JOURNAL OF CELLULAR PHYSIOLOGY 144:175-182 (1990)
Role of Messenger RNA Subcellular Localization in the Posttranscriptional Regulation of Human Histone Gene Expression G . ZAMBETTI, J, STEIN, AND G. STEIN* Department of Cell Biology, Univenity of Ma5sachusetts Medical Center, Worccster, Massachusetts 0 1655 Histone mRNAs are naturally localized on non-membrane-bound polysomes and selectively destabilized during inhibition of DNA replication. Targeting histone mRNA to membrane-bound polysomes, by incorporating sequences coding for a signal peptide into the message, results in the stabilization of the histone fusion mRNA when D N A synthesis i s interrupted (Zambetti et at.: Proceedings of the National Academy of Sciences o/' the Uniled Stdtes of America 84:2683-2687, 1987). A single nucleotide substitution that abolishes the synthesis of the signal peptide results in the localization of the histone fusion mRNA on non-membranebound polysomes to the same extent as endogenous histone mRNA and fully restores the coupling of histone fusion mKNA stability to D N A replication. Signal peptide-histone fusion mRNAs containing two point mutations that result in the incorporation of two positively charged amino acids into the hydrophobic domain of the signal peptide are partially retained on non-membrane-bound polysomes and are partially destabilized during inhibition of D N A synthesis. These data indicate that the degree to which the signal peptide-histone fusion mRNAs are associated with non-membrane-bound polysomes is correlated with the extent to which the mRNAs are degraded during inhibition of DNA synthesis. These results suggest that the subcellular location of histone mRNA plays an important role in the posttranscriptional regulation of histone gene expression.
The histone genes provide a model system for studying the regulation of cell cycle and cell growth-controlled genes. An important characteristic of histone gene expression is that most histone protein synthesis (replication dependent) is temporally and functionally coupled to DNA replication (reviewed in Stein and Stein, 1984). Termination of DNA synthesis with metabolic inhibitors or at the natural end of S phase of the cell cycle results in the selective destabilization of histone mRNA in a coordinate and stoichiometric manner with a concomitant shutdown of histone protein synthesis (Prescott, 1966; Alterman et al., 1984; Baumbach e t al., 1984). The coupling of histone mRNA stability with DNA replication is also evident during terminal differentiation (Bird et al., 1986; Stein et al., 1989). As proliferative activity declines during differentiation, the cellular levels of histone mFtNA decrease in parallel with the rates of DNA synthesis. The primary structure of replication-dependent histone mRNAs is less complex than that of most other messenger RNAs. These histone mRNAs, although capped a t the 5' terminus, do not contain introns and are not polyadenylated (Hentschel and Birnstiel, 1981). Interestingly, all replication-dependent histone mRNAs studied to date contain a region of hyphenated dyad symmetry at the 3' terminus that forms a stemloop structure which is necessary for the coupling of (Q
1990 WILEY-LISS, INC.
histone mRNA stability with DNA synthesis (Hentschel and Birnstiel, 1981). Gene fusion studies have demonstrated that the last 30 nucleotides from the 3' terminus of a murine H3 histone mRNA, which contain the stem-loop structure, confer histone mRNAlike properties to alpha globin mRNA (Pandey and Marzluff, 1987). The globin-histone fusion mRNA is rapidly destabilized during the inhibition of DNA replication. Conversely, replacement of the histone mRNA stem-loop structure with sequences from the 3' end of alpha globin results in the expression of a histoneglobin fusion mRNA that is quite stable when DNA synthesis is inhibited (Levine et al., 1987). The presence of the stem-loop structure in replication-dependent histone mRNAs is not sufficient for proper posttranscriptional control. Previous results indicate that this hairpin structure needs to be in the proper position at the 3' end of the message. Histone mRNAs that contain a n extended 3' terminus due to aberrant processing are stable when DNA synthesis is inhibited, despite the presence of the stem-loop structure at a n internal position (Levine et al., 1987). Fur-
Received January 4, 1990; accepted March 12, 1990. *To whom reprint requestsicorrespondenceshould be addressed.
