BASIC A S P E C T S O F P R O T E I N S Y N T H E S I S IN M U S C L E 1,2
William C. Thompson and Stuart M. Heywood a
University of Connecticut, Storrs 06268
Summary
The embryonic muscle serves as an excellent
tissue for the study of synthesis of specific
proteins in cell-free systems. We have used
homologous and heterologous cell-free systems
to demonstrate specific messenger RNAs present in several size classes of muscle polysomes.
These cell free systems, in which the added
specific mRNAs were forced to compete with
endogenous mRNAs, demonstrated a requirement for initiation factors washed from ribosomes of tissues normally synthesizing the
protein in question. This held true between red
and white muscle initiation factors in the
ability to recognize added mRNA coding for
myoglobin, even though initiation factors from
either muscle source, but not from a nonmuscle source, were effective in the synthesis of
myosin on heterologous ribosomes. The component of the initiation factors which appears to
be specific has occurred in the IF3 fraction
from DEAE cellulose chromatography. This
IF3 preparation has been separated on phosphocellulose columns into components demonstrating specificity towards different mRNA
species, as demonstrated both by binding of
labelled mRNAs to ribosomes and also by their
effectiveness in the synthesis of the respective
proteins in heterologous ceil free systems. The
possibility of post-transcriptional control of
specific protein synthesis suggested by this
work could provide a means of controlling and
coordinating synthesis of groups of special
proteins during differentiation or more stable
cell states.
Introduction
Muscle is a tussie uniquely suited for the
study of the synthesis of specific cellular
proteins. It contains a number of structural
proteins which must be synthesized both in a
controlled temporal and quantitative relationship so as to facilitate their ultimate organization into a complex structural arrangement. In
addition, globular proteins, such as myoglobin,
must be synthesized in certain muscle cells
while in other muscle types its synthesis is
either depressed or absent. The manner by
which muscle controls the qualitative and quantitative synthesis of these proteins is of primary
importance to our understanding of the processes involved in muscle building in developing
systems as well as in the adult organism.
Embryonic chick muscle offers a valuable
source for studying the synthesis of muscle
proteins. It has a low level of free ribonuclease
and the ribosomes are free of membranous
material (Heywood, Dowben and Rich, 1968).
It is very active in protein synthesis and
accumulates, over a relatively short period of
time, a large amount of cell specific protein.
This process can be observed both in vivo
(Herrmann, Heywood and Marchok, 1970)and
in cell culture (Morris, et al., 1972; Heywood,
Havaranis and Herrmann, 1973). Furthermore,
a great deal is known concerning the morphological changes occurring during muscle development (Fischman, 1970). An analysis of the
a This research was supported by NIH Grant No.
controls
of protein synthesis and the proteins
HD 03316-06.
synthesized can, therefore, be correlated to the
2 In~,itational paper presented at the Symposium on
morphological changes occurring during muscle
Protein Synthesis and Muscle Growth held during the
65th Annual Meeting of the American Society of
development.
Animal Science, Lincoln, Nebraska, July 28 to August
Utilizing the proper ionic conditions, intact
1, 1973.
a S.' M. Heywood is a recipient of Career Develop- polysomes can be obtained from embryonic
chick leg muscle (Heywood et al., 1968). These
ment Award No. GM 18904-03.
1050
JOURNAL OF ANIMAL SCIENCE, vol. 38, no. 5, 1974
PROTEIN SYNTHESIS AND MUSCLE GROWTH
polysomes can be separated into different size
classes by sucrose density gradient centrifugation. Avery large polysome, consisting of about
60 ribosomes per mRNA, was found to synthesize the 200,000 dalton subunit of myosin, or
myosin heavy chain (MHC) (Heywood et al.,
1967), while smaller polysomes were observed
to be responsible for the synthesis of actin and
tropomyosin (Heywood and Rich, 1968). It has
been subsequently demonstrated (Sarkar and
Cooke, 1970; Low, Vornakis and Rich, 1971)
that myosin light chains (MLC) were also
synthesized on small polysomes-indicating
that the mRNAs for myosin were monocistronic.
Because the large polysomes synthesizing
MHC can be isolated relatively free of other
polysomes, the mRNA coding for MHC can be
enriched for isolation by collecting these
~olysomes. Upon labelling check embryos with
2p for 90 min. and subsequently isolating
MHC polysomes, a unique radioactive peak,
sedimenting at 26s, is observed which is not
found in RNA extracted from smaller muscle
polysomes (Heywood and Nwagwu, 1969).
