/ . Embryol. exp. Morph. Vol. 56, pp. 139-156, 1980
Printed in Great Britain © Company of Biologists Limited 1980
139
Presence of cap structures in the messenger RNA
of mouse eggs
By G. A. SCHULTZ, 1 J. R. CLOUGH 2 AND
M. H. JOHNSON 3
From the Department of Anatomy, University of Cambridge
SUMMARY
The presence of 5'-terminal 7-methylguanosine cap structures in the messenger RNA
molecules in mouse eggs was assessed by (1) translational inhibition in a message-dependent
reticulocyte cell-free system with the cap analogue 7-methylguanosine-5'-triphosphate, and
(2) enzymatic removal of cap structures with tobacco acid phosphatase followed by endlabelling with y-32P-ATP and polynucleotide kinase. The results suggest that the majority
of mRNA molecules contain cap structures. There was no detectable difference in degree
of capping in unfertilized versus fertilized egg mRNA.
INTRODUCTION
A considerable body of experimental data has recently been accumulated
which indicates that stable messenger RNA (mRNA) molecules are important
templates for protein synthesis during early post-fertilization and cleavage
stages of mouse development. Since there is little evidence for active mRNA
synthesis immediately following fertilization, much of the stable mRNA
may be maternally derived and stored in the oocyte (see Johnson, 1979, for
review). A number of qualitative stage-specific changes in the kinds of proteins
synthesized by these early embryos also occur (Van Blerkom, 1977). Some of
these new proteins first detectable at the late 1-cell to early 2-cell stage are present in translates of mRNA extracted from unfertilized eggs although they are
not normally synthesized by the egg itself (Braude, Pelham, Flach & Lobatto
1979). Furthermore, the appearance of these polypeptides is insensitive to
transcriptional inhibition with a-amanitin. These results lead to the conclusion
that some form of post-transcriptional control is operative for the expression of
at least some of the egg mRNA molecules during early development. The question then arises as to what form such selective mRNA activation might take.
1
Author's address (for reprints): Division of Medical Biochemistry, University of Calgary,
Calgary, Alberta, Canada T2N 1N4
2
Author's address: MRC Mammalian Development unit, Wolfson House, University
College London U.K.
3
Author's address: Department of Anatomy University of Cambridge Downing Street,
Cambridge CB2 3DY, U.K.
140
G. A. SCHULTZ, J. R. CLOUGH AND M. H. JOHNSON
Most eukaryotic mRNAs contain at their 5'-ends a cap structure of the form
m 7 G 5 'ppp 5 'X m p y ... which is added post-transcriptionally to the primary transcript (Shatkin, 1976; Revel & Groner, 1978). This modification protects the
mRNA at its 5'-terminus against enzymatic attack by phosphatases and other
nucleases. It also appears to be functionally important in mRNA translation since
analogues such as m7GMP or m7GTP competitively inhibit translation of capped
message (Canaani, Revel & Groner, 1976; Hickey, Weber & Baglioni, 1976c;
Weber, Hickey & Baglioni, 1976/)). Furthermore, chemical (Muthukrishnan et aL
1975; Both, Banerjee & Shatkin, 1975) and enzymic (Zan-Kowalczewska,
Furuchi, Both & Shatkin, 1977) removal of cap structures reduces or eliminates
translatability of mRNA both in vitro and in vivo (Lockard & Lane, 1978).
Thus, modifications of the 5'-ends of mRNAs provide one way in which
translational control might be exercised. Interestingly, in this context, newly
fertilized mouse eggs incubated in media containing [3H]guanosine appear
to incorporate a small amount of this precursor into cap structures (Young,
1977).
We have, therefore, assayed for the presence of cap structures in mRNA
molecules of mouse eggs through (1) reduction in in vitro translatability in the
presence of the cap analogue m7GTP, and (2) enzymatic cap removal and endlabelling with y-32P-ATP and polynucleotide kinase. The results suggest that the
majority of mRNA in the unfertilized egg is capped. No change in the degree
of capping of mRNA molecules was detected between the unfertilized egg and
fertilized egg stage.
MATERIALS AND METHODS
Collection of eggs
Six-to nine-week-old mice (outbred CFLP, Anglia Laboratory animals)
were superovulated with an intra-peritoneal (i.p.) injection of 5 or. 10 i.u.