ZAMBETTI ET AL
thermore, translation must proceed within close proximity of the natural translation stop codon of histone mRNA for proper posttranscriptional regulation to take place (Capasso et al., 1987; Graves et al., 1987). Frameshift mutations that result in termination of translation either prematurely (>300 nucleotides upstream from the stop codon) or past the normal translation stop codon render the message stable during DNA synthesis inhibition. Destabilization of histone mRNA appears to occur in a stepwise manner initiating at the 3‘ terminus (Ross et al., 1986). The first detectable degradation product lacks 5 nucleotides from the 3’ end (Ross et al., 1986; ROSSand Kobs, 1986) and can be observed in an in vitro mRNA decay system as well as in intact cells (Ross et al., 1986). It remains to be determined whether this product arises from a single endonucleolytic scission or from multiple cuts removing one or more of the nucleotides a t a time. Degradation continues rapidly in a 3’ to 5‘ direction, carried out by an endonuclease that appears to be part of the ribosomeipolysome complex (Ross et al., 1987). The rapid and selective destabilization of histone mRNA during inhibition of DNA replication is strictly dependent on ongoing protein synthesis. Inhibition of protein synthesis with compounds such as cycloheximide or puromycin prior to the interruption of DNA synthesis prevents the destabilization of histone mRNA (Baumbach et al., 1984; Helms et al., 1984). These results are consistent with regulatory models that require the translation of histone mRNA for destabilization of the message during inhibition of DNA synthesis. Subcellular location of histone mRNA may also affect the stability properties of the message when DNA synthesis is interrupted. Histone mRNAs are naturally associated with non-membrane-bound polysomes (Zambetti et al., 1985), as expected for mRNAs that encode nuclear localized proteins. This is consistent with the presence of mRNAs encoding intracellular proteins on non-membrane-bound polysomes and those encoding proteins that are secreted or are components of cellular membranes on the membrane-bound polysomes. We have previously shown that a signal peptide-histone fusion mRNA that is directed to membrane-bound polysomes is stable, as compared to endogenous histone mRNA, when cells were treated with hydroxyurea to inhibit DNA synthesis (Zambetti et al., 1987). The DNA sequence of the signal peptidehistone fusion gene predicted that the encoded histone fusion mRNA contained the stem-loop structure at the appropriate position and that the message was translated correctly. These results suggested that the histone fusion mRNA contained the necessary signals to couple its stability with DNA synthesis and that the fusion mRNA was not in the proper subcellular location for destabilization to occur. However, we could not dismiss the possibility that an alteration in mRNA structure, due to the introduction of sequences coding for the signal peptide, resulted in the stabilization of the histone fusion mRNA during inhibition of DNA synthesis. To distinguish between these possibilities we have introduced point mutations into the signal peptide-histone fusion gene in order to retain the encoded fusion mRNA on non-membrane-bound poly-
