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EVIDENCE FOR A LOCALIZATION SIGNAL IN THE 3′UNTRANSLATED REGION OF MYOSIN HEAVY CHAIN MESSENGER RNA
Cell Biology International, 1997, Vol. 21, No. 4, 243–248
EVIDENCE FOR A LOCALIZATION SIGNAL IN THE 3*UNTRANSLATED REGION OF MYOSIN HEAVY CHAIN MESSENGER RNA JOHN W. WISEMAN1,2, L. ANNE GLOVER1 and JOHN E. HESKETH2* 1
Department of Molecular and Cell Biology, Institute of Medical Sciences, Foresterhill, Aberdeen AB25 2ZD, and 2 Intracellular Targeting Group, Rowett Research Institute, Bucksburn, Aberdeen AB21 9SB, U.K. Accepted 19 February 1997
Localization signals in the 3*untranslated region (3*UTR) of myosin heavy chain mRNA were investigated using hybrid gene constructs. In myoblasts transfected with constructs containing either both coding sequences and 3*UTR of the rabbit â-globin gene or the â-globin coding sequences alone in situ hybridization showed that globin transcripts were distributed throughout the cytoplasm with no localization. In contrast, in myoblasts transfected with â-globin coding sequences linked to the myosin heavy chain 3*UTR there was strong perinuclear localization of the hybrid mRNA; this was maintained in myotubes. We conclude that myosin heavy chain ? 1997 Academic Press Limited 3*UTR contains a localization signal. K: myosin; targeting; 3*untranslated region; messenger RNA; post-transciptional control; myoblasts
INTRODUCTION The targeting of newly-synthesized sarcomeric proteins is crucial for the turnover, growth and repair of the myofibrils (Russell et al., 1992). Theoretically, this could arise from targeting of the protein itself, cotranslational assembly due to targeting motifs in the nascent protein or the mRNA, or a targeting of the appropriate mRNAs so as to achieve local synthesis of the protein close to the final site of function (Russell and Dix, 1992). Although the mechanism of targeting is unknown there is evidence to suggest that myosin is synthesized close to, or in association with, the myofibrils. Firstly, ribosomes are found in the interfibrillary and intrafibrillary cytoplasm (Larsen et al., 1969; Horne and Hesketh, 1990a; Gauthier and Mason-Savas, 1993): furthermore, the association of ribosomes with the myofibrils increased under conditions of increased synthesis of actin and myosin (Horne and Hesketh, 1990b; Gauthier and Mason-Savas, 1993). Secondly, in cultured cells, nascent myosin heavy chains are released from the cell matrix by cytochalasin D (Isaacs and Fulton, 1987), suggesting that myosin synthesis occurs on *To whom correspondence should be addressed. 1065–6995/97/040243+06 $25.00/0/cb970140
polysomes associated with cytoskeletal structures. Thirdly, there is some evidence that the myosin heavy chain mRNA is present in the interfibrillary cytoplasm (Aigner and Pette, 1990; Hesketh et al., 1991; Russell et al., 1992): in one case, after electrical stimulation, the mRNA exhibited a banding pattern suggesting an association with the myofibrils (Aigner and Pette, 1990). Although in situ hybridization studies of myosin heavy chain mRNA localization (Aigner and Pette, 1990; Hesketh et al., 1991; Pomeroy et al., 1991; Russell et al., 1992) have given contradictory data there is increasing evidence that a number of other mRNAs are localized in mammalian cells (Lawrence and Singer, 1986; St Johnston, 1995; Hesketh, 1996), including vimentin mRNA in muscle (Cripe et al., 1993) and actin mRNAs in myoblasts (Sundell and Singer, 1990; Hill and Gunning, 1993). In the case of actin and c-myc mRNAs the 3*untranslated region (3*UTR) of the mRNA has been implicated in the mRNA targeting (Kislauskis et al., 1993; Hesketh et al., 1994; Veyrune et al., 1996). The occurrence of mRNA localization mechanisms together with the presence of myosin heavy chain mRNA and ribosomes in the intermyofibrillary cytoplasm suggests that there may be a ? 1997 Academic Press Limited
244
