Temperature-Specific Expression of a Tetrahymena thermophila ...

Vol. 8, No. 1

MOLECULAR AND CELLULAR BIOLOGY, Jan. 1988, p. 427-432

0270-7306/88/010427-06$02.00/0 Copyright © 1988, American Society for Microbiology

mRNA Stability Plays a Major Role in Regulating the Temperature-Specific Expression of a Tetrahymena thermophila Surface Protein HAROLD D. LOVE, JR.,' AVERIE ALLEN-NASH,' QIAN ZHAO,2 AND GARY A. BANNON'* Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences, Little Rock, Arkansas 72205,1 and Department of Biology, University of Rochester, Rochester, New York 146272 Received 17 August 1987/Accepted 19 October 1987 Synthesis of the serotype H3 (SerH3) surface antigen is temperature dependent and responds within 1 h to a change in incubation conditions (G. A. Bannon, R. Perkins-Dameron, and A. Allen-Nash, Mol. Cell. Biol. 6:3240-3245, 1986). Recently, a Tetrahymena thermophila cDNA clone (pC6; D. W. Martindale and P. J. Bruns, Mol. Cell. Biol. 3:1857-1865, 1983) has been shown to be homologous to a portion of the SerH3 mRNA (F. P. Doerder and R. L. Hallberg, personal communication), and it was shown that the cellular levels of this RNA rapidly decreased when cells were shifted from 30 to 41°C (R. L. Hallberg, K. W. Kraus, and R. C. Findly, Mol. Cell. Biol. 4:2170-2179, 1984). These observations indicate that synthesis of the SerH3 protein is highly regulated in response to temperature and led us to initiate studies to determine the mechanism(s) by which SerH3 gene expression is controlled. Using pC6 as a hybridization probe for the SerH3 mRNA, we have determined that (i) the level of SerH3 protein synthesis is directly correlated with the amount of SerH3 message available for translation; (ii) there is, at most, a twofold difference between the relative transcription rates of SerH3 genes at 30 and 40°C; (iii) the SerH3 mRNA half-life in cells incubated at 30°C is greater than 1 h, whereas the half-life in cells incubated at 40°C is only -3 min. These results demonstrate that Tetrahymena SerH3 surface protein expression is regulated by mRNA abundance. Furthermore, the major mechanism controlling mRNA abundance is a dramatic temperature-dependent change in SerH3 mRNA stability.

tion). These observations indicate that synthesis of the SerH3 protein is highly regulated in response to temperature and led us to initiate studies to determine the mechanism(s) by which SerH3 gene expression is controlled. In this report, we describe the findings that (i) SerH3 protein synthesis is controlled at the level of mRNA abundance, (ii) there is, at most, a twofold difference between the relative transcription rates of SerH3 genes at 30 and 40°C, and (iii) SerH3 mRNA stability changes dramatically when cells incubated at 30 and 40°C are compared.

Gene expression in eucaryotes is regulated at a variety of levels. The recent emphasis on delineating DNA sequence elements and the proteins involved in transcriptional control has resulted in a much better understanding of how genetic expression is regulated. However, transcriptional regulation by itself may not account for all of the rapid changes in gene expression. Response to sudden environmental change is an example where a rapid change in gene expression may occur. Although the responses of procaryotes to environmental stimuli have been extensively investigated, only a few eucaryotic systems are amenable to such studies. Tetrahymena thermophila is capable of producing up to five mutually exclusive, antigenically distinct peptides under different conditions of temperature or incubation media. The immunogenic surface proteins of the Tetrahymena sp. are known as immobilization antigens because antisera raised against them have the ability to stop cell movement when mixed with living cells. There are at least five immunologically distinct serotypes. The expression of three of these serotypes is temperature dependent. Serotype H (SerH) is produced between 20 and 35°C, whereas serotype T is produced at temperatures above 35°C. Serotype L is produced at incubation temperatures below 20°C. SerH has been the most extensively studied. There are four antigenically distinct H types (SerHl through SerH4) resulting from allelic variants of the SerH locus (6). The allelic SerH proteins range in size from 44 to 58 kilodaltons (3, 6). Synthesis of the SerH3 protein is temperature dependent and responds within 1 h to a change in temperature (3, 22). Recently, a Tetrahymena cDNA clone (pC6 [14]) has been shown to code for a portion of the SerH3 mRNA (F. P. Doerder and R. L. Hallberg, personal communica*