somes with minimal possibility of altering mRNA structure. Point mutations that result in the retention of the histone fusion mRNA on non-membrane-bound polysomes restore the coupling of mRNA stability with DNA synthesis. These results suggest that the subcellular location of histone mRNA plays an important role in the posttranscriptional regulation of histone gene expression. MATERIALS AND METHODS Materials [y-”P]ATP (3,000 Ciimmol) was purchased from Amersham; polyvinylsulfonic acid (PVS) and XAR-5 X-ray film were obtained from Eastman Kodak Co.; S1 nuclease, calf intestine alkaline phosphatase, and Escherichiu coli DNA polymerase large fragment (Klenow enzyme) were purchased from Boehringer Mannheim Biochemicals; Joklik-modified minimum essential medium (SMEM), Eagle’s-modified minimum essential medium (EMEM), fetal calf serum, and horse serum were obtained from Gibco. Plasmid DNAs The construction of the signal peptide-histone fusion gene (SPH3E1) was previously described (Zambetti et al. 1987). The mutated signal peptide-histone fusion genes (SPH3ElATG- and SPH3Ela) were prepared by site-directed mutagenesis as described by Zoller and Smith (1983). The mutations were confirmed by Sanger’s dideoxynucleotide DNA sequencing protocol (1977). Cell culture HeLa S3 cells (human cervical carcinoma cell line) were maintained as a suspension culture at 3-6 x lo5 cellsiml in completed SMEM (SMEM containing 5% fetal calf serum, 5% horse serum, 1mM glutamine, 100 pgiml streptomycin, and 100 Uiml penicillin). Transfection protocol HeLa cells grown in suspension culture were seeded at 3 x lo6 cells per 10 cm tissue culture dish in 20 ml completed EMEM (EMEM containing 5% fetal calf serum, 5% horse serum, 1 mM glutamine, 100 pg/ml streptomycin, and 100 Uiml penicillin) and incubated a t 37°C under 5% COz for 12-16 hours. The cells were re-fed with 10 ml fresh completed EMEM and 4 hours later were transfected with plasmid DNA, which had been prepared in a calcium phosphate precipitate (Graham and van der Eb, 1973). Four hours posttransfection, the cells were treated with 2.5 ml completed EMEM containing 15%glyceroliplate for 1minute. The glycerol-shocked cells were washed with fresh EMEM and then cultured in 20 ml completed EMEMiplate a t 37°C under 5% C 0 2 for 46 hours. RNA isolation Total cellular RNA was isolated as previously described (Plumb et al., 1983). The cells were washed in phosphate-buffered saline (PBS; 150 mM NaC1,lO mM sodium phosphate pH 6.8) and resuspended in 300 pl Lysis buffer (2 mM Tris pH 7.4, 1mM EDTA) containing 5 pg/ml PVS. The cells were lysed in 2.4% SDS and 88 pgiml proteinase K a t room temperature for 15 min-
HISTONE mRNA STABILITY: SUBCELLULAR LOCALIZATION
HELA CELLS homogenize in RSB centrifuge 2,000g, 4Oc for 5 '
P L ~ E TA
centrifuge 11,4OOg, 4Oc for 10'
resuspend in TMN Triton X-100 and
centrifuge 5009, 4'~ for 10'
resuspend in TMN, Triton X-100, and NaDOC
centrifuge 7,80Og, 4'C for 10'
centrifuge 2oO,oOOg, 2h at 4OC through 2M sucrose pad
centrifuge 200,0009, 2h at 4'C through 2M sucrose pad
Fig. 1. Outline of procedure for isolation of subcellular polysomal fractions
Utes. The lysates were adjusted to 300 mM NaC1, extracted with phenol and chloroform, and precipitated with 2.5 volumes 95% ethanol a t -20°C. Total nucleic acids were recovered from the ethanol suspension by centrifugation a t 12,OOOg and resuspended in TCM buffer (10 mM Tris pH 7.4, 2 mM CaCl,, 10 mM MgC1,). The samples were digested for 20 minutes a t 37°C with 0.1 mg/ml DNase I, which was pre-treated with proteinase K to remove ribonuclease activity as described by Tullis and Rubin (1980). The RNA samples were extracted with phenol and chloroform and precipitated with 2.5 volumes 95% ethanol and 0.25 M sodium acetate at -20°C overnight. The RNAs were collected at 12,OOOgfor 30 minutes a t 4"C, resuspended in double distilled water, and quantitated by absorbance. ,a, , ;t, ,X, Non-membrane-bound and membrane-bound polysoma1 RNAs were isolated as previously described (Zambetti et al., 1985) and schematically outlined in Figure 1. HeLa cells were washed in PBS and resuspended in RSB (10 mM Tris pH 7.4,lO mM NaC1,2.5 mM MgC1,). The cells were incubated on ice for 20 minutes, disrupted with 15 strokes in a tight-fitting Dounce homogenizer, and centrifuged a t 2,OOOg for 5 minutes. The supernatant (supernatant A) was removed and saved
