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The control of myoglobin synthesis during muscle development
DEVELOPMENTAL
BIOLOGY
The Control
38, 390-?,93 (1974)
of Myoglobin M.
STUART Genetics and Cell Biology
Section,
Synthesis HEYWOOD~
February
Muscle
Development’
S. KENNEDY
AND DORIS
U-125, University Accepted
during
of Connecticut,
Storrs,
Connecticut
06268
4, 1974
The appearance of initiation factors which stimulate myoglobin synthesis in vitro is correlated with the onset of myoglobin synthesis during the development of muscle. The coincident appearance in red muscle of specificity in the IF3 fraction for myoglobin mRNA and myoglobin mRNA in polysomes suggests a concerted control at both transcriptional and translational levels for myoglobin synthesis during development. INTRODUCTION
Messenger RNAs (mRNAs) must compete for the various constituents of the cellular apparatus involved in protein synthesis. In order to make this competition more favorable for specific cell products during cellular differentiation, we have suggested that an mRNA selectivity operates during the initiation steps of protein synthesis (Heywood, 1973; Rourke and Heywood, 1972). This selectivity in the translation of eukaryotic mRNAs was first observed in the translation of myosin and globin mRNA using heterologous cell-free amino acid-incorporating systems (Heywood, 1970; Rourke and Heywood, 1972). Either initiation factor 3 (IF3) or a factor isolated with this fraction was found to be responsible for this messenger selectivity (Heywood, 1970). Recently, a number of reports utilizing globin and viral mRNAs have supported these observations (Wiggle, 1973; Hall and Arnstein, 1973a, b; studies, Nude1 et al., 1973). Additional utilizing both myosin and myoglobin mRNAs as well as initiation factors from red and white muscle, have suggested that muscle contains a multiplicity of messenger recognition factors (Thompson et al., 1973). In this case it was observed that IF3 fraction derived from red (thigh) muscle was effective in the translation of both ’ Supported by NIH Grant HD 03316. ‘Recipient of NIH CDA GM 18904. CopyrIght 0 lSi4 by Academic Press, Inc. All rip-hts of reproduction m any fbrm reserved.
myosin and myoglobin mRNAs while white (breast) muscle IF3 was only effective in the translation of myosin mRNA. Low and Rich (1973) have observed that the myoglobin synthetic ability of chick thigh muscle polysomes increases rapidly after 16 days of development. This increase was shown to correlate well with the appearance and accumulation of myoglobin in tissue. These results suggest that the appearance of myoglobin mRNA in polysomes of chick thigh muscle and the active synthesis of myoglobin occur during a well defined period in the terminal differentiation of red muscle. The experiments reported here were designed to det,ermine whether the specificity of red muscle IF3 could be correlated with the developmental appearance of myoglobin mRNA in polysomes as reported by Low and Rich (1973). The results suggest that the appearance of specificity of the IF3 fraction of red muscle for myoglobin mRNA corresponds to the appearance of the messenger RNA in polysomes. Furthermore, no detectable myoglobin mRNA or myoglobin specific factors were found free or unassociated with ribosomes. EXPERIMENTAL
METHODS
Myoglobin mRNA, initiation factors, myoglobin, and the incubation conditions for the cell-free amino acid-incorporating system were the same as previously described (Rourke and Heywood, 1972). The
391
BRIEF NOTES
muscle S-20 fraction was prepared in the following manner. Thigh muscle from various developmental stages of chick embryos was dissected and allowed to stand in 0.25 M KCl, 0.005 M MgC12, 0.02 M Tris .HCl (pH 7.6), 0.006 Mp-mercaptoethanol, 10% glycerol for 30 min at 0°C. The tissue was then rapidly blotted free of excess buffer and homogenized with a loose-fitting Dounce type homogenizer. Following centrifugation at 20,000 g for 20 min, the middle one-third of the supernatant was used directly as the S-20 fraction. Following the incubation of the various cell-free amino acid-incorporating systems, 1.0 mg carrier myoglobin was added to each incubation mixture. The radioactive myoglobin synthesized in vitro was chemically isolated and its radioactivity determined as et al., previously described (Thompson 1973). RESULTS
AND DISCUSSION
Low and Rich (1973) have demonstrated that there is an abrupt appearance of myoglobin-synthesizing polysomes in chick thigh muscle during late embryogenesis. It can be assumed that this is a result of a rapid increase in the amount of myoglobin mRNA found associated with ribosomes. In order to determine if a similar increase in the specificity of the IF3 fraction for myoglobin mRNA occurs during late differentiation of muscle, IF3 fractions obtained from thigh muscle ribosomes of various stages of development were tested in heterologous cell-free systems for their ability to support myoglobin synthesis in the presence of myoglobin mRNA. The IF3 fraction from 13- and 14-day thigh muscle is ineffective in stimulating myoglobin synthesis and corresponds in activity to later stage (19 day) white muscle IF3 fraction (Fig. 1). However, when IF3 is isolated from thigh muscle at 16 days of development, the time when an abrupt increase in myoglobin polysomes is observed (Low and Rich, 1973), its addition does stimulate myo-
DAYS OF DEVELOPMEN
Appearanceof IF3 fraction specificity for myoglobin mRNA during development.The cell-free FIG.