MATERIALS AND METHODS Cells and culture conditions. Tetrahymena thermophila strains (CU355 and CU427) were grown axenically in enriched proteose peptone (1, 9) at 30 or 40°C. Both strains are homozygous for SerH3 expression. RNA isolation, Northern (RNA) gels, and slot blots. Tetrahymena RNA was isolated according to the methods of Calzone et al. (4). RNA for Northern gels was suspended in a buffer containing 20 mM morpholinepropanesulfonic acid (MOPS) (pH 7.0), 5 mM sodium acetate, 0.5 mM EDTA, 50% formamide, and 5% formaldehyde. RNA was heated to 60°C for 5 min before being fractionated on formaldehydeagarose gels (1% agarose, 1 mM EDTA, 10 mM sodium acetate, 40 mM MOPS [pH 7.0], 2.2 M formaldehyde). After electrophoresis, gels were blotted to nitrocellulose in 20x SSPE (20x SSPE is 20 mM disodium EDTA, 160 mM NaOH, 200 mM NaH2PO4- H20, and 3.6 M NaCl). RNA for slot blots was prepared according to a Schleicher & Schuell Sequences Applications Update. Probes. The SerH3 gene probe used in this study is a cDNA clone (pC6; [14]) isolated from a Tetrahymena conjugation-specific library. pC6 contains 760 base pairs (bp) of the SerH3 mRNA cloned into the PstI site of pBR322. Insert

Corresponding author. 427

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DNA was isolated by electrophoretic elution from agarose gels into dialysis bags (13). XTt2O is a cDNA clone isolated from a Tetrahymena library cloned into Xgtll (25). The -300 bp insert identifies an mRNA of -600 bases whose steadystate levels are indistinguishable in cells at 30 and 40°C (data not shown). Hybridizations. All Northern and slot blots were hybridized either with the SerH3 cDNA insert, XTt2O or oligo(dT). The SerH3 cDNA insert and XTt2O were random primer labeled (7, 8) to a specific activity of 108 cpm/p,g of DNA and oligo (dT) (12 to 18 nucleotides) was kinase labeled (21) with [y-32P]ATP to a specific activity of 105 cpm/,ug of DNA. SerH3 and XTt2O were hybridized to filters at 65°C in 5x SSPE-bovine serum albumin (1 mg/ml)-1 x SPED (lx SPED is 0.1% Ficoll [Pharmacia Fine Chemicals], 0.1% polyvinylpyrrolidone 360, 0.1% bovine serum albumin, 6 mM sodium dodecyl sulfate [SDS], 2 mM disodium pyrophosphate, 2 mM tetrahydro-EDTA). After incubation for >12 h, the filters were washed in six changes of room temperature 2x SSPE-0.1% SDS. A final wash was done at 650C in the same solution. Oligo(dT) was hybridized and washed in the same solution, except all washes were carried out at room temperature. Filters were dried and used to expose Kodak XAR-5 X-ray film. Quantitation of filters. The amount of poly(A)+ RNA on slot blots (see Fig. 2) was quantitated by oligo(dT) hybridization. After hybridization with SerH3 or oligo(dT) probes, filters were autoradiographed and densitometric scans of the X-ray film were performed on an EC densitometer. Areas were calculated by using a Biomed Instruments computer program and those values were used to calculate the relative concentrations of SerH3 mRNAs in cells incubated at 30 and 400C. Actinomycin D experiments. Actinomycin D was used at a final concentration of 25 ,ug/ml in our experiment. (See the legends to-Fig. 4 and 5 for further descriptions.) At this concentration, there was an -95% inhibition of [3H]uridine incorporation into trichloroacetic acid-precipitable material for cells incubated at 30 or 40°C. In vitro nuclear runoff experiments. The in vitro nuclear runoff technique used in this paper was developed by Quian Zhao in Martin Gorovsky's laboratory at the University of Rochester. Briefly, cells were grown overnight at 30 or 400C to a density of -200,000 cells per ml. Cells were collected by pouring them over tubes packed with ice and pelleting them at 250 x g for 5 min. Pellets were suspended in ice-cold extract buffer [0.1 M sucrose, 0.1 M KCl, 2.5 mM MgCI2, 2.5 mM ethylene glycol-bis(P-aminoethyl ether)-N,N,N',N'tetraacetic acid (EGTA), 10 mM HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid) (pH 6.8), 1% Triton X-100] designed to produce cytoskeletal frameworks (23) and incubated on ice for 5 min. Cytoskeletal frameworks were washed twice in ice-cold extract buffer without Triton X-100. The final pellet was suspended in transcription buffer 1 (0.05 M Tris, 0.05 M KCI, 0.005 M MgCl2, 0.001 M spermidine, 0.001 M spermine, 0.001 M CaCl2, 0.002 M 2-mercaptoethanol, 0.1 M sucrose, 25% glycerol [pH 8.0]) at a concentration of 2 x 106 ghosts per ml. Cytoskeletal frameworks (50 ,ul) in transcription buffer 1 were added to 25 ,ul of transcription buffer 2 (0.1 M Tris, 0.1 M KCI, 0.01 M MgCl2, 0.002 M spermidine, 0.002 M spermine, 0.004 M putrescine, 0.006 M 2-mercaptoethanol, 0.002 M CaCl2, 0.0012 M aurintricarboxylic acid [pH 8.0]), 2.5 ,ul each of 20 mM ATP, CTP, and GTP, 40 U of RNasin, and 125 ,uCi of [a-32P]UTP and incubated at 25°C for 40 min. RNA was extracted, and an equal number of trichloroacetic acid-