a t 0°C. The pellet (pellet A) was resuspended in 9 ml TMN (20 mM Tris pH 7.5, 5 mM MgCl,, 25 mM NaCl), adjusted to 1% Triton X-100 and 1%NaDOC, and centrifuged a t 500g for 10 minutes. The supernatant from this centrifugation step (supernatant C) was removed and saved at 0°C. Supernatant A was then centrifuged at 12,000 rpm, 4°C for 10 minutes in a Beckman JA20 rotor. The resulting supernatant (supernatant B) was removed onto ice for subsequent isolation of non-membrane-bound polysomes. The pellet (pellet B) was resuspended in 9 ml TMN buffer, adjusted to 1%Triton X-100 and 1%NaDOC, and centrifuged at 10,000 rpm, 4°C for 10 minutes. The supernatant (supernatant D) was pooled with supernatant C for the isolation of membrane-bound polysomes. Each of the supernatants was layered onto a 3 ml 2 M sucrose pad (prepared in RSB containing 100 pM spermidine) and centrifuged a t 4°C for 2 hours at 48,000 rpm in a Beckman Type 50.2 Ti rotor (200,0009). The polysomal pellets were resuspended in TMN buffer, extracted with phenol and chloroform, and precipitated in 2.5 volumes of 95% ethanol and 250 mM sodium acetate a t - 20°C overnight. The polysomal RNAs were digested with DNase I and quantitated as described above for the isolation of total cellular RNA.
ZAMBETTI E T AL.
S1 nuclease protection analysis Total cellular and polysomal RNA fractions were subjected to S1 nuclease protection analysis a s previously described (Berk and Sharp, 1978; Zambetti et al., 1987). The S1 probes for analysis of RNA from the different transfected cell cultures were prepared from the appropriate fusion gene. The S1 probes were prepared by radiolabeling the 5’ ends of the 450 base pair Sma I fragment of the signal peptide-histone fusion genes. These S1 probes can simultaneously detect signal peptide-histone fusion mRNA and endogenous histone mRNA (280 and 150 nucleotides, respectively). The RNA (10 pg) was hybridized with the radiolabeled S1 probe in 1 x hybridization buffer (0.04 M Pipes pH 6.4, 0.4 M NaCl, 1mM EDTA, 80% formamide) at 56°C for 3 hours. The samples were adjusted to 0.03 M sodium acetate pH 4.6, 0.025M NaC1, 1 mM ZnSo, and then digested with 900 units S1 nuclease at 37°C for 30 minutes. The S1-digested samples were extracted with phenol and chloroform and precipitated with 2.5 volumes 95% ethanol a t -20°C overnight. The Sl-digested samples were electrophoresed through 6% (w/v) acrylamide-8.3 M urea gels, which were dried under vacuum at 80°C for 1 hour and then exposed to pre-flashed XAR5 Kodak film for various lengths of time. The levels of endogenous histone mRNA and signal peptide-histone fusion mRNAs in each fraction were quantitated by scanning laser densitometric analysis of multiple exposures of the autoradiographs. Autoradiographs within the linear response range of the film were used for densitometric analysis.
Analysis of the densitometric results The distribution of endogenous histone mRNA and signal peptide-histone fusion mRNA within the nonmembrane-bound and membrane-bound polysome fractions was determined using equal quantities of RNA per sample. This method of analysis does not take into consideration the unequal distribution of RNA between these fractions. Therefore, the densitometric results obtained from this type of analysis must be adjusted to reflect these differences. The method for adjusting the densitometric results was as follows. Representation of specific mRNA in non-membrane-boundpolysome fraction (X Densitometric units of specific mRNAipg RNA non-membrane-bound polysomal RNA analyzed by S1) (total +g RNA recovered in non-membrane-bound polysoma1 fraction) = A. Representation of specific mRNA in membrane-boundpolysome fraction (Y Densitometric units of specific mRNA/pg RNA membrane-bound polysomal RNA analyzed by Sl) (total p g RNA recovered in membrane-bound polysoma1 fraction) = B. % Distribution of specific mRNA in non-membranebound polysomal fraction = (AILA + El) (100).