1.
systems were prepared as described by Thompson et al. (1973). Each cell-free system contained myoglobin mRNA, white muscle ribosomes, and white muscle IF1 and IF2 initiation factor preparations. IF3 was prepared from red thigh (0-O) and white breast (04) muscle of different age embryonic chicks. Background incorporation into myoglobin in the absence of added mRNA was 45 cpm and was subtracted from each value. Total incorporation using either white muscle IF3 or red muscle IF3 was between 29,000 and 31,000 cpm. Myoglobin synthesis does not occur in this system unless red muscle IF3 is present in the incubation mixture (Thompson et al.. 1973). When myoglobin synthesis is determined after the addition of both red and white muscle IF3, no inhibition of synthesis is observed (a).
globin synthesis. A further increase in the ability of the IF3 fraction to enhance myoglobin synthesis is found from day 16 to day 19. In agreement with our earlier results (Thompson et al., 1973), similar factor preparations from white muscle (16-19 days of development) are ineffective in supporting myoglobin synthesis (Fig. 1). If myoglobin mRNA is omitted from the cell-free system, no myoglobin synthesis is observed when IF3 from thigh muscle is present (Thompson et al., 1973)-indicating that functional myoglobin mRNA is not present in the factor preparations. These results suggest that a coincident increase in the ability of IF3 fraction to stimulate the translation of myoglobin mRNA accompanies the increase in myoglobin synthetic activity of thigh muscle polysomes. Therefore, the abrupt increase
392
DEVELOPMENT
BIOLOGY
in myoglobin synthesis during terminal differentiation of muscle may involve an increase in the amount of myoglobin mRNA as well as in the amount of myoglobin specific translation factors. The fact that myosin mRNA and myosin specific factor are present in both 14- and 19-day embryonic muscle (Rourke and Heywood, 1972; Thompson et al., 1973) while similar components involved in myoglobin synthesis appear only after day 16 suggest that the synthesis of these proteins are independently controlled. The above conclusions are based on the appearance of ribosomal bound components involved in myoglobin synthesis. In order to test whether a discrepancy exists in the temporal appearance of myoglobin mRNA and specific IF3 factors for myoglobin initiation in the cellular sap (nonribosome bound) a number of experiments were performed. If nonribosomal specific factors for myoglobin synthesis are present in the cell sap (S-200 fraction) at different stages of development it would be expected that the thigh muscle IF3 fraction could be replaced by the addition of cell sap to the incubation mixture. As shown in Table 1, the S-200 fraction from 13-, 16-, and 19-day thigh muscle equally supported comparable low levels of incorporation into myoTABLE
1
MYOGLOBIN SYNTHESIS USING AN S-200 FRACTION FROM DIFFERENT DEVELOPMENTAL STAGES OF THIGH MUSCLE S-200 Fraction” Day Day Day Day
13 16 19 19 + IF3’
Total cpm
Myoglobin cpm
Percent myoglobin cpm
34,490 32,400 32,360 34,700
120 160 150 1423
0.3 0.5 0.5 4.1
” The S-200 fraction was prepared as described by Rourke and Heywood (1972). Prepared in this manner, it contains 15-20 mg protein per ml and 2 mg S-200 protein was added to each incubation mixture. “IF3 fraction prepared from 19-day embryonic chick thigh muscle.