targeting of myosin heavy chain mRNA in muscle. Such a targeting would require a localization signal within the mRNA and the aim of the present work was to investigate, using hybrid gene constructs, cell transfection and in situ hybridization, whether the 3*UTR of a myosin heavy chain mRNA does indeed contain localization information. MATERIALS AND METHODS Gene constructs and transfection Three lines of stable transfectants were studied. The lines were produced by cotransfection, using calcium chloride, of a pMClneo plasmid, which encodes neomycin resistance along with a series of â-globin gene constructs under control of the SV40 early promoter (see Fig. 1). pSVglobin-globin contained three exons and the 3*UTR of the rabbit â-globin gene and was produced by inserting a Hind III-KpnI fragment into the SVpoly vector (Stacey and Schnieke, 1990). pSVglobin was derived from this by deletion of the 3*UTR by a BglII-ClaI restriction. pSVglobin-myosin was produced by inserting a PstI-BamHI fragment of the rabbit slow skeletal myosin heavy chain 3*UTR (Accession number EMBL S43506; Brownson et al., 1992) downstream of the globin coding sequences in pSVglobin. Stable transfectants were selected by culture in the presence of 1 mg/ml G418. Cell culture and in situ hybridization C2C12 myoblasts were grown in glass chamber slides using Dulbecco’s minimal Eagle’s medium supplemented with 10% foetal calf serum and in an atmosphere of 5% CO2. Myotube formation was induced by growing the cells in medium supplemented with 2% horse serum. Cells were washed and fixed as described previously (Hesketh et al., 1994; Veyrune et al., 1996), and incubated in 50% formamide. 2#SSC for 10 min at room temperature prior to hybridization overnight at 55)C with 200 ng of digoxigenin-labelled antisense riboprobe generated from a 90 bp ApaI-EcoRI fragment of the last exon of the rabbit â-globin gene (Veyrune et al., 1996). After hybridization the cells were washed and treated with RNAase as described previously (Hesketh et al., 1994; Veyrune et al., 1996) and bound probe was detected by incubation with alkaline phosphatase-linked anti-digoxigenin and incubation with 4-nitro blue tetrazolium. The formazan produced by alkaline phosphatase
Cell Biology International, Vol. 21, No. 4, 1997
activity was quantified using an image analysis system in which images were captured using a Pulnix camera and Fenestra/Cyclops or S1730 software (Kinetic Imaging Ltd, Liverpool and Skye Instruments Ltd, Llandrindod Wells, U.K.). Eight measurements were taken in the perinuclear cytoplasm and eight in the adjacent peripheral cytoplasm of 20 individual cells from each cell line. Within a given cell there was little variation in staining in either region. Values were corrected for slide blanks in cell-free areas. RNA extraction and Northern hybridization Total RNA was extracted by the method of Chomczynski and Sacchi (1987) and RNA species were then separated by electrophoresis through a denaturing 2.2 formaldehyde, 1.2% agarose gel and transferred to nylon membrane (Genescreen, from NEN Dupont Ltd) by capillary blotting. RNA was fixed to the membrane by exposure to UV light and Northern hybridization carried out as described previously (Hesketh et al., 1994; Veyrune et al., 1996). The â-globin probe corresponded to the XbaI-XhoI fragment which was used in the construction of the chimaeric pSVglobin-myosin (Fig. 1) and which contains the three exons of the â-globin coding region. Specific hybridization was detected and quantified using a Packard Instantimager. After data acquisition membranes were washed in 0.1% SDS for 5–7 min at 95)C to remove all bound probe before rehybridization.
RESULTS AND DISCUSSION Stable expression of â-globin transcripts was found in cells transfected with all three constructs. The location of â-globin sequences was investigated by in situ hybridization of stable transfectants containing the â-globin coding sequences linked either to their own 3*UTR (globin-globin), the myosin heavy chain 3*UTR (globin-myosin) or no 3*UTR (globin). In the control cell line containing globin linked to its own 3*UTR there was no localization of the transcripts in the cytoplasm (Fig. 2a) and the mRNA was present throughout the cytoplasm. This confirms our previous observations in fibroblasts (Veyrune et al., 1996). In contrast, cells transfected with pSVglobin-myosin in which the myosin 3*UTR was added to the â-globin coding sequences showed a distinct pattern of â-globin
Cell Biology International, Vol. 21, No. 4, 1997
245
Promoter
3' UTR
Coding region Stop
ATG
pSVglobin-globin
pSV
Exon 1
Exon 2
Exon 3
Hind III
Bgl II
pSV
polyA Exon 1
Exon 2
Exon 3
Hind III
Bgl II
Stop
ATG
pSVglobin-myosin
pSV
BamH I
Stop
ATG
pSVglobin
polyA
Exon 1
Hind III
Exon 2
BamH I
polyA
Exon 3
Bgl II
BamH I
Fig. 1. Chimaeric gene constructs used to investigate the ability of the myosin heavy chain 3*untranslated region to relocalize globin mRNA. 3*UTR, 3*untranslated region; polyA, polyadenylation signal; pSV, SV40 early promoter; , globin coding region; , globin 3*UTR; , myosin heavy chain 3*UTR.