RESULTS It is well documented that histone mRNA stability is tightly coupled to DNA replication; inhibition of DNA
synthesis results in a rapid and selective destabilization of histone mRNAs. Previously, we have determined that histone mRNAs are naturally associated with non-membrane-bound polysomes and are no longer destabilized during inhibition of DNA synthesis when targeted to membrane-bound polysomes with a signal peptide (Zambetti et al., 1987). The stabilization of the histone mRNA on membrane-bound polysomes may be due to the altered subcellular location or to a perturbation in mRNA structure. To distinguish between these possibilities we have introduced point mutations in the fusion gene by site-directed mutagenesis in order to inactivate signal peptide function while preserving mRNA structure as much as possible. Site-directed mutagenesis was carried out a s described by Zoller and Smith (1983), and the wild-type (SPH3E1) and mutant signal peptide-histone fusion mRNA sequences are schematically diagrammed in Figure 2. The SPH3Ela fusion gene (Alpha) contains two point mutations in the region coding for the hydrophobic domain of the signal peptide. These mutations result in the substitution of leucine at amino acid position 10 (GAA-+GTA) and proline a t position 12 ( G G G G T G ) with positively charged histidine residues. The SPH3ElATG- fusion gene (ATG-) contains a single nucleotide substitution which destroys the translation start codon of the signal peptide ( A T G T T G ) . This mutation should result in the initiation of translation a t the natural ATG codon of the histone coding region while abolishing the synthesis of the signal peptide. The effect of the mutations on the association of the signal peptide-histone fusion mRNAs with membranebound polysomes was examined by transfecting the fusion genes into HeLa cells by the calcium phosphate precipitation method of Graham and van der Eb (1973). Forty-six hours after transfection the cells were harvested and non-membrane-bound and membranebound polysomal RNAs were isolated as described previously (Zambetti et al., 1985). The RNA was subjected to S1 nuclease protection analysis so that endogenous histone mRNA and signal peptide-histone fusion mRNA could be simultaneously detected within each sample. As seen in Figure 3, the ATG- fusion mRNA, which does not encode a signal peptide, partitioned into the non-membrane-bound polysome fraction to the same extent as endogenous histone H3 mRNA. Densitometric analysis of the autoradiogram and adjusting for the yields of RNA in each fraction as described in “Materials and Methods” reveals that approximately 82% of the ATG- fusion mRNA and 83%of endogenous histone mRNAs were found associated with non-membrane-bound polysomes. In contrast, wild-type signal peptide-histone fusion mRNA (SPH3E1)was predominantly associated with membrane-bound polysomes (Fig. 3 ) . Approximately 68%of wild-type fusion mRNA was localized in the membrane-bound polysomal RNA fraction. Alpha mRNA, which encodes an altered signal peptide, displayed a more intermediate association with membrane-bound polysomes; approximately 40% of the alpha mRNA was localized in the membranebound polysome fraction and 60% in the nonmembrane-bound polysomal fraction (Fig. 3). These results indicate that the mutation of the translation start codon of the signal peptide results in the synthesis of a
HISTONE mRNA STABILITY: SUBCELLULAR LOCALIZATION 5 0 nt
. . .GGAAGAGU
1 5 10 15 20 Me: Ser I l e Gln H i s Phe A r g V a l A l a Leu I l e P r o Phe P h e A l a A l ? P?e C y s Leu F r o
25 Yd’ P i e 4 1 d M e t A l a AUG AGU AUU CAA CAU UUC CGU GUC GCC CUU AUU CCC UUU UUU GCG GCA UUU UCC CUU CCU GUU UUU C C C AUC GCU
Met S e r I l e G l n His F h e A r g Val A l a H i s I l e H I S Phe Phe A l a A l a P h e C y s Leu P r o V a l Phe A l a Met A l a AUG AGU AUU CAA CAU UUC CGU GUC G C C m A d U U UUU UUU GCG GCA UUU UGC CUU CCU GUU UUU GCC AIJG GCU . .
