- Email: [email protected]
The induction of ALU-sequence transcripts by glucocorticoid in rat liver cells
0022-4731/86 $3.00 + 0.00
J. avoid Biochem.Vol. 25, No. 2, pp. 201-207, 1986 Printedin Great Britain. All rights reserved
Copyright c 1986Pergamon Journals Ltd
THE INDUCTION OF ALU-SEQUENCE TRANSCRIPTS BY GLUCOCORTICOID IN RAT LIVER CELLS LEE-HWEI K.
Department
SUN*
FRED R. FRANKEL~
and
of Microbiology, University of Pennsylvania School of Medicine, Philadelphia, PA 19119, U.S.A. (Received 21 October 1985)
Summary-Two cDNA clones isolated from a library prepared from dexamethasone-treated rat hepatoma cells have permitted us to detect the presence and the induction of heterogeneous, mainly short, RNA molecules in hepatoma cells and in rat liver, but not in several other rat tissues. The induction by dexamethasone is inhibited by 100 x progesterone. Pulse label experiments suggest that it occurs in part at least, at the ievel of transcription and may be mediated by RNA polymerase III. The induction of the RNAs is stimulated by cycloheximide, even in the absence of hormone, but not significantly by other stressful conditions. One line of hepatoma cells spontaneously lost its ability to induce these RNAs and synthesized them constitutively. These altered cells showed proper induction of another dexamethasonemediated response, indicating that the glucocorticoid receptor was functionally normal in these cells. The two clones contain a type 2 Alu-like sequence. The short RNAs can be distinguished from 7SL RNA, which also contains Alu-sequences. We hypothesize that the synthesis of these RNAs may be regulated by an inhibitor of transcription which is inactivated by dexamethasone. Accordingly, cycloheximide relieves the inhibition by preventing synthesis of the inhibitor and the altered cell line has spontaneously lost the function of the inhibitor. The function of these RNAs for the cell is not known. We believe this to be the first report of hormone-regulated tissue specific synthesis of repeat-sequence transcripts.
INTRODUCTION
Nucleotide sequences which are repeated 10’ to lo6 times, and which are interspersed with single-copy sequences can represent as much as 20 or more per cent of the genomes of eukaryotic organisms [l]. Many of these sequences are transcribed into RNA,
some contained within pol II transcripts, others, transcribed by RNA polymerase III [l]. Whether the repeated sequences of most of these transcripts have some active role in the metabolism or gene expression of cells is not known. During the cloning of glucocorticoid-regulated messages of rat hepatoma cells, we isolated two cDNA clones that hybridize to repeated sequence DNA. One group of transcripts that hybridize to these clones are 200-400 bases long and increase in amount 2- to 5-fold after dexamethasone treatment of the hepatoma cells [2]. We have now examined additional aspects of these inducible sequences. EXPERIMENTAL
Clones, cells and animals
The two cDNA clones used in these studies, ~655 and ~333, were isolated and characterised as previously described [2]. The use of either cloned DNA as a probe gave essentially the same results. Most of the data shown in this paper were generated using *Present address: Centocor, Inc. 244 Great Valley Parkway Malvern, PA 19355, U.S.A. tTo whom correspondence should be addressed.
~655. The MI.19 line of rat hepatoma cells, from K. Yamamoto, was maintained in Dulbecco’s minimal essential medium with the addition of 10% horse serum (GIBCO), insulin at lOpg/ml, 0.2 mM glutamine, penicillin at lOOU/ml, and streptomycin at 0.1 pg/ml. Prior to hormonal stimulation, cells were grown for 3 days in the same medium containing 5% horse serum that had been treated with charcoaldextran to reduce endogenous serum steroids. Cells were generally induced with 1.Op M dexamethasone (Sigma) from a stock solution stored at -20°C in 95% (v/v) ethanol. Adrenalectomized SpragueDawley male rats (Taconic Farms, Germantown, N.Y.) were induced for 12 h with 1 mg per 100 g b. wt of dexamethasone in dimethyl sulfoxide by intraperitoneal injection. The alpha,-acid glycoprotein cDNA was obtained as described earlier [2]. The 7SL clone (pUCS-S) was kindly provided by E. Ullu [3]. RNA
isolation and analysis
RNA was isolated by three different methods: (1) Total cytoplasmic RNA was prepared by phenolchloroform extraction of Nonidet P40 (Bethesda Research Labs) supernatants of cells as described previously [2]. The poly(A)-containing fraction was prepared by binding total RNA to oligo(dT)-cellulose (P-L Biochemicals) in 0.5 M NaCl/lO mM Tri-HCl, pH 7.4, and eluting with a no-salt Tris buffer [4], The yield of poly(A)-RNA was l-2% of total RNA. (2) Total cell RNA was prepared by lysis of cells in the guanidinium-isothiocyanate reagent of Chirgwin et aZ.[5] and then isolated by CsCI fractionation as 201
LEE-HWEI K. SUN and FRED R. FRANKEL
202
described in that reference. (3) A new method of mRNA isolation [6] was performed by homogenizing at 22°C tissue culture cells or a small quantity of animal tissue (about 200 mg) in 200 ml solution containing 100mM NaCI, 10 mM Tris, pH 7.5, 1 mM EDTA, 1% sodium dodecyl sulfate and 1 mg/ml predigested pronase. The homogenate was then incubated with stirring at 37°C for 3 h. NaCl was added to 0.4 M. Frequently a viscous mass of DNA was visible and removed before addition of 1 ml of oligo(dT)-cellulose. The suspension was gently shaken at 26°C for 2-16 h. The cellulose was then removed by centrifugation and washed several times in the same solution without pronase. The pellet material was transferred to a column and mRNA was eluted in the usual manner [4]. RNA samples were denatured at 60°C with 50% (v/v) formamide and 2.2 M formaldehyde and electrophoresed on 1% agarose gels containing 2.2 M formaldehyde [7]. The RNA was transfered to nitrocellulose as described by Thomas[8]. Buffers and conditions for prehybridization and hybridization were those described by Wahl et a1.[9]. All hybridizations were carried out with 2 x 10’ to 2 x 10’ dpm of 32P-labeled DNA. For labeling of rat hepatoma cell RNA, cells were grown for 3 days in medium containing stripped serum. 1 FM dexamethasone was added to cultures for 24 h. Alpha-amanitin at l-5 pg/ml was added to some cultures for 3 h. Cultures were then labeled for 20 or 30 min with 0.5 mCi [3H]uridine (40 Ci/mmol) per ml culture medium. RNA was isolated by methods (1) and (2) described above. Specific activities of RNA ranged from 4 x 104-2 x 105cpm/pg. Hybridization conditions were similar to those used above. Filters contained 1 pg of plasmid DNA that had been linearized with EcoRI, denatured with alkali, neutralized and applied to the nitrocellulose. After hybridization, filters were washed with 2 x standard saline-citrate, digested with pancreatic RNase at 20/1g/ml, 37°C and then washed again. Nucleotide
sequencing
Sequencing of Barn HI inserts in ~655 and ~333 was performed by a previously described method [lo]. Briefly, the plasmids were linearized with EcoRI or Hind111 and then annealed with a 14-nucleotide primer (BioLabs) whose 3’ end was one base 5’ of the Barn HI site of pBR322. The insert was sequenced by use of the chain termination procedure [l 11. RESULTS
Induction
in rat hepatoma
cells
The MI-19 line of rat hepatoma cells was treated with 10e6 M dexamethasone for 24 h. RNA, isolated from treated and control cells by two different methods, was analyzed by formaldehyde-agarose gel electrophoresis, blotted to nitrocellulose and probed with nick-translated ~655. Figure 1, a-d, shows that
abed
efgh
Fig. 1. Detection of repeat-sequence transcripts in different RNA preparations from rat hepatoma cells. Lanes (a) and (b). Electrophoresis of 20pg of total cytoplasmic RNA prepared from control (a) and dexamethasone-treated (b) cells by phenol-chloroform extraction of Nonidet P40 cell supernatants. Lanes (c) and (d). Electrophoresis of 20 pg of total cell RNA prepared from control (c) and treated (d) cells by the guanidinium-isothiocyanate reagent. Lanes (e) and (f). Samples used for (c) and (d) were treated with 0.03N NaOH&7m MEDTA at 100°C for IOmin and neutralized prior to electrophoresis. Lanes (g) and (h). Electrophoresis of 2pg of poly(A)+ RNA isolated from cytoplasmic RNA of control (g) and treated (h) cells. RNAs were transferred to nitrocellulose, hybridized to 1.3 x IO’dpm of nick-translated ~655, and exposed for 16 h at 4 C.