Pregnant Mares Serum Gonadotrophin (Folligon, Intervet, Cambridge)
followed 44 to 48 h later by 5 or 10 i.u. of Human Chorionic Gonadotrophin
(HCG, Chorulon, Intervet) given i.p. Fertilized eggs were obtained from females
which had vaginal plugs after being caged singly with CFLP males overnight.
Both mated and unmated animals were sacrificed 13-15 h post-HCG and the
oviducts dissected out into 0-9% saline. Cumulus masses were liberated into a
phosphate-buffered medium (PB1 containing 0-4 % (w/v) bovine serum albumin,
Whittingham & Wales, 1969). This was replaced for 5-10 min by a medium
containing 1 mg/ml hyaluronidase (Koch Light, Colnbrook, Bucks.) and 10 mg/
ml polyvinyl pyrrolidone to remove cumulus cells. Subsequently the eggs were
transferred through two to three washes of PB1+BSA by use of a fine mouth
pipette. Morphologically abnormal ova (or, in the case of fertilized eggs, eggs
not displaying two pronuclei and second polar body extrusion) were discarded,
and the remaining cells counted.
Caps on mouse egg mRNA
141
RNA extraction
Total RNA was extracted essentially as described by Braude & Pelham
(1979). Eggs were transferred in the minimum possible volume to a 1 ml
conical centrifuge tube to which 2 /A of calf liver tRNA (2-5 mg/ml, BoehringerMannheim) was added as carrier to aid subsequent precipitation. Immediately
20 fi\ of phenol saturated with extraction buffer and 20 /A of extraction buffer
(0-2 M-NaCl, 0-001 M EDTA, 0-02 M Tris, pH 7-4) were added. After thorough
mixing, the suspension was drawn up a microhaematocrit tube by capillary
action, one end of the tube was sealed with putty, and the two phases separated
by centrifugation for 1 minute at 5000 rev./min in a haematocrit centrifuge.
The tube was scored above the interface, and the aqueous phase transferred
to a clean centrifuge tube followed by 2 vols. (50-60 /A) of cold absolute ethanol.
The RNA precipitate was recovered by centrifugation at 28000# for 30min
in a Sorvall RC-5 centrifuge after being left overnight at —20 °C. The extraction
procedure was repeated for 2 /A of calf liver tRNA alone to provide a control.
In vitro translation
The translation system used was the cell-free reticulocyte lysate described by
Pelham & Jackson (1976) as adapted by Braude & Pelham (1979). For some
experiments the same lysate was used without prior treatment with staphylococcal nuclease thus leaving endogenous translational activity.
Egg RNA was prepared for translation by dissolving it in [35S]methionine
(1000-1300 Ci/mmole, 6-85 mCi/ml, Amersham); 5/A of methionine were
used for every 20 /tl of lysate required in the incubation. The mixture was freezedried and the requisite volume of lysate added. The resulting translation mix
was distributed in 20 /A aliquots into experimental tubes. Either GTP or the
cap analogue 7-methylguanosine-5'-triphosphate (m7GTP, P-L Biochemicals
Inc., Milwaukee, Wise.) were added to different incubation tubes in the amounts
indicated in the Results section. Since nucleoside triphosphates can chelate
Mg2+, wherever it is stated that m7GTP (or GTP) was added, an equimolar
amount of MgCl2 was also added to restore the magnesium concentration
reduced by chelation. All tubes had a final volume of 22 /A.
After incubation for 1 h at 37 °C, 2 /A of RNAse A (2 mg/ml) in a solution
of 20 mM methionine, 0-1 M EDTA was added. Tubes were incubated for a
further 10 to 15 minutes after which 2 [A samples were taken using a Hamilton
micro-syringe to assess quantitatively incorporation of PS] methionine into
protein. The remainder of the sample was stored at - 7 0 °C until analysed by
polyacrylamide gel electrophoresis. The 2^1 samples were diluted in 250 /A
water. An equal volume of 1 N-NaOH, 0-5 M - H 2 O 2 containing 1 mg/ml methionine was added and the samples incubated until decolorized. Samples were
precipitated with 0-5 ml of 25 % trichloroacetic acid (TCA), filtered on glassfibre filters (Whatman GF/C), washed with 8 % TCA and dried. Filters were
IO
EMB 56
142
G. A. SCHULTZ, J. R. CLOUGH AND M. H. JOHNSON
counted in a toluene-based scintillation cocktail (Cocktail N, Fisons, Loughborough) at an efficiency of approximately 70 %.