distribution in which the transcript was localized predominantly in the perinuclear cytoplasm (Fig. 2b) with essentially an absence of â-globin transcripts in the peripheral cytoplasm: a very tight band of perinuclear staining was seen. This perinuclear localization of the globin-myosin transcript was also evident in myotubes (Fig. 2c) formed by fusion of myoblasts expressing this chimaeric gene. Control, untransfected cells showed no staining (Fig. 2d). Image analysis of the transcript distribution (Table 1) showed that in both pSVglobin-globin and pSVglobin cells the density of staining in the peripheral and perinuclear cytoplasm was similar, with a perinuclear/peripheral staining ratio of approximately 1 in both cell lines. Thus, removal of the â-globin 3*UTR had no apparent effect on mRNA localization, showing that the â-globin 3*UTR contains no signal required for it to be distributed through the cytoplasm. In contrast, quantification of the distribution in the pSVglobin-
myosin cells showed 15-fold higher staining in the perinuclear cytoplasm, confirming the dramatically perinuclear distribution of the chimaeric â-globinmyosin transcripts in these cells. Thus, visual inspection of the in situ hybridization and its quantification both show that the myosin heavy chain 3*UTR causes a redistribution of â-globin coding sequences. Theoretically, this redistribution could be due to either a decreased stability of the mRNA in the cytoplasm or some localization signal within the myosin heavy chain 3*UTR leading to a targeting of the chimaeric transcript to the perinuclear cytoplasm. The stability of the various constructs was assessed by measurements of mRNA levels during actinomycin D chase experiments. Transcription was arrested by additon of actinomycin D (5 ìg/ml) and RNA extracted after 0, 3, 6, 9 and 12 h. The abundance of specific transcripts was measured by Northern hybridization and mRNA stability assessed from the reduction in abundance over the 12 h following
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Fig. 2. In situ hybridization showing the distribution of â-globin transcripts in stably transfected C2C12 cells. Cells transfected with either pSVglobin-globin (a) or pSVglobin-myosin (b,c) constructs were fixed and hybridized with a digoxigenin-labelled riboprobe to detect â-globin coding sequences. Specific labelling was detected using an alkaline phosphatase-linked antidigoxigenin antibody and 4-nitro blue tetrazolium as substrate. Note the staining throughout the cytoplasm, even in the cell periphery, in the pSVglobin-globin cells (a); in contrast, in the pSVglobin-myosin cells the transcripts were restricted to the cytoplasm close to the nucleus in both myoblasts (b) and myotubes (c). Control untransfected cells are shown in (d). Bar represents 20 µm.
Table 1. Quantification of the distribution of â-globin transcripts in cells transfected with control and chimaeric constructs â-globin probe bound (arbitrary intensity units)
Cell line SVglobin-globin SVglobin SVglobin-myosin
Perinuclear cytoplasm
Peripheral cytoplasm
Perinuclear:peripheral
53.2&6.2 64.7&8.9 112.9&11.1
46.7&7.7 56.3&10.2 9.9&7.3
1.3&0.3 1.3&0.3 15.2&9.9
The distribution of transcripts, visualized by in situ hybridization, was quantified at different subcellular locations using image processing and analysis packages which allowed an arbitrary intensity value of between 0 and 255 to be attributed to perinuclear and peripheral cytoplasmic regions of the transfected cells. Results, corrected for background levels, are the means&SEM of 20 cells for each cell line, the results for each cell being the mean of eight perinuclear and eight peripheral cytoplasmic measurements.
the inhibition of transcription. As shown in Fig. 3, all three mRNAs were of similar high stability with a half-life of over 12 h. There was no evidence that these mRNAs were unstable; in comparison the
endogenous c-myc mRNA was highly unstable and rapidly degraded (Fig. 3). The data indicate that the observed effect of myosin heavy chain 3*UTR on globin mRNA localization cannot be explained
Cell Biology International, Vol. 21, No. 4, 1997
Fig. 3. Stability of transcripts containing â-globin coding sequences and modified 3*untranslated regions. The figure shows the levels of â-globin transcripts (a) and c-myc mRNA (b) in pSVglobin-globin (lanes 1–5), pSVglobin-myosin (lanes 6–10) and pSVglobin cells (lanes 11–15) 0, 3, 6, 9 and 12 h after addition of actinomycin D (5 ìg/ml). Total RNA was extracted at each time point and analysed by Northern hybridization. Filters were hybridized with a probe specific to the â-globin coding sequences, stripped and rehybridized with a probe specific to the c-myc mRNA. Note the rapid degradation of c-myc mRNA and the stability of â-globin transcripts. Neither removal of the â-globin 3*UTR or subsequent addition of the myosin heavy chain 3*UTR caused any major change in â-globin transcript stability. The abundance of globin transcripts at 0 h shows that basal expression of the transcripts was different in the three cell lines. This difference reflects the variation in expression seen between transfectants and is probably due to the genes being inserted at different sites in the genome.