a heterogeneous group of small RNAs, in the range of 20@400 nucleotides, hybridizes to the probe in both RNA preparations. The radioautographs were scanned and revealed a 2- to 3-fold induction by dexamethasone of these RNAs. A group of somewhat larger RNA molecules of 1.0-1.2 kb also hybridizes to the probe in some of our preparations. As shown, some cross-hybridization with ribosomal RNA is seen occasionally. In samples prepared by guanidinium-isothiocyanate, a hetergenous distribution of RNAs, in addition to the 200-400 nucleotide molecules, hybridizes to the probe. In the example shown, large molecules of RNA near the top of the gel are seen to hybridize particularly strongly. In the remainder of this paper, we mainly focus on the 20s 400 nucleotide molecules since these are the major components seen in poly(A)-RNA fractions and are enriched in such fractions (see below). That all of the hybridizing molecules are RNA is shown in Fig. 1. e-f. Here, treatment of the guanidinium-isothiocyanate RNA with 0.03 N NaOH-0.7 mM EDTA at 100°C for 10 min prior to electrophoresis eliminated virtually all hybridization. Poly(A)-RNA was selected from the chloroformphenol extracted RNA preparation. Figure I, g, h. shows that 2 pg of polyA-RNA hybridizes that probe much more strongly than 20 pg of total RNA, indicating that the presence of poly(A) sequences has permitted these RNAs to be highly enriched in this fraction. A scan of the radioautograph of this fraction shows a 3-fold induction by dexamethasone of the 2OG400 base molecules and a 6-fold induction of the 1.0-I .2 kb molecules.
Dexamethasone
285
18s
872 603 310 281
b
0
c
Fig. 2. Effect of progesterone on the dexamethasone induction of repeat-sequence transcripts. Electrophoresis of 20 pg of total cytoplasmic RNA isolated from rat hepatoma cells treated with no hormone (a), 5 x IO-’ M dexamethasone (b), or 5 x lo-‘M dexamethasone plus 5 x 10e5 M progesterone for 24 h prior to isolation of RNA Blots were hybridized with 7.5 x 10’dpm of nick-translated ~655 and exposed for 16 h at -20°C.
Progesterone can bind to the glucocorticoid receptor and by competition can prevent dexamethasone binding and dexamethasone-mediated effects. To determine whether the induction of the short RNAs is mediated through the action of the glucocorticoid receptor, cells were treated with 5 x lo-‘M dexamethasone in the presence and absence of 5 x 10m5 M progesterone. Scan of the radioautograph shown in Fig. 2 indicates that the presence of progesterone blocked the 2-fold induction by dexamethasone. The apparent induction of these RNAs could occur as a consequence of the stimulation of their transcription. Alternatively, their accumulation could result from a change in the rate of processing or degradation of these molecules, whithout any change in transcription rate. Labeling newly synthesized RNA sequences during a short pulse relative to the 24-h induction period should exaggerate effects that result from changes in synthesis rather than processing or degradation of the RNA. Therefore, cells growing
Table I. Induction
203
induces Alu-sequence RNAs
in the presence or absence of hormone for 24 h were pulse-labeled at the end of that period for 30 min with [3H]uridine. Total RNA was isolated and hybridized to filters containing bound pBR322 DNA or ~655 DNA. In three experiments (Table I), between 0.01 and 0.002% of the input RNA was bound specifically to the ~655 DNA filters. If it is assumed that the DNA on the filters was in sufficient excess and that hybridization was nearly complete, then 0.01% or less of the RNA synthesized by these cells is represented by these sequences. Treatment with dexamethasone increased the amount of labeled RNA containing these sequences about 3.2-fold (average of 3 expts). This fold induction is similar to the increase in mass of short RNAs accumulated after hormone treatment as assayed by Northern blot hybridization. Although not a rigorous test, since we do not known the actual rates of the various competing reactions, the data support a change in transcription rate to explain the accumulation of repeat-sequence transcripts. Alpha-amanitin, an inhibitor of RNA polymerase II at very low concentrations, was included in some incubations. The data show that it had little inhibitory effect on the synthesis of these RNAs, suggesting that RNA polymerase III may have been responsible for the synthesis. Some hormone-mediated responses have been shown to occur by the direct induction of RNA synthesis without the need for intermediary protein synthesis, while other responses require protein synthesis and are inhibited by drugs that block this reaction [ 121. To examine the role of protein synthesis in the induction of the short RNAs, cells were treated with dexamethasone and/or various concentrations of cycloheximide for 12 h. At I2 h, the induction by hormone is less strong than at 24 h, but can be detected (Fig. 3, a-c). Cycloheximide had a stimulatory effect on the induction (Fig. 3, d&h); 0.2 pg/ml and 2pg/pl of drug had little effect, while lO~gg/ml and 20pg/ml caused a 3-5-fold stimulation of the I2 h level of RNA. Figure 3h shows that this stimulation did not require the presence of hormone. To be certain that this phenomenon was not an artifact of the method of RNA preparation, guanidinuimisothiocyanate was used to prepare RNA from cycloheximide-treated cells. Induction of the short RNAs, as well as RNAs of various other lengths that hybridize to the probe, was detected (Fig. 3, i-k) in
ratios for pulse-labeled RNAs bound specifically sequence clone (~6%)
to a repeat
Dexamethasone plus Exoerimenr* I 2 3
Control I .O (55 cpm) I .o (54 cpm) I .o (70 cpm)
Dexamethasone 5.3 (290 cpm) 2.0(llOcpm) 2.3 (160cpm)
aloha-amanitin 6.3 (345 cpm) I .7 (X9 cpm) 1.6(114cpm)
‘In the three experiments, 3 x 106, 4 x IO6 and I x IO’cpm, respectively, of total RNAs were hybridized with nitrocellulose filters that conkned I pg pBR322 or ~655 DNA. For each RNA sample, radioactivity that bound to the pBR322 DNA filters (approx 20cpm) was subtracted from radioactivity bound lo ~655 DNA, giving the values shown.