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FIG. 1. Northern blot of poly(A)+ RNA isolated from T. thermophila grown at 30 or 40°C for 12 h. RNA was electrophoresed on a 1.0% formaldehyde-agarose gel, blotted to nitrocellulose, and probed with a cDNA clone (pC6) encoding 760 bases of the SerH3 mRNA. No hybridizable SerH3 mRNA was detected in cells grown at 400C.

precipitable counts per minute were added to each hybridization mix. The counts for 32P hybridized to each DNA were quantitated by excising the DNA from the nitrocellulose filter and performing liquid scintillation counting. The counts for 32P hybridized to pBR322 were subtracted from the counts hybridized to pC6 to deternmine specific hybridization to the SerH3 cDNA. RESULTS We had previously shown that synthesis of the SerH3 surface protein was limited to incubation temperatures of 20 to 35°C. At temperatures above 35°C, the SerH3 surface protein was not expressed. Reappearance of the SerH3 protein at 35°C or below is inhibited by actinomycin D, suggesting that new SerH3 mRNA synthesis is required (3). These data were consistent with the idea that SerH3 protein synthesis was regulated, at least in part, by modulating steady-state levels of SerH3 mRNA. When poly(A)+ RNA from cells grown at 30°C was electrophoresed on denaturing agarose gels, blotted, and probed with the SerH3 cDNA (Fig. 1), a single 1.47-kilobase band was observed. When poly(A)+RNA isolated from cells grown at 40°C was probed with the SerH3 cDNA, no hybridization bands were detectable. These results indicate that SerH3 protein production is controlled at the level of mRNA availability. Rapid modulation of SerH3 mRNA steady-state levels in response to environmental temperature. Tetrahymena SerH3 protein synthesis responds within the first hour to a change in incubation temperature (3). To determine whether this rapid response was mediated by changing SerH3 mRNA levels, SerH3 mRNA amounts were determined while a cell population was subjected to a temperature shift. Cytoplasmic RNAs from each time point were fixed to nitrocellulose filters and probed with the SerH3 cDNA. Hybridization with oligo(dT) was used to quantitate the amount of poly(A)+RNA that was loaded on the filters. Changes in

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FIG. 2. Steady-state levels of SerH3 mRNA as cells are subjected to a temperature shift. Cells were incubated at 30°C for 12 h (time 0) and then shifted to 40°C for 12 h (times 0.3, 0.6, 1, and 12). Finally, cells were shifted back to 30°C for 1 h. Total cytoplasmic RNA was isolated at various times, dot blotted to nitrocellulose, and probed with pC6. Poly(A)+ RNA in each dot was quantitated by 32P-oligo(dT) hybridization, and pC6 hybridization was corrected to represent signals from equal amounts of total poly(A)' RNA. This graph represents results from one experiment. This experiment was repeated twice with similar results.

