sequence with high specificity. The binding activity of CSBP was shown to vary during the cell cycle in parallel with the levels of putative target mRNAs. Although disruption of the CSBPA gene resulted in loss of both CSBPA and CSBPB, the putative target message levels still continued to vary during the cell cycle. The presence of an additional and distinct binding activity was revealed in these CSBPA null mutant cells. This activity, termed CSBP II, was also expressed in wild-type Crithidia cells. CSBP II has higher binding specificity for the cycling sequence element than the earlier described CSBP complex. Three polypeptides associated with purified CSBP II show specific binding to the cycling sequence. These proteins may represent a family of sequence-specific RNA-binding proteins involved in post-transcriptional regulation. A consensus sequence present in the 5′- or 3′-untranslated regions of several Crithidia fasciculata messenger RNAs encoding proteins involved in DNA metabolism has been shown to be necessary for the periodic accumulation of these mRNAs during the cell cycle. A protein complex termed cycling sequence-binding protein (CSBP) has two subunits, CSBPA and CSBPB, and binds the consensus sequence with high specificity. The binding activity of CSBP was shown to vary during the cell cycle in parallel with the levels of putative target mRNAs. Although disruption of the CSBPA gene resulted in loss of both CSBPA and CSBPB, the putative target message levels still continued to vary during the cell cycle. The presence of an additional and distinct binding activity was revealed in these CSBPA null mutant cells. This activity, termed CSBP II, was also expressed in wild-type Crithidia cells. CSBP II has higher binding specificity for the cycling sequence element than the earlier described CSBP complex. Three polypeptides associated with purified CSBP II show specific binding to the cycling sequence. These proteins may represent a family of sequence-specific RNA-binding proteins involved in post-transcriptional regulation. Trypanosomes contain a novel mitochondrial DNA network termed kinetoplast DNA consisting of thousands of minicircle DNA molecules and a small number of maxicircle DNA molecules interlocked to form a huge catenated structure (1Simpson L. Annu. Rev. Microbiol. 1987; 41: 363-382Crossref PubMed Google Scholar). Kinetoplast DNA replication occurs through a unique process in which the minicircles are released from the network for replication, and the newly replicated minicircles still containing nicks and gaps are reattached to the network periphery (for recent reviews see Refs. 2Berberof M. Vanhamme L. Tebabi P. Pays A. Jefferies D. Welburn S. Pays E. EMBO J. 1995; 14: 2925-2934Crossref PubMed Scopus (97) Google Scholar and 3Bates E.J. Knuepfer E. Smith D.F. Nucleic Acids Res. 2000; 28: 1211-1220Crossref PubMed Scopus (32) Google Scholar). However, the maxicircles replicate while remaining attached to the network. Unlike in higher eukaryotes where mitochondrial DNA replication occurs throughout the cell cycle, kinetoplast DNA replication in trypanosomes occurs in apparent synchrony with nuclear DNA replication (4Cosgrove W.B. Skeen M.J. J. Protozool. 1970; 17: 172-177Crossref PubMed Scopus (59) Google Scholar). Studies have been undertaken to understand the possible role that cell cycle-dependent coordinated expression of DNA replication genes may play in coordinating nuclear and kinetoplast DNA replication. Regulation of gene expression in trypanosomatids is predominantly post-transcriptional (reviewed in Ref. 5Vanhamme L. Pays E. Microbiol. Rev. 1995; 59: 223-240Crossref PubMed Google Scholar). 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) 1The 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. 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 (6Wilson K. Uyetake L. Boothroyd J. Exp. Parasitol. 