in terms of increased instability of the transcript in the cytoplasm. In conclusion, the present data show that the 3*UTR of myosin heavy chain mRNA contains a localization signal which can target a reporter sequence to the perinuclear cytoplasm in myoblasts and in early myotubes. Since it is in the perinuclear cytoplasm that sarcomere assembly commences in myotubes, the ability of the signal within the myosin 3*UTR to target transcripts to this region of the cytoplasm is compatible with the hypothesis that myosin heavy chain mRNA is localized by a 3*UTR-dependent mechanism in order to provide local synthesis of the protein. However, further studies are required to investigate whether this signal can target transcripts to the myofibrillar cytoplasm (or the myofibrils themselves) in myofibres. However, the existence of a localization signal in the myosin heavy chain 3*UTR, taken with the previously observed distribution of ribosomes (Larsen et al., 1969; Horne and Hesketh, 1990a,b; Gauthier and Mason-Savas, 1993) and myosin heavy chain mRNA (Aigner and Pette, 1990; Hesketh et al., 1991; Russell et al., 1992), suggests that there is a targeting of myosin heavy
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chain mRNA which may allow local synthesis of the protein or cotranslational assembly (Isaacs and Fulton, 1987). While this work was in progress other studies showed that in myoblasts the localization of creatine kinase isoform mRNAs is dependent on 3*UTR sequences (Wilson et al., 1995). Thus, in addition to actin (Kislauskis et al., 1993), c-myc (Hesketh et al., 1994; Veyrune et al., 1996), creatine kinase (Wilson et al., 1995) and tau (Behar et al., 1995) mRNAs, myosin heavy chain mRNA contains a localization signal in the 3*UTR. Thus, the present data support the hypothesis that mRNA localization in differentiated mammalian cells involves targeting information in the 3*UTR.
ACKNOWLEDGEMENTS This work was supported by the Scottish Office Agriculture, Environment and Fisheries Department (SOAEFD). JW was the recipient of an SERC Case studentship. REFERENCES A S, P D, 1990. In situ hybridization of slow myosin heavy chain mRNA in normal and transforming rabbit muscles with the use of a nonradioactively labeled cRNA. Histochemistry 95: 11–18. B L, M R, S E, B J, G I, 1995. cis-Acting signals and trans-acting proteins are involved in tau mRNA targeting into neurites of differentiating neuronal cells. Int J Dev Neurosci 13: 113–127. B C, L P, J JC, S S, 1992. Reciprocal changes in myosin isoform mRNAs of rabbit skeletal muscle in response to the initiation and cessation of chronic electrical stimulation. Muscle Nerve 15: 694–700. C P, S N, 1987. Single-step method of RNA isolation by acid guanidinium thiocynate-phenolchloroform extraction. Anal Biochem 162: 156–159. C L, M E, F AB, 1993. Vimentin mRNA location changes during muscle development. Proc Natl Acad Sci USA 90: 2724–2728. G GF, M-S A, 1993. Ribosomes in the skeletal muscle filament lattice. Anat Rec 237: 149–156. H JE, 1996. Sorting of messenger RNAs in the cytoplasm: mRNA localization and the cytoskeleton. Exp Cell Res 225: 219–236. H JE, C GP, L N, 1991. Myosin heavy chain mRNA is present in both myofibrillar and subsarcolemmal regions of muscle fibres. Biochem J 279: 309–310. H JE, C GP, P M, B J-M, 1994. Targeting of c-myc and â-globin coding sequences to cytoskeletal-bound polysomes by c-myc 3* untranslated region. Biochem J 298: 143–148.