___ _ _ _ _ _ _ ___ Fig. 2. Schematic diagram and nucleotide sequence of signal peptide-histone fusion mRNAs. Mutated codons are enclosed in boxed area and indicated by lower case letters. Abbreviations: CAP, 5’ terminus; ATG, signal peptide translation start codon; atg, histone translation start codon; Sma, Sma 1 restriction endonuclease site used
_ _ _ _ _ _ _ _ _~ _ _ _ ~ _ _ _ _ _ _ _ _ _
Met A l a
for preparing DNA probes for S1 assays; TAA, translation stop codon; SPH3E1, wild-type signal peptide-histone fusion mRNA; Alpha, SPH3Ela mRNA; ATG-, SPH3ElATG mRNA; vertical arrow, signal peptide-histone protein junction.
Fig. 3. Distribution of the signal peptide-histone fusion mRNAs in non-membrane-bound and membrane-bound polysomal RNA fractions. HeLa cells were transfected with the signal peptide-histone fusion genes (Graham and van der Eb, 1973). Forty-six hours after transfection, non-membrane-bound and membrane-bound polysomes were isolated as described in “Materials and Methods.” Equal quantities of RNA from non-membrane-bound and membrane-bound polysoma1 fractions were subjected to S1 nuclease protection analysis (Berk and Sharp, 1978; Zambetti et al., 1987). L a n e 1 is non-membrane-bound polysomal RNA and lane 2 is membrane-bound polysoma1 RNA. H3 represents endogenous H3 histone mRNA and fusion represents signal peptide-histone fusion mHNA (see “Materials and Methods” for quantitation by densitometric analysis).
from non-membrane-bound polysomes to membranebound polysomes (Fig. 3). To study the stability of the mutated signal peptidehistone fusion mRNAs during inhibition of DNA synthesis, the histone fusion genes (SPH3E1, alpha, and ATG-) were transfected into HeLa monolayer cell cultures. Forty-six hours posttransfection, the cells were treated with 1 mM hydroxyurea and samples were taken at 20 minute time intervals. Total cellular RNA was isolated and assayed for signal peptide-histone fusion mRNA and endogenous H3 histone mRNA content by S1 nuclease protection analysis. As seen in Figure 4A, inhibition of DNA synthesis for 1hour resulted in the destabilization of only 46% of the wild-type signal peptide-histone fusion mRNA. In sharp contrast, the ATG- fusion mRNA was destabilized by 94%, which is to the same extent as measured for endogenous H3 histone mRNA (Fig. 4B). Under the same conditions approximately 70% of the alpha mRNA was destabilized, a value which is intermediate to that observed for wild-type signal peptide-histone fusion mRNA and ATG- fusion mRNA (Fig. 4C). The densitometric results from autoradiographs of multiple S1 assays (n 2 3), a s presented in Figure 4AC, are summarized in Figure 5. These data indicate that the degree to which the signal peptide-histone fusion mRNAs are degraded during inhibition of DNA synthesis is correlated with the extent to which the mRNA is associated with non-membrane-bound polysomes. These results suggest that histone mRNA subcellular localization plays a significant role in the coupling of histone mRNA stability to DNA replication.
signal peptide-histone fusion mRNA (ATG-) that is retained on non-membrane-bound polysomes. I n addition, the incorporation of two histidine amino acid residues into the hydrophobic domain of the signal peptide interferes with the translocation of the fusion mRNA
DISCUSSION Previous studies demonstrated that bacterial secretory proteins that were mutated in the hydrophobic domain of the signal peptide, either by insertion of charged amino acids or by deletion, were not secreted and accumulated in the cytoplasm of the bacterium (reviewed in Emr e t al., 1980). Presumably, the muta-
SPH3 1 2
ALPHA 1 2
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Fig. 4. The effect of hydroxyurea on the cellular levels of signal peptide-histone fusion mRNA. HeLa cells were transfected with pSPH3E1, pSPH3ElATG-, or pSPH3Elu DNA and 46 hours later were treated with 1 mM hydroxyurea (HU). Cell samples were collected in 20 minute intervals, and total cellular RNA was isolated and subjected to S1 nuclease protection analysis as described in “Materials and Methods.” Each lane represents a n independently transfected cell culture. A Wild-type signal peptide-histone fusion mRNA. B ATG- fusion mRNA. C : Alpha fusion mRh’A. Lanes 1,Z: Untreated. Lanes 3,4:HU 20 min. Lanes 5,6: HI7 40 min. Lanes 7 , s HU 60 minutes. H3 represents endogenous H3 histone mRNA and fusion represents signal peptide-histone fusion mRNAs. MW is radiolabeled HpaII-digested pBR322 DNA.