1999; 91: 222-230Crossref PubMed Scopus (6) Google Scholar, 7Furger A. Schurch N. Kurath U. Roditi I. Mol. Cell. Biol. 1997; 17: 4372-4380Crossref PubMed Scopus (135) Google Scholar) and the variable surface glycoprotein gene transcripts in Trypanosoma brucei (2Berberof M. Vanhamme L. Tebabi P. Pays A. Jefferies D. Welburn S. Pays E. EMBO J. 1995; 14: 2925-2934Crossref PubMed Scopus (97) Google Scholar, 8Jefferies D. Tebabi P. Pays E. Mol. Cell. Biol. 1991; 11: 338-343Crossref PubMed Scopus (98) Google Scholar) and those of mucin (9D'Orso I. Frasch A.C. J. Biol. Chem. 2001; 276: 15783-15793Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar), amastin (10Coughlin B.C. Teixeira S.M. Kirchhoff L.V. Donelson J.E. J. Biol. Chem. 2000; 275: 12051-12060Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar), and H2A histone (11Maranon C. Thomas M.C. Puerta C. Alonso C. Lopez M.C. Biochim. Biophys. Acta. 2000; 1490: 1-10Crossref PubMed Scopus (20) Google Scholar) 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 (12D'Orso I. Frasch A.C. J. Biol. Chem. 2001; 276: 34801-34809Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar). The transacting factor 1 protein recognizes 44-nucleotide instability elements in the 3′-UTR region of mucin SMUG mRNA (13Di Noia J.M. D'Orso I. Sanchez D.O. Frasch A.C. J. Biol. Chem. 2000; 275: 10218-10227Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar) as well as GU-rich sequences. Homologs of the poly(A)-binding protein 1 have been cloned from T. brucei (14Hotchkiss T.L. Nerantzakis G.E. Dills S.C. Shang L. Read L.K. Mol. Biochem. Parasitol. 1999; 98: 117-129Crossref PubMed Scopus (23) Google Scholar) and Leishmania major (3Bates E.J. Knuepfer E. Smith D.F. Nucleic Acids Res. 2000; 28: 1211-1220Crossref PubMed Scopus (32) Google Scholar). 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 (15Pasion S.G. Brown G.W. Brown L.M. Ray D.S. J. Cell Sci. 1994; 107: 3515-3520Crossref PubMed Google Scholar). 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 (16Pasion S.G. Hines J.C. Ou X. Mahmood R. Ray D.S. Mol. Cell. Biol. 1996; 16: 6724-6735Crossref PubMed Scopus (40) Google Scholar, 17Brown L.M. Ray D.S. Nucleic Acids Res. 1997; 25: 3281-3289Crossref PubMed Scopus (28) Google Scholar). 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 (16Pasion S.G. Hines J.C. Ou X. Mahmood R. Ray D.S. Mol. Cell. Biol. 1996; 16: 6724-6735Crossref PubMed Scopus (40) Google Scholar). Introduction of six copies of the octamer sequence (6× octamer RNA) in the 5′-UTR of a gene that does not cycle under normal conditions resulted in cycling of the message (18Mahmood R. Hines J.C. Ray D.S. Mol. Cell. Biol. 1999; 19: 6174-6182Crossref PubMed Google Scholar). 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 (18Mahmood R. Hines J.C. Ray D.S. Mol. Cell. Biol. 1999; 19: 6174-6182Crossref PubMed Google Scholar, 19Mahmood R. Ray D.S. J. Biol. Chem. 1998; 273: 23729-23734Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar, 20Mahmood R. Mittra B. Hines J.C. Ray D.S. Mol. Cell. Biol. 2001; 21: 4453-4459Crossref PubMed Scopus (26) Google Scholar). The binding of CSBP to the TOP2 5′-UTR RNA and to the 6× octamer RNA probe varied during the cell cycle in parallel with the levels of the putative target mRNAs (18Mahmood R. Hines J.C. Ray D.S. Mol. Cell. Biol. 1999; 19: 6174-6182Crossref PubMed Google Scholar). 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 (20Mahmood R. Mittra B. Hines J.C. Ray D.S. Mol. Cell. Biol. 2001; 21: 4453-4459Crossref PubMed Scopus (26) Google Scholar), 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. Knock-out of CSBPA Gene by Gene Replacement—The CSBPA gene was targeted by homologous recombination. Targeting constructs were created to replace both alleles of the gene. Homologous sequences that were used in the targeting constructs were PCR-amplified from the plasmid pJH3748 (20Mahmood R. Mittra B. Hines J.C. Ray D.S. Mol. Cell. Biol. 2001; 21: 4453-4459Crossref PubMed Scopus (26) Google Scholar) with TaqDNA polymerase (Invitrogen) following conditions given by the supplier. The targeting sequences flanking the CSBPA gene were amplified with the primer pairs G17 (5′-GATATCTGCGCCGCGTGGGAA-3′) and F104 (5′ CTCGGGATCCACACCTCGAGTGTGTGTCAGCTTGTCGG-3′) and F65 (5′-CTCGGGATCCCATGCGAGGCGGGAAGGAGAGC-3′) and F66 (5′-GATATCCGCAGCCTCCGTCGCCGCT-3′), respectively. The amplified products were cloned separately in plasmid pCRII-TOPO using a TOPO TA cloning kit (Invitrogen), yielding the plasmids pMS38.1 and pMS38.2, respectively. The hygromycin phosphotransferase expression cassette was released from the plasmid pX63HYG (21Cruz A. Coburn C.M. Beverley S.M. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 7170-7174Crossref PubMed Scopus (280) Google Scholar) as a SalI-BglII fragment and ligated under cohesive-end ligation conditions with EcoRV-XhoI and BamHI-EcoRV fragments from pMS38.1 and pMS38.2, respectively. The linear ligation product was gel-purified and cloned at the EcoRV site of pGEM5 to give pMS38.6. Digestion of this plasmid construct yielded the EcoRV fragment b (Fig.1A) used for electroporation of Crithidia as described previously (15Pasion S.G. Brown G.W. Brown L.M. Ray D.S. J. Cell Sci. 1994; 107: 3515-3520Crossref PubMed Google Scholar). For targeting the second allele of CSBPA, the 5′-flanking sequence of the CSBPA gene was PCR-amplified using the primers G17 and F104, whereas the 3′-flanking sequence was amplified using the primer pair F105 (5′-CTCGGGATCCGCTTCGCTTCACGCTTCA-3′) and G18 (5′-GATATCTCTCCTCTCTCTCTG-3′). The amplified 5′-flanking sequence was digested with XhoI, and the 3′-flanking sequence was digested with BamHI and gel-purified. The purified products were ligated with a XhoI-BamHI neomycin phosphotransferase expression cassette derived from pX.2KO (15Pasion S.G. Brown G.W. Brown L.M. Ray D.S. J. Cell Sci. 1994; 107: 3515-3520Crossref PubMed Google Scholar). The linear ligation product was then cloned using a TOPO TA cloning kit to give pMS38.7. Digestion of pMS38.7 with EcoRV produced construct c (Fig. 1A), which was used for disruption of the remaining allele of CSBPA. Plasmid DNA (10 μg) containing the targeting constructs was digested with appropriate restriction enzymes to release the targeting fragments. The digested DNA was electroporated in C. fasciculata cells as described previously (15Pasion S.G. Brown G.W. Brown L.M. Ray D.S. J. Cell Sci. 1994; 107: 3515-3520Crossref PubMed Google Scholar). Electroporated cells were selected for drug resistance on agar plates containing brain heart infusion medium (Invitrogen) plus hemin (20 μg/ml) and streptomycin (100 μg/ml) in the presence of the appropriate drug. Genomic DNA was isolated from putative mutant strains and analyzed by Southern blot analysis to confirm each gene replacement. CSBPA Expression Construct—To 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 (22Brooks D.R. McCulloch R. Coombs G.H. Mottram J.C. FEMS Microbiol. Lett. 2000; 186: 287-291Crossref PubMed Google Scholar) 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 (15Pasion S.G. Brown G.W. Brown L.M. Ray D.S. J. Cell Sci. 1994; 107: 3515-3520Crossref PubMed Google Scholar) 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 Analysis—RNA was prepared from 2 × 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 Blotting—C. fasciculata cell lysate from 3 × 106 cells was fractionated by 10% SDS-PAGE and immunoblotted as described previously (15Pasion S.G. Brown G.W. Brown L.M. Ray D.S. J. Cell Sci. 1994; 107: 3515-3520Crossref PubMed Google Scholar). Blots were probed with polyclonal anti-CSBPA and anti-CSBPB sera at 1:5000 dilution. Preparation of RNA Probes—Radioactive 32P-labeled RNA probes were prepared by in vitro transcription reactions from the T7 polymerase promoter as described previously (18Mahmood R. Hines J.C. Ray D.S. Mol. Cell. Biol. 1999; 19: 6174-6182Crossref PubMed Google Scholar) using the Maxiscript kit (Ambion). Plasmids pRM16 and pRM23 (18Mahmood R. Hines J.C. Ray D.S. Mol. Cell. Biol. 1999; 19: 6174-6182Crossref PubMed Google Scholar) linearized with NotI were used as templates for preparation of the 6× wild-type (CAUAGAAG) and mutant (CAUAGcAG) octamer probes. TOP2 wild-type or mutant probes were prepared as described previously (19Mahmood R. Ray D.S. J. Biol. Chem. 1998; 273: 23729-23734Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar). 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 Assays—Cycling sequence binding activity was monitored by performing binding assays (19Mahmood R. Ray D.S. J. Biol. Chem. 1998; 273: 23729-23734Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar) 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 0.5× 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-linking—Binding reactions were performed as described previously with DE52 purified cell extracts or RNA affinity column-purified CSBP II activity using 32P-labeled 6× 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 4× Laemmli buffer (23Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207478) Google Scholar) 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 Extracts—Wild 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 5–7 × 107 cells/ml from 4 liters of culture. Cell extracts were prepared from the cells essentially as described previously (19Mahmood R. Ray D.S. J. Biol. Chem. 1998; 273: 23729-23734Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar). 