204
LEE-HWEI K. SUN and FRED R. FRANKEL
abcdefgh
1
i
k
Fig. 3. Effect of cycloheximide on the induction of repeat-sequence transcripts. Electrophoresis of 20 pg of total cytoplasmic RNAs (lanes (a) to (h) or total cells RNAs (lanes (i) to (k). Lanes (a) to (i). RNA from control cells. Lane (b). RNA from cells treated with 10e6 M dexamethasone for 24 h. Lanes (c) to (g) and (j). RNA from cells treated with 10m6M dexamethasone for 12 h. Cycloheximide was added to some of these cultures for 12 h at the following concentrations: (d) 0.2 pg/ml; (e) 2 pg/ml; (f) and (k) lO~g/ml; (g) and (h) 20pg/ml. Blots were hybridized with 8 x IO’dpm of nick-translated ~655.
this sample as well. The effect of cycloheximide could again be seen in the absence of hormone. Induction by cycloheximide was also seen when poly(A)-RNA was examined. Because of the effect of cycloheximide, we wondered whether these RNAs might be induced by various other stressful environmental conditions. Therefore, cultures of cells were treated in the following ways: 50 PM sodium arsenite for 3 or 6 h prior to RNA isolation; 6% ethanol for 17 h; pH 8 medium for 17 h; growth at 42°C for 11 h followed by 37°C for 6 h. RNA was prepared from these cultures by both isolation methods used above. None of these conditions produced the stimulation shown by cycloheximide, although arsenite and the 42°C incubation caused an increase in the short RNAs of about 3&50% (data not shown). During the course of these studies, we observed that a culture of the rat hepatoma cells had abruptly lost its ability to be induced for these RNAs by dexamethasone. In these cells the RNA was expressed constitutively (Fig. 4, a, b). All cells derived from this culture also showed constitutive synthesis of the short RNAs. To determine whether this reflected an alteration of the glucocortical receptor in these cells, we tested another well-characterized response of the cells to dexamethasone, the induction of alpha, -acid glycoprotein mRNA [2]. Figure 4. c, d shows that induction of this message was completely normal and did not show constitutivity. That the alteration had occurred in the cells themselves, and not in other aspects of the experimental protocol, was shown by growing an aliquot of the original frozen culture from which the altered cells had been derived. RNA isolated from these hormone-treated or control cells shows that the cells responded normally to the hormone (Fig. 4, e, f). The inserts present in the two repeat-sequence clones ~655 and ~333 are only 6&70 nucleotides
long [2]. We have determined their sequence by the Sanger dideoxynucleotide method, directly from the double stranded plasmid, using a primer complementary to the region at the Barn HI site of pBR322. The sequence of ~655 is shown in Fig. 5a. The sequence found for ~333 is shown in Fig. 5b. It can be seen that these two sequences are almost perfect complements of each other, as shown in Fig. SC. The ~655 sequences shows 96% homology with a rat type 2 Ah sequence [2] (Fig. 5d). A well-characterized short RNA containing an Ah sequence is 7SL RNA [3]. This RNA is present in the cytoplasm of all cells thus far studied, and functions in protein secretion [13]. We have examined our RNA preparations for this material to determine whether its concentration is affected by dexamethasone and whether it shares any resemblance to our short
a b
c d
e
f
Fig. 4. Altered rat hepatoma culture shows constitutive synthesis of repeat sequence transcripts but normal induction of alpha,-acid glycoprotein mRNA. Total cytoplsmic RNA was isolated from an altered culture of cells [lanes (a) to (d)l or from the original normal culture [lanes (e). (f)]. The RNA of lanes (a). (b), (e) and (f) was hybridized with nick-translated R655.‘ The ‘RNA of lanes (i) and (d) was hybridized with nick-translated ~1394, a cDNA clone for alpha,-acid glycoprotein.
Dexamethasone
205
induces Mu-sequence RNAs
o
GATGCCCTCT
TCTGGATCTA TCTGAAGACA GCTACAGTGT ACTTATATAC
b
TTTTATTTAT
TTTATGTATA TGAGTACACC ATAGCTGTCT TCAGACACAC
CAGAAGACCX;
GCATCA
c
-GATGCCC-TCT ACTAC’XGCAGA
TCTGGATGTa TCTGAAGACA GCTAcAGTGT ACTtATATAC AC&KC-ACAc AGACTTCTGT CGATdCACA TGAgTATATG
TATTTTATTT
ATTTT
GATGCCCTCT
d
TCTGG-TGTA TCTGAAGACA GCTACAGTGT ACTTATTAT
Fig. 5. Nucleotide sequence of the inserts of two repeat-sequence cDNA clones. a. ~655 sequence. b. ~333 sequence. c. ~655 and ~333 show complementarity. d. Portion of rat type 2 Ah sequence, from Fig. 2 of ref. 1. The 5’ terminus is on the left.
RNAs. In order to unambiguously distinguish the 7SL RNA from our Alu-containing RNAs, we have used as probe on internal segment of the 7S molecule which is not homologous to the Ah sequence. Figure 6, a, b, shows that 7SL RNA is not present in poly(A)-RNA preparations from uninduced or hormone-treated rat hepatoma cells. This shows that our short RNAs, which are enriched in such poly(A)RNA preparations, are not homologous with 7SL RNA. Total RNA preparations do contain 7SL RNA (Fig. 6, c, d). When these fadioautographs were scanned, we found a 1.4- to 2-fold increase in amount of the RNA in response to dexamethasone. The size of this RNA on our gels is about 450 nucleotides, slightly larger and more homogeneous than our short RNAs. Induction
was injected i.p. with 1 mg per 100 g b. wt of dexamethasone. Twelve hours later, mRNA was isolated from a variety of tissues, fractionated by agarose gel electrophoresis, transferred to nitrocellulose and probed with ~655. The results are shown in Fig. 7. Figure 7, a, b shows that rat liver mRNA contains 300-500 nucleotide molecules that hybridize to the probe, and which are induced by dexamethasone 2-3-fold. Such molecules are absent from mRNA prepared from kidney or heart, both before and after hormone treatment (Fig. 7, c-f). Alkali treatment of the liver mRNAs prior to electrophoresis eliminated all hybridization (Fig. 7, g, h). We do not known the significance of the high molecular weight material in the kidney and heartpreparations that hybridize with the probe.
in rat tissues DISCUSSION
A group of adult Sprague-Dawley rats was adrenalectomized. After 7 days, half of the group
Ah sequences represent the most abundant family of human and rodent middle repetitive DNAs. They occur at approx 3-5 x lo5 copies per haploid genome
28s -
28s Ii% -
18s
0
b
c
d
Fig. 6. Detection of 7SL RNA in rat hepatoma cell RNA preparations. Electrophoresis of 2pg of poly(A)+ RNA [lanes (a) and (b)] and 2Opg of total cytoplasmic RNA [lanes (c) and (d)] prepared from control [(a) and (c)] or dexamethasone-treated [(b) and (d)] cells. Blots were probed with nick-translated pUC8-S, a plasmid containing an internal segment of 7SL cDNA that is not homologous to the Ah sequence [ref. 31.
abcdef
9
h
Fig. 7. Detection of short repeat-sequence transcripts in RNA preparations from rat tissues. Poly(A)+ RNA was isolated by the pronase_SDS procedure from liver (a, b) heart (c, d) or kidney (e, f) of adrenalectomized control rats (a, c, e, g) or such rats treated with dexamethasone for 12 h (b, d, f, h). Liver RNAs were also treated with alkali and neutralized prior to electrophoresis (g, h). The RNAs were hybridized with 1.3 x lo8 dpm of nick-translated ~655.