steady-state levels of the SerH3 mRNA occurred rapidly after a change in incubation temperature (Fig. 2). By 40 min after a shift from 30 to 40°C, SerH3 mRNAs were reduced by 70%. This decline continued until SerH3 mRNAs were undetectable. If this population of cells was then shifted back to 30°C, the steady-state level of SerH3 mRNAs approached 100% by 60 min after the temperature shift. These results show that the rapid response of SerH3 protein synthesis to a change in temperature is correlated with the rapid modulation of SerH3 mRNA steady-state levels. SerH3 transcription rates do not explain different SerH3 mRNA levels of cells at 30 and 40°C. Changing SerH3 transcription levels would be an obvious mechanism which could result in different levels of a specific mRNA. We determined the relative transcription rates of SerH3 genes from cells incubated for 12 h at 30 or 40°C by in vitro nuclear runoff experiments. Nuclear transcripts were labeled with [a-32P]UTP, and RNA was isolated and used to probe slot blots containing pC6 and pBR322 (Fig. 3). Surprisingly,

there was, at most, a twofold difference between the relative transcription rates of SerH3 genes from cells incubated at 30 or 40°C. This difference cannot account for the dramatic change in steady-state levels of the SerH3 message observed

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FIG. 3. Transcription of the SerH3 gene at 30 and 40°C. RNAs were elongated in vitro in the presence of [a-32P]UTP by using cytoskeletal frameworks isolated from cells at 30 or 40°C. The labeled RNAs were isolated and hybridized to denatured pC6 and pBR322 (pBR) plasmid DNAs.

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FIG. 4. SerH3 mRNA stability is different at 30 and 40°C. SerH3 message stability was determined at the two temperatures by addition of actinomycin D (25 ,ug/ml) to cells growing at 30°C and then either immediately shifting the cells to 40°C or continuing incubation at 30°C. Total cytoplasmic RNA was isolated at 0, 10, 20, 40, and 60 min after actinomycin D addition, blotted to nitrocellulose, and probed either with the SerH3 cDNA (pC6) or with a control cDNA (XTt2O, a clone isolated from a Tetrahymena cDNA library that codes for an mRNA that has similar steady-state levels in cells at both 30 and 40°C). Numbers across the top of the figure represent minutes after actinomycin D addition.

between cells incubated at 30 or 40°C and indicates that a posttranscriptional mechanism must play a key role in controlling SerH3 expression. Different stability of SerH3 mRNA at 30 and 40°C. A marked change in the stability of the SerH3 mRNA could be a possible mechanism to explain the rapid modulation of SerH3 mRNA steady-state levels observed as cells are shifted between 30 and 40°C. Cells were treated with an RNA synthesis inhibitor, actinomycin D (25 ,ug/ml), to determine whether the stability of SerH3 mRNAs changed under different temperature conditions. Total cytoplasmic RNA was isolated every 10 min for 1 h after the addition of the RNA synthesis inhibitor, blotted to nitrocellulose, and then probed with pC6 insert or XTt20 (Fig. 4A). The SerH3 mRNA at 30°C was much more stable than the SerH3 mRNA at 40°C. Significant quantities of the SerH3 message were still present 60 min after transcription was inhibited in 30°C cells, whereas the SerH3 mRNA was practically undetectable after only 10 min in cells incubated at 40°C. In contrast, a message present in equal levels in cells incubated at 30 and 40°C (data not shown) showed no stability change (Fig. 4B). In kinetic analyses of similar experiments, SerH3 mRNA half-lives were calculated in cells incubated at 30 or 40°C (Fig. 5A and B). The half-life of SerH3 mRNA in cells incubated at 30°C was greater than 1 h. The half-life of the

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FIG. 5. SerH3 mRNA half-lives at 30 and 400C. Slot blots were prepared exactly described in the legend to Fig. 3 except that more samples were taken during the early times for SerH3 half-life determination at 40°C. (A) Stability of the SerH3 mRNA at 30 and 40°C over a 20-min time period. (B) Stability of the SerH3 mRNA at 300C over a 2-h time period. For both panels, each point represents the average of at least three experiments. Bars represent standard error of the mean. as