Polyacrylamide gel electrophoresis
Samples of translation products were analyzed qualitatively in one dimension
on SDS polyacrylamide slab gels exactly as described by Braude and Pelham
(1979) except that the separating gel was composed of a uniform 10% acrylamide rather than a gradient of 7-15 %. Radioactive proteins were visualized by
fluorography (Laskey & Mills, 1975) using preflashed Fuji Rx X-ray film and
exposure for 3-14 days.
Enzymatic decapping and end-group labelling of RNA
Because the terminal nucleotide of the cap structure has an inverted 5'-5'
linkage relative to the normal 3'-5' phosphodiester bonds in the rest of the
polynucleotide chain, capped RNA molecules are not capable of being endlabelled with polynucleotide kinase and ATP. Non-capped RNA species with
5'-phosphates can be labelled after removal of the terminal phosphate with
alkaline phosphatase. However the enzyme tobacco acid phosphatase (TAP)
can cleave the cap structures to yield 5'-phosphate termini which subsequently
can also be end-labelled. Therefore, by end-labelling RNA molecules with and
without treatment with TAP, a method is provided to discriminate between
capped and non-capped RNA molecules. This approach was applied to analysis
of RNA extracted from unfertilized and fertilized eggs in the presence of tRNA
carrier.
Removal of 5'-end cap structures, dephosphorylation, and subsequent endgroup labelling with y-32P-ATP and polynucleotide kinase was performed
in the same reaction mix using slight modifications of the procedure of Efstratiadis et ah (1977). Egg RNA or tRNA control preparations were divided into
two equal aliquots and incubated in a 10 /A reaction mixture of 25 mM sodium
acetate, pH 6-0, 10 mM mercaptoethanol, 0-2 mM EDTA in the presence
(+ TAP) or absence (-TAP) of 1 unit of tobacco acid phosphatase at 37 °C for
60 min. Subsequent procedures were identical for both the decapped (+TAP) and
sham ( - T A P ) aliquots. To each tube was added 2 /A of 0-5 M-Tris-HCl, pH 8-3,
and 0-2 unit of bacterial alkaline phosphatase (Bethesda Research Laboratories,
Inc., Rockville, Md.). Dephosphorylation was allowed to continue at 37 °C
for 30 min after which 6 [A of a solution containing 33 mM MgCl2 and 33 mM
dithiothreitol (DTT) was added. The alkaline phosphatase was inhibited by
addition of 1 jA of 50 mM potassium phosphate, pH 9-5 and the entire mixture
transferred to a clean tube in which 250 [iC\ (approximately 80 pmoles) of
y-32P-ATP (3000 Ci/m-mole, Amersham) had been taken to dryness by lyophilization. The reaction mix was brought to a final volume of 25 /A with
H 2 O and phosphorylation initiated by the addition of 1 /A (4 units) of poly-
Caps on mouse egg mRNA
143
nucleotide kinase. Following further incubation at 37 °C for 30 min, the reaction
was terminated by addition of 5 /i\ of calf liver tRNA (2-5 mg/ml), 5 /A 2-4 M
ammonium acetate and 100/d ethanol. The mixture was allowed to precipitate
overnight at - 20 °C and recovered by centrifugation.
Isolation of end-group-label/ed RNA bearing poly(A) tracts
The end-group-labelled RNA pellets were dissolved in 0-25 ml of 0-5 M
ammonium acetate and applied to small columns (0-25 ml bed volume) of
oligo-dT cellulose (Boehringer-Mannheim) made in cut-off Pasteur pipettes
and equilibrated in the same buffer. The columns were washed with five 0-5 ml
aliquots of 0-5 M ammonium acetate followed by four 0-5 ml aliquots of 0-1 M
ammonium acetate such that no further radioactive material was eluted. Poly
(A)-containing RNA (poly(A) + RNA) bound to the affinity column was then
eluted with 1 ml of H 2 O. For analysis on Polyacrylamide gels, RNA was
recovered by lyophilization. For quantitative analysis, the RNA was precipitated with an equal volume of 10% TCA after addition of 100/tg carrier
yeast RNA. The precipitates were trapped on GF/C filters, washed with 5 %
TCA, dried and counted.
Materials
Tobacco mosaic virus (TMV), Cowpea mosaic virus (CPMV), and Tobacco
rattle virus (TRV) mRNAs were kindly provided by Dr H. R. B. Pelham,
Department of Biochemistry, University of Cambridge. Tobacco acid phosphatase was a gift from Dr A. Efstatiadis, Biological Laboratories, Harvard University, and polyniicleotide kinase was a gift from Drs G. Chaconas and H. van
de Sande, Division of Medical Biochemistry, University of Calgary.