VOLUME 38, 1974
TABLE
2
MYOGLOBIN SYNTHESIS USING AN S-20 FRACTION FROM DIFFERENT DEVELOPMENTAL STAGES OF THIGH MUSCLE WITH AND WITHOUT ADDED IF3 FRACTION
S-20 Fraction
Day Day Day Day Day Day
13 13 16 16 19 19
IF3 Fraction
Myoglobin cpm per mg ribosomes”
+
280 210 690 810 1370 1280
+ +
Percent myoglobin wm 0.4 0.6 1.4 1.1 2.7 3.2
“The amount of ribosomes in lysates at different days of development varies; therefore, radioactivity was standardized to the ribosome content of each cell-free system.
globin. Only the addition of 19-day thigh muscle IF3 fraction resulted in a substantial increase in myoglobin synthesis. These results suggest that no myoglobin specific IF3 factor exists free of ribosomes from 13 to 19 days of development. Therefore, the results presented in Fig. 1 are indeed illustrative of the developmental appearance of IF3 specificity for myoglobin mRNA. To test the possibility that nonribosomal bound myoglobin mRNA may be present in cytoplasm as a direct result of an inefficient translation due to the lack of specific IF3, muscle S-20 preparations were prepared from various days of development. These preparations were then incubated with and without added IF3 fraction from 19-day thigh muscle. As shown in Table 2 the addition of 19-day IF3 had no effect on the incorporation of amino acids into myoglobin at any stage of development. Therefore, it is unlikely that the lack of specific IF3 is limiting the translation of any cytoplasmic myoglobin mRNA. Taken together the results suggest that both myoglobin mRNA and myoglobin specific factor appear in the cytoplasm in complementary amounts so that neither is limiting to myoglobin synthesis. In considering these two components,
BRIEF NOTES
mRNA and a selective initiation factor, as those specifically required in the cytoplasm for myoglobin synthesis, it is conluded that the appearance of myoglobin synthesis in the cell is brought about by the simultaneous appearance of both these components at a particular stage of development. These results suggest the possibility that during development the timing of synthesis of myoglobin is brought about by the appearance of myoglobin mRNA in the cytoplasm while quantitative control of gene expression or translational efficiency of mRNA may result from the simultaneous appearance of specific factors involved with initiation of protein synthesis. Such a concerted mechanism would thereby ensure a precise qualitative and quantitative control over protein synthesis during development. REFERENCES HALL, N. D., and ARNSTEIN, H. R. (19’73a). Specificity of reticulocyte initiation factors for the translation
393
of globin messenger RNA. Biochem. Biophys. Res. Commun. 54, 1489-1497. HALL, N. D., and ARNSTEIN, H. R. (1973b). Differential translation of o- and fl-globin messenger in a cell-free system. FEBS (Fed. Eur. Biochem. Sot.) Lett. 35, 45-50. HEYWOOD, S. M. (1970). Specificity of mRNA binding factor in eukaryotes. Proc. Nat. Acad. Sci. U.S. 67, 178221788. HEYWOOD, S. M. (1973). Is there specificity in the initiation of protein synthesis in eukaryotic cells? Deuelop. Biol. 31, fl-f3. Low, R. B., and RICH, A. (1973). Myoglobin biosynthesis in the embryonic chick. Biochemistr?: 12, 4555-4559. NUDEL, V., LEBLEU, B., and REVEL, M. (1973). Discrimination between messenger ribonucleic acids by a mammalian translation initiation factor. Proc. Nut. Acad. Sci. U.S. 70, 2139-2144. ROURKE, A., and HEYWOOD, S. M. (1972). Myosin synthesis and specificity of eukaryotic initiation factors. Biochemistry 11, 2061-2066. THOMPSON, W. C., BUZASH, E. A.. and HEYWOOD,S. M. (1973). Translation of myoglobin messenger ribonucleic acid. Biochemistry 12, 4559-4565. WIGGLE, D. T. (1973). Purification of messengerspecific initiation factor from ascites-cell supernatant. Eur. J. Biochem. 35, 11-17.