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SerH3 mRNA in 40°C cells was only -3 min. These results, coupled with the in vitro nuclear runoff results, indicate that the mechanism operating to change SerH3 surface protein expression in T. thermophila is a significant change in the stability of the SerH3 surface protein message at 30 and

400C. DISCUSSION The rapidity with which T. thermophila regulates expression of SerH3 surface protein synthesis (3, 22) during a SerH to serotype T transformation requires a mechanism that is capable of responding to a change in incubation temperature in a matter of minutes. We have demonstrated that T. thermophila SerH3 surface protein synthesis is controlled largely by mRNA abundance. Furthermore, the major factor controlling SerH3 mRNA abundance involves changes in SerH3 mRNA stability, whereas transcriptional regulation of SerH3 genes is a minor component. Surface protein synthesis in another ciliated protozoan, a Paramecium sp., is also controlled by changes in the environment and is regulated largely at the level of mRNA abundance (15, 17). However, it is still to be determined whether message abundance is being controlled by a transcriptional or posttranscriptional mechanism. Changing the stability of a specific message as a result of a change in cellular environment as a means of controlling gene expression is not unique to free-living ciliated protozoa. The human lymphokine colony-stimulating factor (granulocyte-macrophage colony-stimulating factor) is transiently synthesized and then secreted by T cells as a result of lectin or antigen activation (10, 20). Recently, an A-U-rich sequence in the granulocyte-macrophage colony-stimulating factor mRNA has been demonstrated to be responsible for the transient expression of granulocyte-macrophage colonystimulating factor by making it susceptible to rapid degradation (19). This sequence is located in the 3'-untranslated region and is 51 bases long (24). Shaw and Kamen (19) have noted that similar long runs of A-U occur in the 3'-untranslated regions of other transiently expressed mRNAs (lymphokines, cytokines, and proto-oncogenes), leading them to speculate that a signal received at the cell surface may cause a temporary block in specific mRNA degradation, resulting in increased expression. Clemens (5) has noted a striking complementarity between these A-U-rich motifs and a sequence at the 3' ends of a family of B2 RNAs found in the mouse. B2 RNAs are transcribed from highly repetitive sequences in the mouse genome and accumulate to high steady-state levels in cells transformed by tumor viruses or chemical carcinogens and in embryonic cells (16, 18, 20). The correlation of B2 RNA accumulation with the increased expression of proto-oncogene and autocrine growth factor mRNAs has led Clemens (5) to speculate that the hybrid which could be formed between the 3' ends of these messages could lead to its protection from degradation. To our knowledge, this hypothesis has not been tested experimen-

tally.

It is unlikely that a similar A-U motif will be identified in the 3' end of Tetrahymena SerH3 messages. This is due to the high A+T content of transcribed, but not translated, regions of Tetrahymena genes (2, 11). However, this type of mechanism is particularly attractive when one considers the specificity and rapidity of a change in Tetrahymena surface protein expression. Experiments are currently in progress to determine how SerH3 message stability is changed as a result of a change in environmental temperature.