RESULTS
Inhibition of translation with cap analogue
Reticulocyte lysate with endogenous message activity was used to characterize
a system to assess degree of capping of mRNA molecules by inhibition of
translation with the cap analogue m7GTP. When increasing concentrations
of m7GTP were added to this lysate, translation of mRNA into protein was
reduced to 20 % of control values with concentrations as low as 0-5 mM m7GTP
(Fig. 1). This reduction in translation was not simply due to addition of guanosine nucleoside triphosphate since equivalent concentrations of GTP did not
affect translatability and at concentrations greater than 0-5 mM, even stimulated
protein synthesis. Polyacrylamide gel separations of the translates were fluorographed to provide a qualitative visualization of the synthesized polypeptides
(Fig. 2). Globin polypeptides which contain greater than 90 % of the incorporated [35S]methionine in these translations have been run off the bottom of the
gel to allow clearer visualization of the remaining translation products. Clearly
144
G. A. SCHULTZ, J. R. CLOUGH AND M. H. JOHNSON
a
GTP
100 -
50 -
\
m7GTP
\
\
.
J
—
•
.
•
n
10
GTPorm7GTP[niM]
20
Fig. 1. Effect of m7GTP and GTP on translation of endogenous mRNA from
reticulocyte lysate. Incubations with varying final concentrations of m'GTP or
GTP were carried out as described in Materials and Methods. Protein synthesis
is expressed as a percentage of the control value which was 4-06 x 105 c.p.m./2/*l
sample. Background was determined using labelled lysate kept on ice and was
9-85 x 103 c.p.m./2 fil sample.
a reduction in the synthesis of all types of polypeptides coded for by the endogenous message occurs in the presence of m7GTP when compared with the
equivalent effect of GTP.
Further characterization of the system was accomplished in message-dependent lysate by testing the effects of m7GTP on the translation of TMV mRNA
which is known to be capped (Kieth & Fraenkel-Conrat, 1975) and on the translation of CPMV mRNA which is totally non-capped (Klootwijk, Klein, Zabel &
Van Kammen 1977). Translation of the capped TMV mRNA was reduced to
about 30 % of control values using 0-5 or 1*0 mM m'GTP while the uncapped
CPMV mRNA was virtually unaffected (Table 2).
The translation of mRNA extracted from unfertilized eggs was also reduced
to about 20 % of control values in the presence of 0-5 to 1-0 mM m7GTP (Fig. 3).
Qualitative analysis of the products on polyacrylamide gels indicated that the
synthesis of all polypeptides coded for by the egg mRNA population was affected by the m7GTP (Fig. 4). The one radioactive band remaining even in the tRNA
background sample is the consequence of addition of [35S]methionine to an
endogenous reticulocyte polypeptide by a ribosome-independent reaction
(Pelham & Jackson, 1976), therefore, explaining its insensitivity to m 7 GTP.
The quantitative aspects of experiments on other unfertilized egg mRNA
Caps on mouse egg mRNA
B
C
D
E
145
F
Fig. 2. Fluorograph of one-dimensional SDS polyacrylamide gel of the [35S]methionine products from a 2 fi\ sample of the in vitro translations shown in Figure 1. A,
B and C are from lysates containing 0-5, 10 and 20 ITIM GTP, respectively. D, E
and F are from lysates containing 0-5, 1 0 and 20 mM m7GTP, respectively. Exposure
time was 3 days.
146
G. A. SCHULTZ, J. R. CLOUGH AND M. H. JOHNSON
0-5
m7GTP (niM)
Fig. 3. Effect of m7GTP on translation of unfertilized egg RNA. RNA was prepared from 1097 eggs and after addition of [35S]methionine and lysate was split
into four equal fractions to which were added 0, 02, 05 and 1 0 mM m'GTP, respectively. Protein synthesis is expressed as a percentage of control value (.25605
c.p.m./2/d sample) after the tRNA background (11634 c.p.m./2. [A sample) was
subtracted.
preparations are summarized in Table 1 along with data on the non-inhibitory
effect of GTP.
End-labelling before and after TAP treatment
End-labelling procedures with and without treatment with TAP were utilized
to discriminate between capped and non-capped mRNA molecules as described
in Materials and Methods. To assess that the enzymes and reaction conditions
employed did not lead to mRNA degradation, end-labelling was performed with
TAP removal of cap structures of tobacco rattle virus (TRV) mRNA and the
electrophoretic mobility of this labelled material as detected by autoradiography
compared to that of untreated TRV mRNA detected by staining procedures
(channels A and B, Fig. 5). With the gel conditions utilized, two high molecular
v/eight mRNA components are resolved in the stained pattern. These two mRNA
species remain intact with identical mobility to the starting material after the
end-labelling procedures (Fig. 5).