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H MA, G P, 1993. Beta and gamma actin mRNAs are differentially located in myoblasts. J Cell Biol 122: 825–832. H Z, H J, 1990a. Immunological localization of ribosomes in striated rat muscle. Evidence for myofibrillar association and ontological changes in the subsarcolemmal: myofibrillar distribution. Biochem J 268: 231–236. H Z, H J, 1990b. Increased association of ribosomes with myofibrils during the skeletal-muscle hypertrophy induced either by the beta-adrenoceptor agonist clenbuterol or by tenotomy. Biochem J 272: 831–833. I W, F AB, 1987. Cotranslational assembly of myosin heavy chain in developing cultured skeletal muscle. Proc Natl Acad Sci USA 84: 6174–6178. K EH, L Z, T KL, S RH, 1993. Isoformspecific 3*untranslated sequences sort a-cardiac and â-cytoplasmic actin messenger RNAs to different cytoplasmic compartments. J Cell Biol 123: 165–172. L PF, H P, W JN, 1969. Morphological relationship of polyribosomes and myosin filaments in developing and regenerating skeletal muscle. Nature 222: 1168–1169. L JB, S RH, 1986. Intracellular localization of mRNAs for cytoskeletal proteins. Cell 45: 407–415. P ME, L JB, S RH, BG, 1991. Distribution of myosin heavy chain
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mRNA in embryonic muscle tissue visualized by ultrastructural in situ hybridization. Dev Biol 143: 58–67. R B, D DJ, 1992. Mechanisms for intracellular distribution of mRNA: in situ hybridization studies in muscle. Am J Physiol 262: C1–C8. R B, D DJ, H DL, J-E J, 1992. Repair of injured skeletal muscle: a molecular approach. Med Sci Sports Exerc 24: 189–196. S J D, 1995. The intracellular localization of messenger RNAs. Cell 81: 161–170. S A, S A, 1990. SVpoly: a versatile mammalian expression vector. Nucl Acids Res 18: 28–29. S CL, S RH, 1990. Actin mRNA localizes in the absence of protein synthesis. J Cell Biol 111: 2397–2403. V J-L, C GP, W J, B J-M, H JE, 1996. A localisation signal in the 3*untranslated region of c-myc mRNA targets c-myc mRNA and â-globin reporter sequences to the perinuclear cytoplasm and cytoskeletal-bound polysomes. J Cell Sci 109: 1185– 1194. W IA, B KM, F AM, 1995. Differential localisation of the mRNA of the M and B forms of creatine kinase in myoblasts. Biochem J 308: 599–605.
EVIDENCE FOR A LOCALIZATION SIGNAL IN THE 3*UNTRANSLATED REGION OF MYOSIN HEAVY CHAIN MESSENGER RNA JOHN W. WISEMAN1,2, L. ANNE GLOVER1 and JOHN E. HESKETH2* 1
Department of Molecular and Cell Biology, Institute of Medical Sciences, Foresterhill, Aberdeen AB25 2ZD, and 2 Intracellular Targeting Group, Rowett Research Institute, Bucksburn, Aberdeen AB21 9SB, U.K. Accepted 19 February 1997
Localization signals in the 3*untranslated region (3*UTR) of myosin heavy chain mRNA were investigated using hybrid gene constructs. In myoblasts transfected with constructs containing either both coding sequences and 3*UTR of the rabbit â-globin gene or the â-globin coding sequences alone in situ hybridization showed that globin transcripts were distributed throughout the cytoplasm with no localization. In contrast, in myoblasts transfected with â-globin coding sequences linked to the myosin heavy chain 3*UTR there was strong perinuclear localization of the hybrid mRNA; this was maintained in myotubes. We conclude that myosin heavy chain ? 1997 Academic Press Limited 3*UTR contains a localization signal. K: myosin; targeting; 3*untranslated region; messenger RNA; post-transciptional control; myoblasts
INTRODUCTION The targeting of newly-synthesized sarcomeric proteins is crucial for the turnover, growth and repair of the myofibrils (Russell et al., 1992). Theoretically, this could arise from targeting of the protein itself, cotranslational assembly due to targeting motifs in the nascent protein or the mRNA, or a targeting of the appropriate mRNAs so as to achieve local synthesis of the protein close to the final site of function (Russell and Dix, 1992). Although the mechanism of targeting is unknown there is evidence to suggest that myosin is synthesized close to, or in association with, the myofibrils. Firstly, ribosomes are found in the interfibrillary and intrafibrillary cytoplasm (Larsen et al., 1969; Horne and Hesketh, 1990a; Gauthier and Mason-Savas, 1993): furthermore, the association of ribosomes with the myofibrils increased under conditions of increased synthesis of actin and myosin (Horne and Hesketh, 1990b; Gauthier and Mason-Savas, 1993). Secondly, in cultured cells, nascent myosin heavy chains are released from the cell matrix by cytochalasin D (Isaacs and Fulton, 1987), suggesting that myosin synthesis occurs on *To whom correspondence should be addressed. 1065–6995/97/040243+06 $25.00/0/cb970140