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ACKNOWLEDGMENTS

The authors thank Paul Doerder and Richard Hallberg for supplying us with pC6 and sharing their data with us prior to publication, Qian Zhao and Martin A. Gorovsky for sharing their technique for the in vitro nuclear runoff experiments with us prior to its publication, and Martin A. Gorovsky and Richard Hallberg for their critical review of this manuscript. This work was supported by grant DCB-8427599 from the National Science Foundation to G.A.B. and Public Health Service grant GM26973 from the National Institutes of Health to M. A. Gorovsky. LITERATURE CITED 1. Allen, S. A., T. C. White, J. P. Langmore, and M. A. Swancutt. 1983. Highly purified micro- and macronuclei from Tetrahymena thermophila isolated by percoll gradients. J. Protozool. 30:21-29. 2. Bannon, G. A., J. K. Bowen, M. C. Yao, and M. A. Gorovsky. 1984. Tetrahymena H4 genes: structure, evolution and organization in macro- and micronuclei. Nucleic Acids Res. 12: 1%1-1975. 3. Bannon, G. A., R. Perkins-Dameron, and A. Allen-Nash. 1986. Structure and expression of two temperature-specific surface proteins in the ciliated protozoan Tetrahymena thermophila. Mol. Cell. Biol. 6:3240-3245. 4. Calzone, F. J., R. C. Angerer, and M. A. Gorovsky. 1982. Regulation of protein synthesis in Tetrahymena: isolation and characterization of polysomes by gel filtration and precipitation at pH 5.3. Nucleic Acids Res. 10:2145-2161. 5. Clemens, M. J. 1987. A potential role for RNA transcribed from B2 repeats in the regulation of mRNA stability. Cell 49:157-158. 6. Doerder, F. P., and M. S. Berkowitz. 1986. Purification and partial characterization of the H immobilization antigens of Tetrahymena thermophila. J. Protozool. 33:204-207. 7. Feinberg, A. P., and B. Vogelstein. 1983. A technique for radiolabelling DNA restriction endonuclease fragments to high specific activity. Anal. Biochem. 132:6-13. 8. Feinberg, A. P., and B. Vogelstein. 1984. A technique for radiolabelling DNA restriction endonuclease fragments to high specific activity: addendum. Anal. Biochem. 137:266-267. 9. Gorovsky, M. A., M. C. Yao, J. B. Keevert, and G. L. Pleger. 1975. Isolation of macro- and micronuclei of Tetrahymena pyriformis. Methods Cell Biol. 9:311-327. 10. Gough, N. M., J. Gough, D. Metcalf, A. Kelso, D. Grail, N. A. Nicola, A. W. Burgess, and A. R. Dunn. 1984. Molecular cloning of cDNA encoding a murine haematopoietic growth regulator, granulocyte-macrophage colony-stimulatingfactor. Nature (London) 309:763-767. 11. Horowitz, S. H., J. K. Bowen, G. A. Bannon, and M. A. Gorovsky. 1987. Unusual features of transcribed and translated regions of the histone H4 gene family of Tetrahymena thermophila. Nucleic Acids Res 15:141-160. 12. Kelso, A., and D. Metcalf. 1985. Characteristics of colonystimulating factor production by murine T-lymphocyte clones. Exp. Hematol. (New York) 13:7-15. 13. Maniatis, T., E. F. Fritsch, and J. Sambrook. 1982. Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. 14. Martindale, D. W., and P. J. Bruns. 1983. Cloning of abundant mRNA species present during conjugation of Tetrahymena thermophila: identification of mRNA species present exclusively during meiosis. Mol. Cell. Biol. 3:1857-1865. 15. Meyer, E., F. Caron, and B. Guiard. 1984. Blocking of in vitro translation of Paramecium messenger RNAs is due to messenger RNA primary structure. Biochimie 66:403-412. 16. Murphy, D., P. M. Brickell, D. S. Latchman, K. Wilson, and P. W. J. Rigby. 1983. Transcripts regulated during normal embryonic development and oncogenic transformation share a repetitive element. Cell 35:865-871. 17. Preer, J. R., L. B. Preer, and B. M. Rudman. 1981. mRNAs for the immobilization antigens of Paramecium. Proc. Natl. Acad. Sci. USA 78:6776-6778.

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Tetrahymena thermophila. Mol. Cell. Biol. 5: 1925-1932. 23. Wolfe, J. 1985. Cytoskeletal reorganization and plasma membrane fusion in conjugating Tetrahymena. J. Cell Sci. 73:6985. 24. Wong, G. G., J. S. Witek, P. A. Temple, K. M. Wilkens, A. C. Leary, D. P. Luxenberg, S. S. Jones, E. L. Brown, R. M. Kay, E. C. Orr, C. Shoemaker, D. W. Golde, R. J. Kaufman, R. M. Hewick, E. A. Wang, and S. C. Clark. 1985. Human GM-CSF: molecular cloning of the complementary DNA and purification of the natural and recombinant proteins. Science 228:810815. 25. Young, R. A., and R. W. Davies. 1983. Efficient isolation of genes by using antibody probes. Proc. Natl. Acad. Sci. USA 80:1194-1198. mation in