BIOLOGY
The Control
38, 390-?,93 (1974)
of Myoglobin M.
STUART Genetics and Cell Biology
Section,
Synthesis HEYWOOD~
February
Muscle
Development’
S. KENNEDY
AND DORIS
U-125, University Accepted
during
of Connecticut,
Storrs,
Connecticut
06268
4, 1974
The appearance of initiation factors which stimulate myoglobin synthesis in vitro is correlated with the onset of myoglobin synthesis during the development of muscle. The coincident appearance in red muscle of specificity in the IF3 fraction for myoglobin mRNA and myoglobin mRNA in polysomes suggests a concerted control at both transcriptional and translational levels for myoglobin synthesis during development. INTRODUCTION
Messenger RNAs (mRNAs) must compete for the various constituents of the cellular apparatus involved in protein synthesis. In order to make this competition more favorable for specific cell products during cellular differentiation, we have suggested that an mRNA selectivity operates during the initiation steps of protein synthesis (Heywood, 1973; Rourke and Heywood, 1972). This selectivity in the translation of eukaryotic mRNAs was first observed in the translation of myosin and globin mRNA using heterologous cell-free amino acid-incorporating systems (Heywood, 1970; Rourke and Heywood, 1972). Either initiation factor 3 (IF3) or a factor isolated with this fraction was found to be responsible for this messenger selectivity (Heywood, 1970). Recently, a number of reports utilizing globin and viral mRNAs have supported these observations (Wiggle, 1973; Hall and Arnstein, 1973a, b; studies, Nude1 et al., 1973). Additional utilizing both myosin and myoglobin mRNAs as well as initiation factors from red and white muscle, have suggested that muscle contains a multiplicity of messenger recognition factors (Thompson et al., 1973). In this case it was observed that IF3 fraction derived from red (thigh) muscle was effective in the translation of both ’ Supported by NIH Grant HD 03316. ‘Recipient of NIH CDA GM 18904. CopyrIght 0 lSi4 by Academic Press, Inc. All rip-hts of reproduction m any fbrm reserved.
myosin and myoglobin mRNAs while white (breast) muscle IF3 was only effective in the translation of myosin mRNA. Low and Rich (1973) have observed that the myoglobin synthetic ability of chick thigh muscle polysomes increases rapidly after 16 days of development. This increase was shown to correlate well with the appearance and accumulation of myoglobin in tissue. These results suggest that the appearance of myoglobin mRNA in polysomes of chick thigh muscle and the active synthesis of myoglobin occur during a well defined period in the terminal differentiation of red muscle. The experiments reported here were designed to det,ermine whether the specificity of red muscle IF3 could be correlated with the developmental appearance of myoglobin mRNA in polysomes as reported by Low and Rich (1973). The results suggest that the appearance of specificity of the IF3 fraction of red muscle for myoglobin mRNA corresponds to the appearance of the messenger RNA in polysomes. Furthermore, no detectable myoglobin mRNA or myoglobin specific factors were found free or unassociated with ribosomes. EXPERIMENTAL
METHODS
Myoglobin mRNA, initiation factors, myoglobin, and the incubation conditions for the cell-free amino acid-incorporating system were the same as previously described (Rourke and Heywood, 1972). The
391
BRIEF NOTES
muscle S-20 fraction was prepared in the following manner. Thigh muscle from various developmental stages of chick embryos was dissected and allowed to stand in 0.25 M KCl, 0.005 M MgC12, 0.02 M Tris .HCl (pH 7.6), 0.006 Mp-mercaptoethanol, 10% glycerol for 30 min at 0°C. The tissue was then rapidly blotted free of excess buffer and homogenized with a loose-fitting Dounce type homogenizer. Following centrifugation at 20,000 g for 20 min, the middle one-third of the supernatant was used directly as the S-20 fraction. Following the incubation of the various cell-free amino acid-incorporating systems, 1.0 mg carrier myoglobin was added to each incubation mixture. The radioactive myoglobin synthesized in vitro was chemically isolated and its radioactivity determined as et al., previously described (Thompson 1973). RESULTS
AND DISCUSSION
Low and Rich (1973) have demonstrated that there is an abrupt appearance of myoglobin-synthesizing polysomes in chick thigh muscle during late embryogenesis. It can be assumed that this is a result of a rapid increase in the amount of myoglobin mRNA found associated with ribosomes. In order to determine if a similar increase in the specificity of the IF3 fraction for myoglobin mRNA occurs during late differentiation of muscle, IF3 fractions obtained from thigh muscle ribosomes of various stages of development were tested in heterologous cell-free systems for their ability to support myoglobin synthesis in the presence of myoglobin mRNA. The IF3 fraction from 13- and 14-day thigh muscle is ineffective in stimulating myoglobin synthesis and corresponds in activity to later stage (19 day) white muscle IF3 fraction (Fig. 1). However, when IF3 is isolated from thigh muscle at 16 days of development, the time when an abrupt increase in myoglobin polysomes is observed (Low and Rich, 1973), its addition does stimulate myo-
DAYS OF DEVELOPMEN
Appearanceof IF3 fraction specificity for myoglobin mRNA during development.The cell-free FIG.