206
LEE-HWEI K. SUN and FRED
[l]. A similar sequence is also present in the Drosophila genome, in the form of two copies of 7SL RNA genes, suggesting that sequences within 7SL DNA may have served as the origin for this abundant family of repeats [ 141. RNA transcripts containing Ah sequences occur in mammalian cells in a variety of forms. The simplest are the small, discrete RNAs, such as 5.4s RNA and 7SL RNA, transcribed by RNA polymerase III. These small RNAs are often found associated with longer nuclear or cytoplasmic RNA transcripts, and it has been shown that 7SL forms an essential part of the signal recognition particle required for transport of secreted proteins across the endoplasmic reticulum 1131. Ah sequences are also contained within the heterogeneous nuclear RNAs of mammalian cells, presumably transcribed by RNA polymerase II [15]. They are present to a much lesser extent in cytoplasmic mRNAs [15]. This suggests that they are mainly present within introns and are deleted during the processing of HnRNA. Some, however, must reside in the 5’ or 3’ untranslated regions of mRNAs or perhaps in the coding regions, and are therefore retained on processing. Some recent experiments point to the presence of very short (200400 nucleotide), heterogeneous cytoplasmic RNAs of rodent cells that contain Ah sequences [ 151.These could be molecules produced by the processing of HnRNA or by further cleavage of mRNA. However, since Ah sequences contain pol III promoter sequences, these short RNAs could also be primary pol III transcripts [16]. In this report, we present evidence that a group of short, heterogeneous RNAs occurs in rat hepatoma cells, and that the accumulation of these molecules is controlled by a steroid hormone. Our studies suggest that this control may occur at the level of transcription, since 3.2-fold more radioactive RNA hybridizes to our repeated sequence cDNA clones when the cells are labeled for 30-min in the presence of dexamethasone. Growth of the cells in the presence of dexamethasone plus progesterone prevented the induction. This provides support for the role of glucocorticoid receptor in the induction phenomenon, since at the concentration used, progesterone is able to displace dexamethasone from the receptor, but does not itself produce a glucocorticoid response [ 171. That the induction was not restricted to the tissue culture system was shown by the presence of similar RNA molecules in the liver of adult rats. These molecules were induced several-fold by administration of hormone to the rats. The RNAs were neither present nor inducible in the heart or kidney of these rats. Thus, this appears to be the first report of the induction of tissue-specific repeat sequence transcripts by a hormone. During tests to determine whether protein synthesis was necessary for the induction, we discovered that inhibiting protein synthesis significantly enhanced
R. FRANKEL
the induction, even in the absence of hormone. One interpretation of such an effect is that a labile inhibitory protein negatively regulates the transcription of these RNAs. According to such a model, RNA induction could occur either by inactivation of the inhibitor, presumably by the glucocorticoid-receptor complex, or by preventing its synthesis, by cycloheximide. Interestingly, induction of tyrosine aminotransferase by dexamethasone in rat hepatoma cells has recently been shown to be negatively regulated by the product of a non-syntenic gene [18]. Also, it has been shown in at least one system that cycloheximide can increase the rate of transcription of a gene [ 191. An alternative explanation for the cycloheximide result, however, is that the drug may stabilize the transcripts from degradation. We found that one culture of rat hepatoma cells spontaneously acquired the property of synthesizing high levels of the short RNAs in the absence of dexamethasone. This property was stable in the progeny of these cells. In such cultures, the hormone caused no additional induction, although cycloheximide caused a slight enhancement of the accumulation of the RNAs. In these cultures, the induction of an unrelated glucocorticoid-regulated mRNA (for alpha, -acid glycoprotein) appeared normal, indicating that the receptor pathway had not been altered. A plausible explanation, in the light of the cycloheximide effect, is that the putative inhibitory protein had been altered in these cells and no longer restricted synthesis of repeat sequence transcripts. In this paper we have not examined the higher molecular weight species of RNA that hybridize to the repeat sequence probes that we have seen in the total cell RNA preparations. These presumably represent nuclear species. Whether they are precursors of the short molecules or are independent transcripts is not known. Whether the repeat sequence transcripts have some role in cell differentiation or regulated gene expression or gene product function remains to be determined. However, the tissue-specific, dexamethasone-regulated accumulation of repeat sequence transcripts suggests some role for these molecules in the complex response of cells to hormone. Acknowledgement-These
studies were supported by NIH
grant CA17301.
REFERENCES
Jelinek W. R. and Schmid C. W.: Repetitive sequences in eukaryotic DNA and their expression, A. Rev. Biochew 51 (1982) 813-844. Feinberg R. F., Sun L-H. K., Ordahl C. P. and Frankel F. R.: Identification of glucocorticoid-induced genes in rat hepatoma cells by isolation of cloned cDNA sequences. Proc. natn. Acad. Sci. U.S.A. 80 (1983) 5042-5046. Ullu E., Murphy S. and Melli M.: Human 7SL RNA consists of a 140 nucleotide middle-repetitive sequence inserted in an Alu sequence. Ceil 29 (1982) 195-202.
Dexamethasone
induces Alu-sequence RNAs
4. Aviv J. and Leder P.: Purification of biologically active globin mRNA by chromatography on oligothymidili~ acid-cellulose. Proc. n&t, Acud. Sci. U.S.A. 69 (1972) 1408-1412. 5. Chirgwin J. M., Prezybyla A. E., MacDonald R. J. and Rutter W. J.: Isolation of biologically active RNA from sources enriched in ribonuclease. Biochemisfry 18 (1979) 5294-5299. 6. Gonda T. J., Sheiness D. K. and Bishop J. M.: Transcripts from the cellular homologs of retroviral oncogenes: Distribution among chicken tissues. Molec. cell. ffiol. 2 (1982) 617624. 7. Rave N., Crkvenjakov R. and Boedtker H.: Identification of procollagen mRNAs transferrred to diazobenzyloxymethyl paper from formaldehyde-agarose gels. Nucl. Acid. Res. 6 (1979) 35593567. 8. Thomas P. S.: Hybridization of denatured RNA and small DNA fragments transferred to nitrocellulose. Proc. natn. Acad. Sci. U.S.A. 77 (1980) 5201-5205.
9. Wahl G. M., Stern M. and Stark G. R.: Efficient transfer of large DNA fragments from agarose gels to diazo~nzyloxyme~yl paper and rapid hybridization by using dextran sulfate. Pruc. natn. Acad. Sci. U.S.A. 76 (1979) 3683-3687. 10. Wallace R. B., Johnson M. J., Suggs S. V., Miyoshi K., Bhatt R. and Itakura K.: A set of synthetic oligonucleotide primers for DNA sequencing in the plasmid vector pBR322. Gene 16 (1981) 21-26. 11. Sanger F., Nicklen S. and Coulson A. R.: DNA sequencing with chain-terminating inhibitors. Proc. nacn. Acad. Sci. U.S.A. 74 (1977) 5463-5467. 12. Ashburner M., Chihara C., Meltzer P. and Richards G.:
207
Temporal control of puffing activity in polytene chromosomes. Cold Spring Harb. Symp. quant. Bio2.38 (1973) 6.55662.
13. Walter P. and Blobel G.: Signal recognition particle contains a 7S RNA essential for protein translocation across the endoplasmic reticulum. Nature 299 (1982) 691498.
14. Ullu E. and Tschudi C.: Ah sequences are processed 7SL RNA genes. Nature 312 (1984) 171-172: 1.5 Kramerov D. A.. Lekakh I. V.. Samarina 0. P. and Ryskov A. P.: The sequences homologous to major interspersed repeats Bl and B2 of mouse genome are present in mRNA and small cytoplasmic pofy(A)+ RNA. 1vuci. Acid. Rex 11 (1982) 7477-7481. 16. Haynes S. R. and Jelinek W. R.: Low molecular weight RNAs transcribed in vitro by RNA polymerase from A/u-type dispersed repeats in Chinese hamster DNA are also found in viva Proc. nain. Acad. Sci. U.S.A. 78 (1981) 6130-6134.
17. Baxter J. D. and Tomkins G. M.: Specific cytoplasmic glucocorticoid receptors in hepatoma tissue culture cells. Pro&. natn. Acad. Sci. U.S.A. 68 (1971) 923-937. 18. Peterson T. C., Killory A. M. and Foumier R. E. K.: Chromosomal assignment and trans regulation of the tyrosine aminotransferase structural gene in hepatoma hybrid cells. Molec. cell. Biol. 5 (1985) 2491.-2494. 19. Ringold G. M., Dieckmann B., Vannice J. L., Trahey M. and McCormick F.: Inhibition of protein synthesis stimulates transcription of human beta-interferon genes in Chinese hamster ovary cells. Proc. natn. Acad. Sci. U.S.A. 81 (1984) 39643968.