More recent analysis of this RNA species from
polysomes obtained from muscle cell cultures
have shown it to migrate slower on acrylamide
gel electrophoresis than 28S mRNA and be
approximately 32-34S (Morris et al., 1972).
The difference in its behavior on sucrose
gradient analysis and dectrophoretic analysis is
presumably a result of its more extended
configuration under the conditions used than
that of the rRNA marker. An mRNA coding for
a 200,000 dalton polypeptide would have
about 6,000 nucleotides, thereby having a
molecular weight itself of approximately 2.0 x
I 0 ~. A polynudeotide of this molecular weight
would be expected to sediment close to 28S
rRNA. Therefore, a general agreement exists
between the size of this RNA species (28S on
sucrose gradients and 32-34S on acrylamide
gels) and MHC.
Utilizing both homologous (muscle ribosomes) and heterologous (erythroblast ribosomes) cell-free amino acid incorporating systems (Heywood and Nwagwu, 1969; Heywood,
1969; Rourke and Heywood, 1972), it has been
demonstrated that the 26S RNA is indeed the
mRNA coding for MHC. This has been demonstrated by urea and SDS acrylamide gel electrophoresis, antigen-antibody precipitation, and
peptide analysis of in vitro synthesized MHC.
These experiments demonstrate that myosin
mRNA can be translated with a high degree of
fidelity in a cell-free system.
The myosin mRNA is one of the largest
1051
eukaryotic mRNAs that has been used, thus far,
to program a cell-free system. Once its translation is initiated, a considerable lag would be
expected as the nascent chain grows, before
completed myosin chains are released. Kinetic
studies have revealed that 7 to 8 min. elapse
before completed myosin polypeptides are released (Morris et al., 1972). Assuming initiation
occurs upon addition of mRNA to the incubation mixture, MHC growth occurs at the rate of
4 to 5 amino acids per second. This is comparable to the rate estimated for the translation of
hemoglobin mRNA (Hunt, Hunter and Munro,
1969). Therefore, it appears as if the efficiency
of translation of myosin mRNA, once initiation
has occurred, is comparable to that of hemoglobin translational systems.
The homologous and heterologous translational systems we have utilized to study the
synthesis of myosin and, more recently, myoglobin have contained endogenous mRNA on
the polysomes and have entailed the addition of
relatively small amounts of added messenger
RNA. Thus, the added mRNA fraction must
compete with endogenous mRNA for its share
o f the proteih~ynthetic machinery. Under
these conditions we have observed that the
translation of myosin mRNA and globin mRNA
on heterologous ribosomes requires ribosomal
factors or initiation factors of the same cell
type from which the messengers are derived
(Heywood, 1970; Rourke and Heywood,
1972). The factor responsible for the specificity
in translation is either initiation factor 3 (IF3)
or a co-factor isolated with IF3 since both the
mRNA binding activity and mRNA recognition
activity are found in fractions which are
chromatographically indistinguishable on
DEAE cellulose columns (Heywood and
Thompson, 1971; Heywood, 1970). Our IF3
fraction is likely a mixture of Staehelin's E3
and E4 initiation factors and Anderson's M3
and Mza ,factors (personal communication). In
all cases these factors have the function of
aligning the small ribosomal subunit, initiator
tRNA, and mRNA during the formation of the
initiation complex.