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 Analysis—All 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 0–40% (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 0–40% 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 0–0.35 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 6× octamer RNA affinity column. The column was prepared as described earlier (14Hotchkiss T.L. Nerantzakis G.E. Dills S.C. Shang L. Read L.K. Mol. Biochem. Parasitol. 1999; 98: 117-129Crossref PubMed Scopus (23) Google Scholar) by attaching a RNA with six copies of the wild-type octamer cycling sequence (6× 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 Analyses—UNO 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 10–30% glycerol gradient sedimentation as described previously (20Mahmood R. Mittra B. Hines J.C. Ray D.S. Mol. Cell. Biol. 2001; 21: 4453-4459Crossref PubMed Scopus (26) Google Scholar). Another 50-μl aliquot of CSBP II was subjected to gel-sieving chromatography on a Superose 12 column (Amersham Biosciences) as detailed earlier (20Mahmood R. Mittra B. Hines J.C. Ray D.S. Mol. Cell. Biol. 2001; 21: 4453-4459Crossref PubMed Scopus (26) Google Scholar). CSBPA Gene Knock-out Results in the Loss of Both CSBPA and CSBPB Protein Expression—The CSBPA gene was disrupted by targeted gene replacement through homologous recombination. Constructs b and c (Fig 1A) were used to sequentially replace the two alleles of the CSBPA gene (construct a), thereby generating clones heterozygous for CSBPA gene (A+/–) and CSBPA null mutants (A–/–), respectively. Southern blots of the wild-type, CSBP(A +/–), and CSBP(A –/–) cells confirmed replacement of both alleles of the gene by drug cassettes (data not shown). Western blot analyses were performed with cell lysates from wild-type and mutant cells to confirm the absence of CSBPA protein in the mutant cells (Fig. 1B). The blot was probed with rabbit anti-CSBPA and anti-CSBPB antisera. Levels of both CSBPA and CSBPB were decreased in the heterozygote as compared with the level detected in wild-type cells. CSBPA was totally absent in extracts from CSBPA null mutant cells, but surprisingly, the CSBPB protein was also observed to be absent, even upon prolonged exposure of the blot. Thus, the knock-out of the CSBPA gene eventually depleted the cells of both CSBPA and CSBPB proteins. Northern blots of total RNA from CSBPA null mutant cells (Fig. 2A) show that the CSBPB mRNA level in the mutant cells is similar to that of wild-type cells. Also, episomal expression of CSBPA protein in the CSBPA mutant cells restored wild-type levels of both CSBPA and CSBPB proteins (Fig. 2B). These results indicate that the loss of expression of CSBPA affects CSBPB expression at a post-transcriptional level, possibly at the level of protein stability or synthesis. Although the mechanism of the apparent co-regulation of these proteins is unknown, the mutant cell line has allowed us to search for and identify a second protein complex that specifically binds the octamer consensus sequence. Target Message Levels Continue to Cycle in the Absence of CSBPA and CSBPB Proteins—We 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 180–240-min time period corresponding to the highest levels of DNA synthesis (15Pasion S.G. Brown G.W. Brown L.M. Ray D.S. J. Cell Sci. 1994; 107: 3515-3520Crossref PubMed Google Scholar). 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. Presence of a Cycling Sequence Binding Activity in CSBPA Null Mutant Cells—Evidence 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 (19Mahmood R. Ray D.S. J. Biol. Chem. 1998; 273: 23729-23734Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar). 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 (19Mahmood R. Ray D.S. J. Biol. Chem. 1998; 273: 23729-23734Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar). 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. Cycling Sequence Binding Activity in CSBPA Null Mutant Cells Is Sequence-specific—To 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" @default.
• W2015646332 created "2016-06-24" @default.
• W2015646332 creator A5022590898 @default.
• W2015646332 creator A5045871906 @default.
• W2015646332 creator A5071169022 @default.
• W2015646332 creator A5087832026 @default.
• W2015646332 date "2003-07-01" @default.
• W2015646332 modified "2023-09-26" @default.
• W2015646332 title "Presence of Multiple mRNA Cycling Sequence Element-binding Proteins in Crithidia fasciculata" @default.
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