polysomes associated with cytoskeletal structures. Thirdly, there is some evidence that the myosin heavy chain mRNA is present in the interfibrillary cytoplasm (Aigner and Pette, 1990; Hesketh et al., 1991; Russell et al., 1992): in one case, after electrical stimulation, the mRNA exhibited a banding pattern suggesting an association with the myofibrils (Aigner and Pette, 1990). Although in situ hybridization studies of myosin heavy chain mRNA localization (Aigner and Pette, 1990; Hesketh et al., 1991; Pomeroy et al., 1991; Russell et al., 1992) have given contradictory data there is increasing evidence that a number of other mRNAs are localized in mammalian cells (Lawrence and Singer, 1986; St Johnston, 1995; Hesketh, 1996), including vimentin mRNA in muscle (Cripe et al., 1993) and actin mRNAs in myoblasts (Sundell and Singer, 1990; Hill and Gunning, 1993). In the case of actin and c-myc mRNAs the 3*untranslated region (3*UTR) of the mRNA has been implicated in the mRNA targeting (Kislauskis et al., 1993; Hesketh et al., 1994; Veyrune et al., 1996). The occurrence of mRNA localization mechanisms together with the presence of myosin heavy chain mRNA and ribosomes in the intermyofibrillary cytoplasm suggests that there may be a ? 1997 Academic Press Limited
244
targeting of myosin heavy chain mRNA in muscle. Such a targeting would require a localization signal within the mRNA and the aim of the present work was to investigate, using hybrid gene constructs, cell transfection and in situ hybridization, whether the 3*UTR of a myosin heavy chain mRNA does indeed contain localization information. MATERIALS AND METHODS Gene constructs and transfection Three lines of stable transfectants were studied. The lines were produced by cotransfection, using calcium chloride, of a pMClneo plasmid, which encodes neomycin resistance along with a series of â-globin gene constructs under control of the SV40 early promoter (see Fig. 1). pSVglobin-globin contained three exons and the 3*UTR of the rabbit â-globin gene and was produced by inserting a Hind III-KpnI fragment into the SVpoly vector (Stacey and Schnieke, 1990). pSVglobin was derived from this by deletion of the 3*UTR by a BglII-ClaI restriction. pSVglobin-myosin was produced by inserting a PstI-BamHI fragment of the rabbit slow skeletal myosin heavy chain 3*UTR (Accession number EMBL S43506; Brownson et al., 1992) downstream of the globin coding sequences in pSVglobin. Stable transfectants were selected by culture in the presence of 1 mg/ml G418. Cell culture and in situ hybridization C2C12 myoblasts were grown in glass chamber slides using Dulbecco’s minimal Eagle’s medium supplemented with 10% foetal calf serum and in an atmosphere of 5% CO2. Myotube formation was induced by growing the cells in medium supplemented with 2% horse serum. Cells were washed and fixed as described previously (Hesketh et al., 1994; Veyrune et al., 1996), and incubated in 50% formamide. 2#SSC for 10 min at room temperature prior to hybridization overnight at 55)C with 200 ng of digoxigenin-labelled antisense riboprobe generated from a 90 bp ApaI-EcoRI fragment of the last exon of the rabbit â-globin gene (Veyrune et al., 1996). After hybridization the cells were washed and treated with RNAase as described previously (Hesketh et al., 1994; Veyrune et al., 1996) and bound probe was detected by incubation with alkaline phosphatase-linked anti-digoxigenin and incubation with 4-nitro blue tetrazolium. The formazan produced by alkaline phosphatase
Cell Biology International, Vol. 21, No. 4, 1997
activity was quantified using an image analysis system in which images were captured using a Pulnix camera and Fenestra/Cyclops or S1730 software (Kinetic Imaging Ltd, Liverpool and Skye Instruments Ltd, Llandrindod Wells, U.K.). Eight measurements were taken in the perinuclear cytoplasm and eight in the adjacent peripheral cytoplasm of 20 individual cells from each cell line. Within a given cell there was little variation in staining in either region. Values were corrected for slide blanks in cell-free areas. RNA extraction and Northern hybridization Total RNA was extracted by the method of Chomczynski and Sacchi (1987) and RNA species were then separated by electrophoresis through a denaturing 2.2 formaldehyde, 1.2% agarose gel and transferred to nylon membrane (Genescreen, from NEN Dupont Ltd) by capillary blotting. RNA was fixed to the membrane by exposure to UV light and Northern hybridization carried out as described previously (Hesketh et al., 1994; Veyrune et al., 1996). The â-globin probe corresponded to the XbaI-XhoI fragment which was used in the construction of the chimaeric pSVglobin-myosin (Fig. 1) and which contains the three exons of the â-globin coding region. Specific hybridization was detected and quantified using a Packard Instantimager. After data acquisition membranes were washed in 0.1% SDS for 5–7 min at 95)C to remove all bound probe before rehybridization.