The same procedures were then applied to analysis of RNA extracted from
unfertilized and fertilized eggs in the presence of tRNA carrier. Under the conditions utilized, about 20 x 106 c.p.m. of y-32P-ATP were transferred to 5'-ends
of RNA molecules although clearly the predominant transfer was into the tRNA
carrier which was in large excess to that of egg RNA (Table 2). When this material was subjected to affinity chromatography on oligo-dT cellulose to purify
Caps on mouse egg mRNA
A
B
C
147
D
•(-)
Fig. 4. Fluorograph of one dimensional SDS polyacrylamide gel of the [35S]methionine products of a 4/tl sample of the in vitro translations of Figure 3. t is the
background activity of the lysate with tRNA but no egg RNA. A is the control
without m7GTP. B, C and D are translations in the presence of 0-2, 0-5 and 1 0 mM
nrGTP, respectively. Exposure time was 8 days.
the poly(A) + RNA (putative message) component of the egg RNA, less than
0-1 % was bound and this varied with the number of eggs extracted. An example
of an experiment conducted on RNA extracted from 616 unfertilized eggs is
summarized in Table 2. End-labelled tRNA alone (whether TAP treated or not)
binds non-specifically to the oligo-dT cellulose at the level of 0-012 and 0-011 %,
148
G. A. SCHULTZ, J. R. CLOUGH AND M. H. JOHNSON
Table 1. Inhibition ofmRNA
translation by 'cap' analogue
[35S]methionine incorporation (c.p.m.)
tRNA
Control
background (—analogue)
TMV*
8527
123018
CPMV*
8 385
113 925
10454
11234
12383
13052
27 305
25 355
20268
22144
Egg (460)t
Egg (546)
Egg (490)
Egg (412)
Experimental (+analogue)
45346 (0-5 mM m7GTP)
32720 (10 mMm'GTP)
121 809 (0-5 mM m7GTP)
106795 (1-0 mMm'GTP)
10665 (10mMm 7 GTP)
13 834 (10 mMm'GTP)
22400 (10 ITIMGTP)
23020(l-0mMGTP)
% of
control J
32-0
30-7
107-5
93-2
1-3
18-4
1270
109-6
* TMV and CPMV mRNA were present at a concentration of 0-4 /tg/20 /A lysate.
f Number in parentheses designates number of eggs extracted.
% Calculated after subtraction of tRNA background.
respectively. Over several experiments this background value varied from 0-009%
to 0014%. Egg RNA extracted in the presence of the tRNA carrier bound
to the level of 0-088 % above the background value when end-labelled after
'cappase' treatment with TAP. In contrast, in the absence of 'cappase' treatment, only 0-021% of the 32P-label purified with poly(A) + RNA. Assuming
the EGG+TAP sample represents end-labelled poly(A) + RNA molecules
which were both capped and uncapped while E G G - T A P represents endlabelling of only uncapped molecules, the ratio between the two values leads to
the calculation that 76-1 % of the poly(A) + RNA molecules (putative mRNA)
of this unfertilized egg RNA preparation are capped (Table 2). The amount of
radioactivity bound to the oligo-dT cellulose was converted to percentages to
correct for minor differences in total radioactivity applied to the column from
one sample to the next. The results of a number of such analyses performed on
sets of 400-700 unfertilized or fertilized eggs are summarized in Table 3. In
each case, the proportion of poly(A)-containing RNA which was end-labelled
after decapping procedures was always much higher than in the absence of
decapping procedures.