When IF3 from muscle (DEAE cellulose
purified) is fractionated on phosphocellulose
columns by a step gradient, fractions are eluted
(PC-l, table 1) which appear to be nonspecific
in binding mRNA as well as fractions which
show specificity towards different mRNAs
(PC-2 binds a 12-17S RNA containing actin and
tubulin mRNA, and PC-3 which binds myosin
26S mRNA, table 1). The fractions found to be
most active in binding each messenger RNA to
ribosomes were also found to be-most effective
1052
THOMPSON AND HEYWOOD
TABLE 1. PHOSPHOCELLULOSEFRACTIONATION OF MUSCLEIF3 INTO FRACTIONS
WHICHSHOWSPECIFICITYIN BINDING AND TRANSLATING DIFFERENT
mRNAsa
P-C
Fraction
mRNA
% cpm
Bound
1
26 S
25
2
26 S
4
3
26 S
60
4
26 S
4
cpm Incorporated into specific protein
(Myosin)
300
80
570
75
(Actin and
1
2
3
4
12 - 17
12- 17
12 - 17
12 - 17
S
S
S
S
30
48
12
3
Tubulin)
1075
2600
1100
100
aMuscle I F 3 w a s first p u r i f i e d b y D E A E c e l l u l o s e
chromatography (Heywood, 1970) and then loaded
o n t o a c o l u m n o f p h o s p b o c e l l u l o s e , l ~ r a c t i o n PC-1 w a s
e l u t e d w i t h 0o05M p o t a s s i u m p h o s p h a t e b u f f e r , p H
7 . 8 , PC-2 a t 0 . 2 M p h o s p h a t e , PC-3 a t 0 . 3 5 M
p h o s p h a t e and PC-4 at 0 . 5 M p h o s p h a t e , m R N A
b i n d i n g w a s p e r f o r m e d as p r e v i o u s l y d e s c r i b e d ( H e y wood, 1970). Myosin and actin-tubulin synthesis were
d e t e r m i n e d b y a c r y l a m i d e gel e l e c t r o p h o r e s i s a f t e r
p u r i f i c a t i o n o f t h e p r o d u c t s as p r e v i o u s l y d e s c r i b e d
( R o u r k e and H e y w o o d , 1 9 7 2 ; M o r r i s e t al., 1 9 7 2 ) .
m R N A , i n i t i a t i o n f a c t o r s , and soluble e n z y m e s w e r e
prepared f r o m 1 4 day e m b r y o n i c c h i c k m u s c l e w h i l e
r i b o s o m e s w e r e f r o m c h i c k e r y t h r o b l a s t s . In t h e
binding experiments, 210 cpm per assay of a2p
m y o s i n m R N A w a s used and 5 0 0 c p m p e r assay o f
a 2 P']-2-17S mRNAo
in the translation of that mRNA in corresponding cell free systems (table 1). Although differences are noted, the electrophoretic patterns on
SDS-acrylamide gels of the various phosphocellulose fractions are generally quite similar (unpublished data). The active component of IF3
which is responsible for the specificity observed
in the cell free systems has not yet been
isolated. Nevertheless, these results using myosin mRNA, globin mRNA, and a 12-17S muscle
mRNA fraction suggest that cell specific or
message specific factors may be involved in a
message selection process during initiation of
protein synthesis in eukaryotic cells.
In order to define the limits of mRNA
selection more narrowly, we have investigated
the translation of a myoglobin mRMA fraction
obtained from 19-day embryonic red muscle
(Thompson, Buzash and Heywood, 1973).
Myoglobin, an oxygen binding protein of
17,000 daltons, is present in red muscle cells
but is absent or present only in very low
amounts in white muscle cells. Myosin, on the
other hand, is synthesized in both red and
white muscle. The cell-free amino acid incorporating systems utilized contained ribosomes and
initiation factors from red and white muscle. It
is observed that an 8-12S RNA fraction from
small polysomes (4 to 8 ribosomes per polysome) contains myoglobin mRNA and is capable of directing the synthesis of myoglobin
using white muscle ribosomes. The myoglobin
synthesized was found to co-purify and migrate
as a single band on both urea and SDS-acrylamide gel electrophoresis. Moreover, a comparison between the tryptic peptides of in vivo and
in vitro synthesized myoglobin attested to the
fact that the mRNA is translated with a high
degree of fidelity in the cell-free system.
Of particular importance is the fmding that
while both red and white muscle initiation
factors are effective in translating myosin
mRNA on erythroblast ribosomes, myoglobin
synthesis is strongly dependent on ribosomal
factors isolated from red m u s c l e - specifically
the IF3 fraction which, thus far, has been
shown to be involved with specificity in myosin
synthesis. These results suggest that there may
be a multiplicity of specific factors in the IF3
fraction derived from the same cell type.
Although a great many questions are still left
unanswered as to the manner by which the IF3
fraction gains its specificity and the precise
manner by which it acts in the messenger
selection process, we think it timely to suggest
that such a control mechanism operating during
the initiation of protein synthesis may provide
a fine tuning in post-transcriptional regulation
of gene expression in eukaryotic cells. This
messenger selection process could not only
regulate the onset of synthesis of specific
proteins, but also influence the amount of
cell-specific proteins to be synthesized at a
specific time during the development of muscle.
Such a mechanism could operate by the addition of a "specificity" molecule to cellular IF3,
thereby channeling this initiation factor into
the synthesis of specific proteins. Under this
particular cell state other mRNAs would compete less favorably for the protein synthetic
machinery of the cell.
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