RESULTS AND DISCUSSION Stable expression of â-globin transcripts was found in cells transfected with all three constructs. The location of â-globin sequences was investigated by in situ hybridization of stable transfectants containing the â-globin coding sequences linked either to their own 3*UTR (globin-globin), the myosin heavy chain 3*UTR (globin-myosin) or no 3*UTR (globin). In the control cell line containing globin linked to its own 3*UTR there was no localization of the transcripts in the cytoplasm (Fig. 2a) and the mRNA was present throughout the cytoplasm. This confirms our previous observations in fibroblasts (Veyrune et al., 1996). In contrast, cells transfected with pSVglobin-myosin in which the myosin 3*UTR was added to the â-globin coding sequences showed a distinct pattern of â-globin
Cell Biology International, Vol. 21, No. 4, 1997
245
Promoter
3' UTR
Coding region Stop
ATG
pSVglobin-globin
pSV
Exon 1
Exon 2
Exon 3
Hind III
Bgl II
pSV
polyA Exon 1
Exon 2
Exon 3
Hind III
Bgl II
Stop
ATG
pSVglobin-myosin
pSV
BamH I
Stop
ATG
pSVglobin
polyA
Exon 1
Hind III
Exon 2
BamH I
polyA
Exon 3
Bgl II
BamH I
Fig. 1. Chimaeric gene constructs used to investigate the ability of the myosin heavy chain 3*untranslated region to relocalize globin mRNA. 3*UTR, 3*untranslated region; polyA, polyadenylation signal; pSV, SV40 early promoter; , globin coding region; , globin 3*UTR; , myosin heavy chain 3*UTR.
distribution in which the transcript was localized predominantly in the perinuclear cytoplasm (Fig. 2b) with essentially an absence of â-globin transcripts in the peripheral cytoplasm: a very tight band of perinuclear staining was seen. This perinuclear localization of the globin-myosin transcript was also evident in myotubes (Fig. 2c) formed by fusion of myoblasts expressing this chimaeric gene. Control, untransfected cells showed no staining (Fig. 2d). Image analysis of the transcript distribution (Table 1) showed that in both pSVglobin-globin and pSVglobin cells the density of staining in the peripheral and perinuclear cytoplasm was similar, with a perinuclear/peripheral staining ratio of approximately 1 in both cell lines. Thus, removal of the â-globin 3*UTR had no apparent effect on mRNA localization, showing that the â-globin 3*UTR contains no signal required for it to be distributed through the cytoplasm. In contrast, quantification of the distribution in the pSVglobin-
myosin cells showed 15-fold higher staining in the perinuclear cytoplasm, confirming the dramatically perinuclear distribution of the chimaeric â-globinmyosin transcripts in these cells. Thus, visual inspection of the in situ hybridization and its quantification both show that the myosin heavy chain 3*UTR causes a redistribution of â-globin coding sequences. Theoretically, this redistribution could be due to either a decreased stability of the mRNA in the cytoplasm or some localization signal within the myosin heavy chain 3*UTR leading to a targeting of the chimaeric transcript to the perinuclear cytoplasm. The stability of the various constructs was assessed by measurements of mRNA levels during actinomycin D chase experiments. Transcription was arrested by additon of actinomycin D (5 ìg/ml) and RNA extracted after 0, 3, 6, 9 and 12 h. The abundance of specific transcripts was measured by Northern hybridization and mRNA stability assessed from the reduction in abundance over the 12 h following
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Fig. 2. In situ hybridization showing the distribution of â-globin transcripts in stably transfected C2C12 cells. Cells transfected with either pSVglobin-globin (a) or pSVglobin-myosin (b,c) constructs were fixed and hybridized with a digoxigenin-labelled riboprobe to detect â-globin coding sequences. Specific labelling was detected using an alkaline phosphatase-linked antidigoxigenin antibody and 4-nitro blue tetrazolium as substrate. Note the staining throughout the cytoplasm, even in the cell periphery, in the pSVglobin-globin cells (a); in contrast, in the pSVglobin-myosin cells the transcripts were restricted to the cytoplasm close to the nucleus in both myoblasts (b) and myotubes (c). Control untransfected cells are shown in (d). Bar represents 20 µm.