J. avoid Biochem.Vol. 25, No. 2, pp. 201-207, 1986 Printedin Great Britain. All rights reserved
Copyright c 1986Pergamon Journals Ltd
THE INDUCTION OF ALU-SEQUENCE TRANSCRIPTS BY GLUCOCORTICOID IN RAT LIVER CELLS LEE-HWEI K.
Department
SUN*
FRED R. FRANKEL~
and
of Microbiology, University of Pennsylvania School of Medicine, Philadelphia, PA 19119, U.S.A. (Received 21 October 1985)
Summary-Two cDNA clones isolated from a library prepared from dexamethasone-treated rat hepatoma cells have permitted us to detect the presence and the induction of heterogeneous, mainly short, RNA molecules in hepatoma cells and in rat liver, but not in several other rat tissues. The induction by dexamethasone is inhibited by 100 x progesterone. Pulse label experiments suggest that it occurs in part at least, at the ievel of transcription and may be mediated by RNA polymerase III. The induction of the RNAs is stimulated by cycloheximide, even in the absence of hormone, but not significantly by other stressful conditions. One line of hepatoma cells spontaneously lost its ability to induce these RNAs and synthesized them constitutively. These altered cells showed proper induction of another dexamethasonemediated response, indicating that the glucocorticoid receptor was functionally normal in these cells. The two clones contain a type 2 Alu-like sequence. The short RNAs can be distinguished from 7SL RNA, which also contains Alu-sequences. We hypothesize that the synthesis of these RNAs may be regulated by an inhibitor of transcription which is inactivated by dexamethasone. Accordingly, cycloheximide relieves the inhibition by preventing synthesis of the inhibitor and the altered cell line has spontaneously lost the function of the inhibitor. The function of these RNAs for the cell is not known. We believe this to be the first report of hormone-regulated tissue specific synthesis of repeat-sequence transcripts.
INTRODUCTION
Nucleotide sequences which are repeated 10’ to lo6 times, and which are interspersed with single-copy sequences can represent as much as 20 or more per cent of the genomes of eukaryotic organisms [l]. Many of these sequences are transcribed into RNA,
some contained within pol II transcripts, others, transcribed by RNA polymerase III [l]. Whether the repeated sequences of most of these transcripts have some active role in the metabolism or gene expression of cells is not known. During the cloning of glucocorticoid-regulated messages of rat hepatoma cells, we isolated two cDNA clones that hybridize to repeated sequence DNA. One group of transcripts that hybridize to these clones are 200-400 bases long and increase in amount 2- to 5-fold after dexamethasone treatment of the hepatoma cells [2]. We have now examined additional aspects of these inducible sequences. EXPERIMENTAL
Clones, cells and animals
The two cDNA clones used in these studies, ~655 and ~333, were isolated and characterised as previously described [2]. The use of either cloned DNA as a probe gave essentially the same results. Most of the data shown in this paper were generated using *Present address: Centocor, Inc. 244 Great Valley Parkway Malvern, PA 19355, U.S.A. tTo whom correspondence should be addressed.
~655. The MI.19 line of rat hepatoma cells, from K. Yamamoto, was maintained in Dulbecco’s minimal essential medium with the addition of 10% horse serum (GIBCO), insulin at lOpg/ml, 0.2 mM glutamine, penicillin at lOOU/ml, and streptomycin at 0.1 pg/ml. Prior to hormonal stimulation, cells were grown for 3 days in the same medium containing 5% horse serum that had been treated with charcoaldextran to reduce endogenous serum steroids. Cells were generally induced with 1.Op M dexamethasone (Sigma) from a stock solution stored at -20°C in 95% (v/v) ethanol. Adrenalectomized SpragueDawley male rats (Taconic Farms, Germantown, N.Y.) were induced for 12 h with 1 mg per 100 g b. wt of dexamethasone in dimethyl sulfoxide by intraperitoneal injection. The alpha,-acid glycoprotein cDNA was obtained as described earlier [2]. The 7SL clone (pUCS-S) was kindly provided by E. Ullu [3]. RNA
isolation and analysis
RNA was isolated by three different methods: (1) Total cytoplasmic RNA was prepared by phenolchloroform extraction of Nonidet P40 (Bethesda Research Labs) supernatants of cells as described previously [2]. The poly(A)-containing fraction was prepared by binding total RNA to oligo(dT)-cellulose (P-L Biochemicals) in 0.5 M NaCl/lO mM Tri-HCl, pH 7.4, and eluting with a no-salt Tris buffer [4], The yield of poly(A)-RNA was l-2% of total RNA. (2) Total cell RNA was prepared by lysis of cells in the guanidinium-isothiocyanate reagent of Chirgwin et aZ.[5] and then isolated by CsCI fractionation as 201
LEE-HWEI K. SUN and FRED R. FRANKEL
202
described in that reference. (3) A new method of mRNA isolation [6] was performed by homogenizing at 22°C tissue culture cells or a small quantity of animal tissue (about 200 mg) in 200 ml solution containing 100mM NaCI, 10 mM Tris, pH 7.5, 1 mM EDTA, 1% sodium dodecyl sulfate and 1 mg/ml predigested pronase. The homogenate was then incubated with stirring at 37°C for 3 h. NaCl was added to 0.4 M. Frequently a viscous mass of DNA was visible and removed before addition of 1 ml of oligo(dT)-cellulose. The suspension was gently shaken at 26°C for 2-16 h. The cellulose was then removed by centrifugation and washed several times in the same solution without pronase. The pellet material was transferred to a column and mRNA was eluted in the usual manner [4]. RNA samples were denatured at 60°C with 50% (v/v) formamide and 2.2 M formaldehyde and electrophoresed on 1% agarose gels containing 2.2 M formaldehyde [7]. The RNA was transfered to nitrocellulose as described by Thomas[8]. Buffers and conditions for prehybridization and hybridization were those described by Wahl et a1.[9]. All hybridizations were carried out with 2 x 10’ to 2 x 10’ dpm of 32P-labeled DNA. For labeling of rat hepatoma cell RNA, cells were grown for 3 days in medium containing stripped serum. 1 FM dexamethasone was added to cultures for 24 h. Alpha-amanitin at l-5 pg/ml was added to some cultures for 3 h. Cultures were then labeled for 20 or 30 min with 0.5 mCi [3H]uridine (40 Ci/mmol) per ml culture medium. RNA was isolated by methods (1) and (2) described above. Specific activities of RNA ranged from 4 x 104-2 x 105cpm/pg. Hybridization conditions were similar to those used above. Filters contained 1 pg of plasmid DNA that had been linearized with EcoRI, denatured with alkali, neutralized and applied to the nitrocellulose. After hybridization, filters were washed with 2 x standard saline-citrate, digested with pancreatic RNase at 20/1g/ml, 37°C and then washed again. Nucleotide
sequencing
Sequencing of Barn HI inserts in ~655 and ~333 was performed by a previously described method [lo]. Briefly, the plasmids were linearized with EcoRI or Hind111 and then annealed with a 14-nucleotide primer (BioLabs) whose 3’ end was one base 5’ of the Barn HI site of pBR322. The insert was sequenced by use of the chain termination procedure [l 11. RESULTS
Induction
in rat hepatoma
cells
The MI-19 line of rat hepatoma cells was treated with 10e6 M dexamethasone for 24 h. RNA, isolated from treated and control cells by two different methods, was analyzed by formaldehyde-agarose gel electrophoresis, blotted to nitrocellulose and probed with nick-translated ~655. Figure 1, a-d, shows that
abed
efgh
Fig. 1. Detection of repeat-sequence transcripts in different RNA preparations from rat hepatoma cells. Lanes (a) and (b). Electrophoresis of 20pg of total cytoplasmic RNA prepared from control (a) and dexamethasone-treated (b) cells by phenol-chloroform extraction of Nonidet P40 cell supernatants. Lanes (c) and (d). Electrophoresis of 20 pg of total cell RNA prepared from control (c) and treated (d) cells by the guanidinium-isothiocyanate reagent. Lanes (e) and (f). Samples used for (c) and (d) were treated with 0.03N NaOH&7m MEDTA at 100°C for IOmin and neutralized prior to electrophoresis. Lanes (g) and (h). Electrophoresis of 2pg of poly(A)+ RNA isolated from cytoplasmic RNA of control (g) and treated (h) cells. RNAs were transferred to nitrocellulose, hybridized to 1.3 x IO’dpm of nick-translated ~655, and exposed for 16 h at 4 C.