1.
systems were prepared as described by Thompson et al. (1973). Each cell-free system contained myoglobin mRNA, white muscle ribosomes, and white muscle IF1 and IF2 initiation factor preparations. IF3 was prepared from red thigh (0-O) and white breast (04) muscle of different age embryonic chicks. Background incorporation into myoglobin in the absence of added mRNA was 45 cpm and was subtracted from each value. Total incorporation using either white muscle IF3 or red muscle IF3 was between 29,000 and 31,000 cpm. Myoglobin synthesis does not occur in this system unless red muscle IF3 is present in the incubation mixture (Thompson et al.. 1973). When myoglobin synthesis is determined after the addition of both red and white muscle IF3, no inhibition of synthesis is observed (a).
globin synthesis. A further increase in the ability of the IF3 fraction to enhance myoglobin synthesis is found from day 16 to day 19. In agreement with our earlier results (Thompson et al., 1973), similar factor preparations from white muscle (16-19 days of development) are ineffective in supporting myoglobin synthesis (Fig. 1). If myoglobin mRNA is omitted from the cell-free system, no myoglobin synthesis is observed when IF3 from thigh muscle is present (Thompson et al., 1973)-indicating that functional myoglobin mRNA is not present in the factor preparations. These results suggest that a coincident increase in the ability of IF3 fraction to stimulate the translation of myoglobin mRNA accompanies the increase in myoglobin synthetic activity of thigh muscle polysomes. Therefore, the abrupt increase
392
DEVELOPMENT
BIOLOGY
in myoglobin synthesis during terminal differentiation of muscle may involve an increase in the amount of myoglobin mRNA as well as in the amount of myoglobin specific translation factors. The fact that myosin mRNA and myosin specific factor are present in both 14- and 19-day embryonic muscle (Rourke and Heywood, 1972; Thompson et al., 1973) while similar components involved in myoglobin synthesis appear only after day 16 suggest that the synthesis of these proteins are independently controlled. The above conclusions are based on the appearance of ribosomal bound components involved in myoglobin synthesis. In order to test whether a discrepancy exists in the temporal appearance of myoglobin mRNA and specific IF3 factors for myoglobin initiation in the cellular sap (nonribosome bound) a number of experiments were performed. If nonribosomal specific factors for myoglobin synthesis are present in the cell sap (S-200 fraction) at different stages of development it would be expected that the thigh muscle IF3 fraction could be replaced by the addition of cell sap to the incubation mixture. As shown in Table 1, the S-200 fraction from 13-, 16-, and 19-day thigh muscle equally supported comparable low levels of incorporation into myoTABLE
1
MYOGLOBIN SYNTHESIS USING AN S-200 FRACTION FROM DIFFERENT DEVELOPMENTAL STAGES OF THIGH MUSCLE S-200 Fraction” Day Day Day Day
13 16 19 19 + IF3’
Total cpm
Myoglobin cpm
Percent myoglobin cpm
34,490 32,400 32,360 34,700
120 160 150 1423
0.3 0.5 0.5 4.1
” The S-200 fraction was prepared as described by Rourke and Heywood (1972). Prepared in this manner, it contains 15-20 mg protein per ml and 2 mg S-200 protein was added to each incubation mixture. “IF3 fraction prepared from 19-day embryonic chick thigh muscle.