IIISTONE mRNA STABILITY: SUBCELLULAR LOCALIZATION 100
lation start codon was mutated (ATG- fusion mRNA) which prevented the synthesis of the signal peptide and therefore completely inactivated signal peptide function (Fig. 3). Destabilization of ATG- fusion mRNA during inhibition of DNA synthesis with essentially the same ki80 netics and to the same extent as endogenous histone mRNA indicates that this fusion mRNA is structurally competent to be properly regulated. Based on sequence data, the wild-type signal peptide-histone fusion mRNA should also be structurally competent to couple its stability with DNA replication since it differs from 60 the ATG fusion mRNA a t only one nucleotide. In addition, the wild-type signal peptide-histone fusion m mRNA terminates translation at the same site and has m an identical 3’ terminus as ATG- fusion mRNA. The finding that the wild-type signal peptide-histone fusion e 0 mRNA is relatively stable on membrane-bound polyap somes as compared t o ATG- fusion mRNA on non40 membrane-bound polysomes during inhibition of DNA synthesis suggests that subcellular location is important in the pnsttranscriptional regulation of histone gene expression. The relative stability of the wild-type signal peptidehistone fusion mRNA during inhibition of DNA syn20 thesis may also reflect a perturbation of histone protein structure due to the signal peptide (note: the ATG- mutant codes for a normal histone protein that does not contain a signal peptide). This possibility does not appear likely based on the observations that the 3‘ terminus of the message, and not the nascent histone polypeptide, is required for coupling message stability 0 40 60 2o Tirne(HU) with DNA replication (Capasso et al., 1987; Levine et Fig. 5. Densitometric analysis of the hydroxyurea effect on the cel- al., 1987; Pandey and Marzluff, 1987). lular levels of the signal peptide-histone fusion mRNAs. Multiple exThese results indicate that the stability of wild-type posures of representative autoradiographs of S1 assays, as presented signal peptide-histone fusion mRNA during the inhiin Figure 4A-C, were analyzed by scanning laser densitometry. The results are reported as percent decrease in mRNA levels during hy- bition of DNA replication is functionally related to the droxyurea treatment as compared to control samples (0, endogenous change in the subcellular location of the mRNA. It is H3 histone mRNA; *, wild-type signal peptide-histone fusion mRNA; interesting to speculate that the differential stability of D, ATG- fusion mRNA 0, alpha fusion mRNA). wild-type signal peptide-histone fusion mRNA and ATG- fusion mRNA may be due to qualitative differences in the composition of membrane-bound and nontions in the hydrophobic region of the bacterial signal membrane-bound polysomes; however, there is no peptide prevented the translocation of the polysomes previous evidence to support this possibility. Alternainvolved in the synthesis of the defective secretory pro- tively, the subcellular location of the wild-type signal teins to the cell membrane. Based on these studies, peptide-histone fusion mRNA may be deficient in the since no genetic information concerning the targeting factors that are involved in the selective degradation of of mRNA t o membrane-bound polysomes in eukaryotes histone mRNA during inhibition of DNA synthesis. was available at the time our studies were initiated, Several groups have postulated that histone protein the hydrophobic region of the signal peptide was mu- synthesis is autogenously regulated a t the posttrantated in an attempt to prevent the translocation of the scriptional level and that a critical concentration of signal peptide-histone fusion mRNA to membrane- unbound histone protein may be required to destabilize bound polysomes. The incorporation of two positively histone mRNA selectively during inhibition of DNA charged histidine residues in the hydrophobic domain synthesis (Butler and Mueller, 1973; Stein and Stein, of the signal peptide of the alpha mutant resulted in 1984; Peltz and Ross, 1987). This hypothesis is suponly a partial block in the association of mutated signal ported by the finding that free histone proteins can peptide-histone fusion mRNA with membrane-bound selectively enhance histone mRNA degradation in an polysomes (Fig. 3). This result is not surprising in light in vitro mRNA decay system (Peltz and Ross, 1987).In of more recent studies on protein export in eukaryotes, situ hybridization analysis of human WI-38 fibroblast which demonstrate that the relationship between the cells demonstrated that histone mRNAs are localized primary sequence of the signal peptide and its ability in “grape-like clusters” throughout the cytoplasm to function as a secretory signal is quite variable and (Lawrence et al., 1988). Localization of histone mRNA can withstand substantial insertion, substitution, or in clusters may create pockets within the cytoplasm in deletion mutations (Kaiser et al., 1987; Randall and which unbound histone protein could reach the necesHardy, 1989). Subsequently, the signal peptide trans- sary concentration for the destabilization of histone .D
ZAMBETTI ET AL.