a heterogeneous group of small RNAs, in the range of 20@400 nucleotides, hybridizes to the probe in both RNA preparations. The radioautographs were scanned and revealed a 2- to 3-fold induction by dexamethasone of these RNAs. A group of somewhat larger RNA molecules of 1.0-1.2 kb also hybridizes to the probe in some of our preparations. As shown, some cross-hybridization with ribosomal RNA is seen occasionally. In samples prepared by guanidinium-isothiocyanate, a hetergenous distribution of RNAs, in addition to the 200-400 nucleotide molecules, hybridizes to the probe. In the example shown, large molecules of RNA near the top of the gel are seen to hybridize particularly strongly. In the remainder of this paper, we mainly focus on the 20s 400 nucleotide molecules since these are the major components seen in poly(A)-RNA fractions and are enriched in such fractions (see below). That all of the hybridizing molecules are RNA is shown in Fig. 1. e-f. Here, treatment of the guanidinium-isothiocyanate RNA with 0.03 N NaOH-0.7 mM EDTA at 100°C for 10 min prior to electrophoresis eliminated virtually all hybridization. Poly(A)-RNA was selected from the chloroformphenol extracted RNA preparation. Figure I, g, h. shows that 2 pg of polyA-RNA hybridizes that probe much more strongly than 20 pg of total RNA, indicating that the presence of poly(A) sequences has permitted these RNAs to be highly enriched in this fraction. A scan of the radioautograph of this fraction shows a 3-fold induction by dexamethasone of the 2OG400 base molecules and a 6-fold induction of the 1.0-I .2 kb molecules.
Dexamethasone
285
18s
872 603 310 281
b
0
c
Fig. 2. Effect of progesterone on the dexamethasone induction of repeat-sequence transcripts. Electrophoresis of 20 pg of total cytoplasmic RNA isolated from rat hepatoma cells treated with no hormone (a), 5 x IO-’ M dexamethasone (b), or 5 x lo-‘M dexamethasone plus 5 x 10e5 M progesterone for 24 h prior to isolation of RNA Blots were hybridized with 7.5 x 10’dpm of nick-translated ~655 and exposed for 16 h at -20°C.
Progesterone can bind to the glucocorticoid receptor and by competition can prevent dexamethasone binding and dexamethasone-mediated effects. To determine whether the induction of the short RNAs is mediated through the action of the glucocorticoid receptor, cells were treated with 5 x lo-‘M dexamethasone in the presence and absence of 5 x 10m5 M progesterone. Scan of the radioautograph shown in Fig. 2 indicates that the presence of progesterone blocked the 2-fold induction by dexamethasone. The apparent induction of these RNAs could occur as a consequence of the stimulation of their transcription. Alternatively, their accumulation could result from a change in the rate of processing or degradation of these molecules, whithout any change in transcription rate. Labeling newly synthesized RNA sequences during a short pulse relative to the 24-h induction period should exaggerate effects that result from changes in synthesis rather than processing or degradation of the RNA. Therefore, cells growing
Table I. Induction
203
induces Alu-sequence RNAs
in the presence or absence of hormone for 24 h were pulse-labeled at the end of that period for 30 min with [3H]uridine. Total RNA was isolated and hybridized to filters containing bound pBR322 DNA or ~655 DNA. In three experiments (Table I), between 0.01 and 0.002% of the input RNA was bound specifically to the ~655 DNA filters. If it is assumed that the DNA on the filters was in sufficient excess and that hybridization was nearly complete, then 0.01% or less of the RNA synthesized by these cells is represented by these sequences. Treatment with dexamethasone increased the amount of labeled RNA containing these sequences about 3.2-fold (average of 3 expts). This fold induction is similar to the increase in mass of short RNAs accumulated after hormone treatment as assayed by Northern blot hybridization. Although not a rigorous test, since we do not known the actual rates of the various competing reactions, the data support a change in transcription rate to explain the accumulation of repeat-sequence transcripts. Alpha-amanitin, an inhibitor of RNA polymerase II at very low concentrations, was included in some incubations. The data show that it had little inhibitory effect on the synthesis of these RNAs, suggesting that RNA polymerase III may have been responsible for the synthesis. Some hormone-mediated responses have been shown to occur by the direct induction of RNA synthesis without the need for intermediary protein synthesis, while other responses require protein synthesis and are inhibited by drugs that block this reaction [ 121. To examine the role of protein synthesis in the induction of the short RNAs, cells were treated with dexamethasone and/or various concentrations of cycloheximide for 12 h. At I2 h, the induction by hormone is less strong than at 24 h, but can be detected (Fig. 3, a-c). Cycloheximide had a stimulatory effect on the induction (Fig. 3, d&h); 0.2 pg/ml and 2pg/pl of drug had little effect, while lO~gg/ml and 20pg/ml caused a 3-5-fold stimulation of the I2 h level of RNA. Figure 3h shows that this stimulation did not require the presence of hormone. To be certain that this phenomenon was not an artifact of the method of RNA preparation, guanidinuimisothiocyanate was used to prepare RNA from cycloheximide-treated cells. Induction of the short RNAs, as well as RNAs of various other lengths that hybridize to the probe, was detected (Fig. 3, i-k) in
ratios for pulse-labeled RNAs bound specifically sequence clone (~6%)
to a repeat
Dexamethasone plus Exoerimenr* I 2 3
Control I .O (55 cpm) I .o (54 cpm) I .o (70 cpm)
Dexamethasone 5.3 (290 cpm) 2.0(llOcpm) 2.3 (160cpm)
aloha-amanitin 6.3 (345 cpm) I .7 (X9 cpm) 1.6(114cpm)
‘In the three experiments, 3 x 106, 4 x IO6 and I x IO’cpm, respectively, of total RNAs were hybridized with nitrocellulose filters that conkned I pg pBR322 or ~655 DNA. For each RNA sample, radioactivity that bound to the pBR322 DNA filters (approx 20cpm) was subtracted from radioactivity bound lo ~655 DNA, giving the values shown.