Table 1. Quantification of the distribution of â-globin transcripts in cells transfected with control and chimaeric constructs â-globin probe bound (arbitrary intensity units)
Cell line SVglobin-globin SVglobin SVglobin-myosin
Perinuclear cytoplasm
Peripheral cytoplasm
Perinuclear:peripheral
53.2&6.2 64.7&8.9 112.9&11.1
46.7&7.7 56.3&10.2 9.9&7.3
1.3&0.3 1.3&0.3 15.2&9.9
The distribution of transcripts, visualized by in situ hybridization, was quantified at different subcellular locations using image processing and analysis packages which allowed an arbitrary intensity value of between 0 and 255 to be attributed to perinuclear and peripheral cytoplasmic regions of the transfected cells. Results, corrected for background levels, are the means&SEM of 20 cells for each cell line, the results for each cell being the mean of eight perinuclear and eight peripheral cytoplasmic measurements.
the inhibition of transcription. As shown in Fig. 3, all three mRNAs were of similar high stability with a half-life of over 12 h. There was no evidence that these mRNAs were unstable; in comparison the
endogenous c-myc mRNA was highly unstable and rapidly degraded (Fig. 3). The data indicate that the observed effect of myosin heavy chain 3*UTR on globin mRNA localization cannot be explained
Cell Biology International, Vol. 21, No. 4, 1997
Fig. 3. Stability of transcripts containing â-globin coding sequences and modified 3*untranslated regions. The figure shows the levels of â-globin transcripts (a) and c-myc mRNA (b) in pSVglobin-globin (lanes 1–5), pSVglobin-myosin (lanes 6–10) and pSVglobin cells (lanes 11–15) 0, 3, 6, 9 and 12 h after addition of actinomycin D (5 ìg/ml). Total RNA was extracted at each time point and analysed by Northern hybridization. Filters were hybridized with a probe specific to the â-globin coding sequences, stripped and rehybridized with a probe specific to the c-myc mRNA. Note the rapid degradation of c-myc mRNA and the stability of â-globin transcripts. Neither removal of the â-globin 3*UTR or subsequent addition of the myosin heavy chain 3*UTR caused any major change in â-globin transcript stability. The abundance of globin transcripts at 0 h shows that basal expression of the transcripts was different in the three cell lines. This difference reflects the variation in expression seen between transfectants and is probably due to the genes being inserted at different sites in the genome.
in terms of increased instability of the transcript in the cytoplasm. In conclusion, the present data show that the 3*UTR of myosin heavy chain mRNA contains a localization signal which can target a reporter sequence to the perinuclear cytoplasm in myoblasts and in early myotubes. Since it is in the perinuclear cytoplasm that sarcomere assembly commences in myotubes, the ability of the signal within the myosin 3*UTR to target transcripts to this region of the cytoplasm is compatible with the hypothesis that myosin heavy chain mRNA is localized by a 3*UTR-dependent mechanism in order to provide local synthesis of the protein. However, further studies are required to investigate whether this signal can target transcripts to the myofibrillar cytoplasm (or the myofibrils themselves) in myofibres. However, the existence of a localization signal in the myosin heavy chain 3*UTR, taken with the previously observed distribution of ribosomes (Larsen et al., 1969; Horne and Hesketh, 1990a,b; Gauthier and Mason-Savas, 1993) and myosin heavy chain mRNA (Aigner and Pette, 1990; Hesketh et al., 1991; Russell et al., 1992), suggests that there is a targeting of myosin heavy
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chain mRNA which may allow local synthesis of the protein or cotranslational assembly (Isaacs and Fulton, 1987). While this work was in progress other studies showed that in myoblasts the localization of creatine kinase isoform mRNAs is dependent on 3*UTR sequences (Wilson et al., 1995). Thus, in addition to actin (Kislauskis et al., 1993), c-myc (Hesketh et al., 1994; Veyrune et al., 1996), creatine kinase (Wilson et al., 1995) and tau (Behar et al., 1995) mRNAs, myosin heavy chain mRNA contains a localization signal in the 3*UTR. Thus, the present data support the hypothesis that mRNA localization in differentiated mammalian cells involves targeting information in the 3*UTR.
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