VOLUME 38, 1974
TABLE
2
MYOGLOBIN SYNTHESIS USING AN S-20 FRACTION FROM DIFFERENT DEVELOPMENTAL STAGES OF THIGH MUSCLE WITH AND WITHOUT ADDED IF3 FRACTION
S-20 Fraction
Day Day Day Day Day Day
13 13 16 16 19 19
IF3 Fraction
Myoglobin cpm per mg ribosomes”
+
280 210 690 810 1370 1280
+ +
Percent myoglobin wm 0.4 0.6 1.4 1.1 2.7 3.2
“The amount of ribosomes in lysates at different days of development varies; therefore, radioactivity was standardized to the ribosome content of each cell-free system.
globin. Only the addition of 19-day thigh muscle IF3 fraction resulted in a substantial increase in myoglobin synthesis. These results suggest that no myoglobin specific IF3 factor exists free of ribosomes from 13 to 19 days of development. Therefore, the results presented in Fig. 1 are indeed illustrative of the developmental appearance of IF3 specificity for myoglobin mRNA. To test the possibility that nonribosomal bound myoglobin mRNA may be present in cytoplasm as a direct result of an inefficient translation due to the lack of specific IF3, muscle S-20 preparations were prepared from various days of development. These preparations were then incubated with and without added IF3 fraction from 19-day thigh muscle. As shown in Table 2 the addition of 19-day IF3 had no effect on the incorporation of amino acids into myoglobin at any stage of development. Therefore, it is unlikely that the lack of specific IF3 is limiting the translation of any cytoplasmic myoglobin mRNA. Taken together the results suggest that both myoglobin mRNA and myoglobin specific factor appear in the cytoplasm in complementary amounts so that neither is limiting to myoglobin synthesis. In considering these two components,
BRIEF NOTES
mRNA and a selective initiation factor, as those specifically required in the cytoplasm for myoglobin synthesis, it is conluded that the appearance of myoglobin synthesis in the cell is brought about by the simultaneous appearance of both these components at a particular stage of development. These results suggest the possibility that during development the timing of synthesis of myoglobin is brought about by the appearance of myoglobin mRNA in the cytoplasm while quantitative control of gene expression or translational efficiency of mRNA may result from the simultaneous appearance of specific factors involved with initiation of protein synthesis. Such a concerted mechanism would thereby ensure a precise qualitative and quantitative control over protein synthesis during development. REFERENCES HALL, N. D., and ARNSTEIN, H. R. (19’73a). Specificity of reticulocyte initiation factors for the translation
393
of globin messenger RNA. Biochem. Biophys. Res. Commun. 54, 1489-1497. HALL, N. D., and ARNSTEIN, H. R. (1973b). Differential translation of o- and fl-globin messenger in a cell-free system. FEBS (Fed. Eur. Biochem. Sot.) Lett. 35, 45-50. HEYWOOD, S. M. (1970). Specificity of mRNA binding factor in eukaryotes. Proc. Nat. Acad. Sci. U.S. 67, 178221788. HEYWOOD, S. M. (1973). Is there specificity in the initiation of protein synthesis in eukaryotic cells? Deuelop. Biol. 31, fl-f3. Low, R. B., and RICH, A. (1973). Myoglobin biosynthesis in the embryonic chick. Biochemistr?: 12, 4555-4559. NUDEL, V., LEBLEU, B., and REVEL, M. (1973). Discrimination between messenger ribonucleic acids by a mammalian translation initiation factor. Proc. Nut. Acad. Sci. U.S. 70, 2139-2144. ROURKE, A., and HEYWOOD, S. M. (1972). Myosin synthesis and specificity of eukaryotic initiation factors. Biochemistry 11, 2061-2066. THOMPSON, W. C., BUZASH, E. A.. and HEYWOOD,S. M. (1973). Translation of myoglobin messenger ribonucleic acid. Biochemistry 12, 4559-4565. WIGGLE, D. T. (1973). Purification of messengerspecific initiation factor from ascites-cell supernatant. Eur. J. Biochem. 35, 11-17.