204
LEE-HWEI K. SUN and FRED R. FRANKEL
abcdefgh
1
i
k
Fig. 3. Effect of cycloheximide on the induction of repeat-sequence transcripts. Electrophoresis of 20 pg of total cytoplasmic RNAs (lanes (a) to (h) or total cells RNAs (lanes (i) to (k). Lanes (a) to (i). RNA from control cells. Lane (b). RNA from cells treated with 10e6 M dexamethasone for 24 h. Lanes (c) to (g) and (j). RNA from cells treated with 10m6M dexamethasone for 12 h. Cycloheximide was added to some of these cultures for 12 h at the following concentrations: (d) 0.2 pg/ml; (e) 2 pg/ml; (f) and (k) lO~g/ml; (g) and (h) 20pg/ml. Blots were hybridized with 8 x IO’dpm of nick-translated ~655.
this sample as well. The effect of cycloheximide could again be seen in the absence of hormone. Induction by cycloheximide was also seen when poly(A)-RNA was examined. Because of the effect of cycloheximide, we wondered whether these RNAs might be induced by various other stressful environmental conditions. Therefore, cultures of cells were treated in the following ways: 50 PM sodium arsenite for 3 or 6 h prior to RNA isolation; 6% ethanol for 17 h; pH 8 medium for 17 h; growth at 42°C for 11 h followed by 37°C for 6 h. RNA was prepared from these cultures by both isolation methods used above. None of these conditions produced the stimulation shown by cycloheximide, although arsenite and the 42°C incubation caused an increase in the short RNAs of about 3&50% (data not shown). During the course of these studies, we observed that a culture of the rat hepatoma cells had abruptly lost its ability to be induced for these RNAs by dexamethasone. In these cells the RNA was expressed constitutively (Fig. 4, a, b). All cells derived from this culture also showed constitutive synthesis of the short RNAs. To determine whether this reflected an alteration of the glucocortical receptor in these cells, we tested another well-characterized response of the cells to dexamethasone, the induction of alpha, -acid glycoprotein mRNA [2]. Figure 4. c, d shows that induction of this message was completely normal and did not show constitutivity. That the alteration had occurred in the cells themselves, and not in other aspects of the experimental protocol, was shown by growing an aliquot of the original frozen culture from which the altered cells had been derived. RNA isolated from these hormone-treated or control cells shows that the cells responded normally to the hormone (Fig. 4, e, f). The inserts present in the two repeat-sequence clones ~655 and ~333 are only 6&70 nucleotides
long [2]. We have determined their sequence by the Sanger dideoxynucleotide method, directly from the double stranded plasmid, using a primer complementary to the region at the Barn HI site of pBR322. The sequence of ~655 is shown in Fig. 5a. The sequence found for ~333 is shown in Fig. 5b. It can be seen that these two sequences are almost perfect complements of each other, as shown in Fig. SC. The ~655 sequences shows 96% homology with a rat type 2 Ah sequence [2] (Fig. 5d). A well-characterized short RNA containing an Ah sequence is 7SL RNA [3]. This RNA is present in the cytoplasm of all cells thus far studied, and functions in protein secretion [13]. We have examined our RNA preparations for this material to determine whether its concentration is affected by dexamethasone and whether it shares any resemblance to our short
a b
c d
e
f
Fig. 4. Altered rat hepatoma culture shows constitutive synthesis of repeat sequence transcripts but normal induction of alpha,-acid glycoprotein mRNA. Total cytoplsmic RNA was isolated from an altered culture of cells [lanes (a) to (d)l or from the original normal culture [lanes (e). (f)]. The RNA of lanes (a). (b), (e) and (f) was hybridized with nick-translated R655.‘ The ‘RNA of lanes (i) and (d) was hybridized with nick-translated ~1394, a cDNA clone for alpha,-acid glycoprotein.
Dexamethasone
205
induces Mu-sequence RNAs
o
GATGCCCTCT
TCTGGATCTA TCTGAAGACA GCTACAGTGT ACTTATATAC
b
TTTTATTTAT
TTTATGTATA TGAGTACACC ATAGCTGTCT TCAGACACAC
CAGAAGACCX;
GCATCA
c
-GATGCCC-TCT ACTAC’XGCAGA
TCTGGATGTa TCTGAAGACA GCTAcAGTGT ACTtATATAC AC&KC-ACAc AGACTTCTGT CGATdCACA TGAgTATATG
TATTTTATTT
ATTTT
GATGCCCTCT
d
TCTGG-TGTA TCTGAAGACA GCTACAGTGT ACTTATTAT
Fig. 5. Nucleotide sequence of the inserts of two repeat-sequence cDNA clones. a. ~655 sequence. b. ~333 sequence. c. ~655 and ~333 show complementarity. d. Portion of rat type 2 Ah sequence, from Fig. 2 of ref. 1. The 5’ terminus is on the left.
RNAs. In order to unambiguously distinguish the 7SL RNA from our Alu-containing RNAs, we have used as probe on internal segment of the 7S molecule which is not homologous to the Ah sequence. Figure 6, a, b, shows that 7SL RNA is not present in poly(A)-RNA preparations from uninduced or hormone-treated rat hepatoma cells. This shows that our short RNAs, which are enriched in such poly(A)RNA preparations, are not homologous with 7SL RNA. Total RNA preparations do contain 7SL RNA (Fig. 6, c, d). When these fadioautographs were scanned, we found a 1.4- to 2-fold increase in amount of the RNA in response to dexamethasone. The size of this RNA on our gels is about 450 nucleotides, slightly larger and more homogeneous than our short RNAs. Induction
was injected i.p. with 1 mg per 100 g b. wt of dexamethasone. Twelve hours later, mRNA was isolated from a variety of tissues, fractionated by agarose gel electrophoresis, transferred to nitrocellulose and probed with ~655. The results are shown in Fig. 7. Figure 7, a, b shows that rat liver mRNA contains 300-500 nucleotide molecules that hybridize to the probe, and which are induced by dexamethasone 2-3-fold. Such molecules are absent from mRNA prepared from kidney or heart, both before and after hormone treatment (Fig. 7, c-f). Alkali treatment of the liver mRNAs prior to electrophoresis eliminated all hybridization (Fig. 7, g, h). We do not known the significance of the high molecular weight material in the kidney and heartpreparations that hybridize with the probe.
in rat tissues DISCUSSION
A group of adult Sprague-Dawley rats was adrenalectomized. After 7 days, half of the group
Ah sequences represent the most abundant family of human and rodent middle repetitive DNAs. They occur at approx 3-5 x lo5 copies per haploid genome
28s -
28s Ii% -
18s
0
b
c
d
Fig. 6. Detection of 7SL RNA in rat hepatoma cell RNA preparations. Electrophoresis of 2pg of poly(A)+ RNA [lanes (a) and (b)] and 2Opg of total cytoplasmic RNA [lanes (c) and (d)] prepared from control [(a) and (c)] or dexamethasone-treated [(b) and (d)] cells. Blots were probed with nick-translated pUC8-S, a plasmid containing an internal segment of 7SL cDNA that is not homologous to the Ah sequence [ref. 31.
abcdef
9
h
Fig. 7. Detection of short repeat-sequence transcripts in RNA preparations from rat tissues. Poly(A)+ RNA was isolated by the pronase_SDS procedure from liver (a, b) heart (c, d) or kidney (e, f) of adrenalectomized control rats (a, c, e, g) or such rats treated with dexamethasone for 12 h (b, d, f, h). Liver RNAs were also treated with alkali and neutralized prior to electrophoresis (g, h). The RNAs were hybridized with 1.3 x lo8 dpm of nick-translated ~655.