mRNA to occur. Directing histone mRNA to membrane-bound polysomes may physically separate the message from these clusters and, therefore, the factors that are involved in the destabilization of histone mRNA during inhibition of DNA synthesis.
ACKNOWLEDGMENTS These studies were supported by grants from the National Institutes of Health (GM 32010 and GM 32381) and the National Science Foundation (DCB 88-96116). G.Z. is a recipient of a Sigma Chi Grant-in-Aid of Research Award. We thank Marie Giorgio for her assistance with the photography, and Andre van Wijnen, Dave Collart, and Tim Morris for their helpful (discussions. LITERATURE CITED Alterman, R., Ganguly, S., Schulze, D., Marzluff, W., Schildkraut, C., and Skoultchi, A. (1984) Cell cycle regulation of mouse H3 histone mRNA metabolism. Mol. Cell. Biol., 4r123-132. Baumbach, L., Marashi, F., Plumb, M., Stein, G., and Stein, J . (1984) Inhibition of DNA replication coordinately reduces cellular levels of core and HI histone mRNA8: Requirement for protein synthesis. Biochemistry, 23r1618-1625. Berk, A,, and Sharp, P. (1978) Spliced early mRNAs of simian virus 40. Proc. Natl. Acad. Sci. U.S.A., 75:1274-1278. Bird, R., Jacobs, F., Stein, G., Stein, J., and Sells, B. (1986) A unique subspecies of histone H4 mRNA from rat myoblasts contains poly(A). Proc. Natl. Acad. Sci. U.S.A., 82:6760-6764. Butler, W., and Mueller, G. (1973) Control of histone synthesis in HeLa cells. Biochim. Biophys. Acta, 294r481-496. Capasso, O., Meeker, G., and Heintz, N. (19871 Sequences controlling histone H4 mRNA abundance. EMBO J., 6:1825-1831. Emr, S., Hall, M., and Silhavy, T. (1980) A mechanism of protein localization: The signal hypothesis and bacteria. J. Cell Biol., 86: 710,711. Graham, F., and van der Eb, A. (1973) A new technique for the assay of infectivity of human adenovirus 5 DNA. Virology, 52,456-467. Graves, R., Pandey, N., Chodchoy, N., and Marzluff, W. (1987) Translation is required for regulation of histone mRNA degradation. Cell, 48t615-626. Helms, S., Baumbach, L., Stein, G., and Stein, J. (1984) Requirement o f protein synthesis for the coupling of histone mRNA levels and DNA replication. FEBS Lett., 168t65-69. Hentschel, C.. and Birnstiel, M. (1981)The organization and expression of histone gene families. Cell, 25r301-313. Kaiser, C., Preuss, D., Grisafi, P., and Botstein, D. (1987) Many random sequences functionally replace the secretion signal sequence of yeast invertase. Science, 236r312-317.
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