206
LEE-HWEI K. SUN and FRED
[l]. A similar sequence is also present in the Drosophila genome, in the form of two copies of 7SL RNA genes, suggesting that sequences within 7SL DNA may have served as the origin for this abundant family of repeats [ 141. RNA transcripts containing Ah sequences occur in mammalian cells in a variety of forms. The simplest are the small, discrete RNAs, such as 5.4s RNA and 7SL RNA, transcribed by RNA polymerase III. These small RNAs are often found associated with longer nuclear or cytoplasmic RNA transcripts, and it has been shown that 7SL forms an essential part of the signal recognition particle required for transport of secreted proteins across the endoplasmic reticulum 1131. Ah sequences are also contained within the heterogeneous nuclear RNAs of mammalian cells, presumably transcribed by RNA polymerase II [15]. They are present to a much lesser extent in cytoplasmic mRNAs [15]. This suggests that they are mainly present within introns and are deleted during the processing of HnRNA. Some, however, must reside in the 5’ or 3’ untranslated regions of mRNAs or perhaps in the coding regions, and are therefore retained on processing. Some recent experiments point to the presence of very short (200400 nucleotide), heterogeneous cytoplasmic RNAs of rodent cells that contain Ah sequences [ 151.These could be molecules produced by the processing of HnRNA or by further cleavage of mRNA. However, since Ah sequences contain pol III promoter sequences, these short RNAs could also be primary pol III transcripts [16]. In this report, we present evidence that a group of short, heterogeneous RNAs occurs in rat hepatoma cells, and that the accumulation of these molecules is controlled by a steroid hormone. Our studies suggest that this control may occur at the level of transcription, since 3.2-fold more radioactive RNA hybridizes to our repeated sequence cDNA clones when the cells are labeled for 30-min in the presence of dexamethasone. Growth of the cells in the presence of dexamethasone plus progesterone prevented the induction. This provides support for the role of glucocorticoid receptor in the induction phenomenon, since at the concentration used, progesterone is able to displace dexamethasone from the receptor, but does not itself produce a glucocorticoid response [ 171. That the induction was not restricted to the tissue culture system was shown by the presence of similar RNA molecules in the liver of adult rats. These molecules were induced several-fold by administration of hormone to the rats. The RNAs were neither present nor inducible in the heart or kidney of these rats. Thus, this appears to be the first report of the induction of tissue-specific repeat sequence transcripts by a hormone. During tests to determine whether protein synthesis was necessary for the induction, we discovered that inhibiting protein synthesis significantly enhanced
R. FRANKEL
the induction, even in the absence of hormone. One interpretation of such an effect is that a labile inhibitory protein negatively regulates the transcription of these RNAs. According to such a model, RNA induction could occur either by inactivation of the inhibitor, presumably by the glucocorticoid-receptor complex, or by preventing its synthesis, by cycloheximide. Interestingly, induction of tyrosine aminotransferase by dexamethasone in rat hepatoma cells has recently been shown to be negatively regulated by the product of a non-syntenic gene [18]. Also, it has been shown in at least one system that cycloheximide can increase the rate of transcription of a gene [ 191. An alternative explanation for the cycloheximide result, however, is that the drug may stabilize the transcripts from degradation. We found that one culture of rat hepatoma cells spontaneously acquired the property of synthesizing high levels of the short RNAs in the absence of dexamethasone. This property was stable in the progeny of these cells. In such cultures, the hormone caused no additional induction, although cycloheximide caused a slight enhancement of the accumulation of the RNAs. In these cultures, the induction of an unrelated glucocorticoid-regulated mRNA (for alpha, -acid glycoprotein) appeared normal, indicating that the receptor pathway had not been altered. A plausible explanation, in the light of the cycloheximide effect, is that the putative inhibitory protein had been altered in these cells and no longer restricted synthesis of repeat sequence transcripts. In this paper we have not examined the higher molecular weight species of RNA that hybridize to the repeat sequence probes that we have seen in the total cell RNA preparations. These presumably represent nuclear species. Whether they are precursors of the short molecules or are independent transcripts is not known. Whether the repeat sequence transcripts have some role in cell differentiation or regulated gene expression or gene product function remains to be determined. However, the tissue-specific, dexamethasone-regulated accumulation of repeat sequence transcripts suggests some role for these molecules in the complex response of cells to hormone. Acknowledgement-These
studies were supported by NIH
grant CA17301.
REFERENCES
Jelinek W. R. and Schmid C. W.: Repetitive sequences in eukaryotic DNA and their expression, A. Rev. Biochew 51 (1982) 813-844. Feinberg R. F., Sun L-H. K., Ordahl C. P. and Frankel F. R.: Identification of glucocorticoid-induced genes in rat hepatoma cells by isolation of cloned cDNA sequences. Proc. natn. Acad. Sci. U.S.A. 80 (1983) 5042-5046. Ullu E., Murphy S. and Melli M.: Human 7SL RNA consists of a 140 nucleotide middle-repetitive sequence inserted in an Alu sequence. Ceil 29 (1982) 195-202.
Dexamethasone
induces Alu-sequence RNAs
4. Aviv J. and Leder P.: Purification of biologically active globin mRNA by chromatography on oligothymidili~ acid-cellulose. Proc. n&t, Acud. Sci. U.S.A. 69 (1972) 1408-1412. 5. Chirgwin J. M., Prezybyla A. E., MacDonald R. J. and Rutter W. J.: Isolation of biologically active RNA from sources enriched in ribonuclease. Biochemisfry 18 (1979) 5294-5299. 6. Gonda T. J., Sheiness D. K. and Bishop J. M.: Transcripts from the cellular homologs of retroviral oncogenes: Distribution among chicken tissues. Molec. cell. ffiol. 2 (1982) 617624. 7. Rave N., Crkvenjakov R. and Boedtker H.: Identification of procollagen mRNAs transferrred to diazobenzyloxymethyl paper from formaldehyde-agarose gels. Nucl. Acid. Res. 6 (1979) 35593567. 8. Thomas P. S.: Hybridization of denatured RNA and small DNA fragments transferred to nitrocellulose. Proc. natn. Acad. Sci. U.S.A. 77 (1980) 5201-5205.
9. Wahl G. M., Stern M. and Stark G. R.: Efficient transfer of large DNA fragments from agarose gels to diazo~nzyloxyme~yl paper and rapid hybridization by using dextran sulfate. Pruc. natn. Acad. Sci. U.S.A. 76 (1979) 3683-3687. 10. Wallace R. B., Johnson M. J., Suggs S. V., Miyoshi K., Bhatt R. and Itakura K.: A set of synthetic oligonucleotide primers for DNA sequencing in the plasmid vector pBR322. Gene 16 (1981) 21-26. 11. Sanger F., Nicklen S. and Coulson A. R.: DNA sequencing with chain-terminating inhibitors. Proc. nacn. Acad. Sci. U.S.A. 74 (1977) 5463-5467. 12. Ashburner M., Chihara C., Meltzer P. and Richards G.:
207
Temporal control of puffing activity in polytene chromosomes. Cold Spring Harb. Symp. quant. Bio2.38 (1973) 6.55662.
13. Walter P. and Blobel G.: Signal recognition particle contains a 7S RNA essential for protein translocation across the endoplasmic reticulum. Nature 299 (1982) 691498.
14. Ullu E. and Tschudi C.: Ah sequences are processed 7SL RNA genes. Nature 312 (1984) 171-172: 1.5 Kramerov D. A.. Lekakh I. V.. Samarina 0. P. and Ryskov A. P.: The sequences homologous to major interspersed repeats Bl and B2 of mouse genome are present in mRNA and small cytoplasmic pofy(A)+ RNA. 1vuci. Acid. Rex 11 (1982) 7477-7481. 16. Haynes S. R. and Jelinek W. R.: Low molecular weight RNAs transcribed in vitro by RNA polymerase from A/u-type dispersed repeats in Chinese hamster DNA are also found in viva Proc. nain. Acad. Sci. U.S.A. 78 (1981) 6130-6134.
17. Baxter J. D. and Tomkins G. M.: Specific cytoplasmic glucocorticoid receptors in hepatoma tissue culture cells. Pro&. natn. Acad. Sci. U.S.A. 68 (1971) 923-937. 18. Peterson T. C., Killory A. M. and Foumier R. E. K.: Chromosomal assignment and trans regulation of the tyrosine aminotransferase structural gene in hepatoma hybrid cells. Molec. cell. Biol. 5 (1985) 2491.-2494. 19. Ringold G. M., Dieckmann B., Vannice J. L., Trahey M. and McCormick F.: Inhibition of protein synthesis stimulates transcription of human beta-interferon genes in Chinese hamster ovary cells. Proc. natn. Acad. Sci. U.S.A. 81 (1984) 39643968.