In order to demonstrate that the quantitative differences in 32P-incorporation
observed in the presence or absence of TAP treatment did not simply reflect
variation in the amount of tRNA carrier bound to oligo-dT cellulose columns,
poly(A)-containing, end-labelled RNAs from an experiment similar to that
summarized in Table 2 were resolved on polyacrylamide gels (Fig. 5). A small
amount of labelled control carrier tRNA does bind to the oligo-dT cellulose
column and is resolved as a few bands in the lower part of the gel (channel E,
Fig. 5). In the EGG + TAP sample, a number of labelled poly(A) + RNA
bands of higher molecular weight than the carrier tRNA are resolved and
Caps on mouse egg mRNA
A
B
149
CD
Fig. 5. Polyacrylamide gel electrophoresis of end-labelled RNA. Polyacrylamide
gels contained 3-5% acrylamide, 0-17% bisacrylamide, 8 M urea, 25 mM Tris
and 192 mM glycine, pH 8-3 and were prepared as described by Sanger & Coulson
(1975). The gels were 35 cm long, 10 cm wide and 0-15 cm thick. RNA samples were
dissolved in 25 /tl formamide, applied to slots in the slab gel, and electrophoresis
conducted at 20 mA for 6 h with 25 mM Tris, 192 mM glycine buffer, pH 8-3 until a
bromophenol blue tracking dye had migrated to the bottom of the gel. (A) Photograph of 18 /tg TRV mRNA not subjected to end-labelling procedures and stained
with methylene blue as described by Peacock & Dingman (1967). (B) Photograph of
autoradiogram (Fuji X-ray film, 24 h at - 2 0 °C) of 3 /tg TRV mRNA end-labelled
after TAP treatment as described in the text and run on adjacent slot to material in
A. (C, D, E) RNA was extracted from 880 unfertilized eggs in the presence of
5 fig of carrier tRNA, divided into two equal aliquots and subjected to end-labelling
in the presence (C) or absence (D) of TAP treatment. Poly(A) + RNA was purified
on oligo-dT cellulose and resolved as described. Carrier tRNA treated identically
to (C) was included as a control (E). Total amounts of radioactive 32P incorporated
into material bound to oligo-dT cellulose were 36273 c.p.m. for C, 5767 c.p.m. for
D and 2743 c.p.m. for E. Autoradiography was for 4 days at —20 °C.
20-58
20-23
20-61
2-47
c.p.m.
32
P-RNA
bound
(xlO- 3 )
OlOOl
0012/
%
bound
0 0 R
.
A%
bound
Ratio
-TAP/
+TAP
%
capped
CJ
"
^
*j
'
N
tr1
o
E
Z
O
0-239
761
EGG-TAP
20-21
6-47
0-032 \
n m '
tRNA-TAP
23-43
2-59
0011/
\
* RNA was prepared from 616 unfertilized eggs along with 5 /<g tRNA carrier and divided into two equal aliquots, half of which was end labelled Z
after being subjected to enzymatic decapping by tobacco acid phosphatase (+TAP) and half which was end-labelled without decapping (-TAP). ^
Background was determined using a control of 5 /ig of carrier tRNA. All samples were subjected to affinity chromatography on oligo-dT cellulose •<£
as described in Materials and Methods.
L
EGG + TAP
tRNA + TAP
RNA*
c.p.m.
32
P-RNA
applied
(xlO~ 6 )
Table 2. Purification of end-labelled poly{A) + RNA from unfertilized mouse eggs on oligo-dT cellulose
in
O
p
Caps on mouse egg mRNA
151
Table 3. Summary of % Poly(A) + RNA which is capped
Stage
% capped Poly(A) + RNA
Unfertilized egg (n = 6)*
X_= 82-27 (74-51 -90-92)f
Fertilized egg (n = 4)
X= 81-97 (68-29-94-51)
* Number of samples analysed as described in Table 2.
f Range of values obtained.
clearly are of egg origin (channel C, Fig. 5). As expected, few labelled poly(A) +
RNA bands are resolved in the E G G - T A P sample under identical conditions
of electrophoresis and autoradiographic exposure (channel D, Fig. 5).
DISCUSSION
Two approaches were utilized to investigate the nature of 5'-terminal structures on mRNAs in mouse eggs. The rationale for the first approach was based
on the evidence that cap structures on mRNA molecules appear to augment
efficiency of initiation in translation and the finding that cap analogues like
m'GTP can reduce the translatability of capped mRNAs in vitro (Shatkin, 1976;
Revel & Groner, 1978; Kozak, 1978). However, the degree of discrimination
between translation of capped and uncapped mRNAs and the specificity of
action of cap analogues for the inhibition of translation of capped messages is
dependent upon a number of parameters including the choice of in vitro system
(Lodish & Rose, 1977), the temperature and concentration of monovalent ions
during translation (Kemper & Stolarsky, 1977; Weber et al. 1978; Chu &
Rhoads, 1978), and sources of initiation factors (Held, West & Gallagher 1977;
Bergmann et al. 1979). The nature of the specificity of inhibition of translation
by the cap analogue m 7GTP in the reticulocyte lysate system utilized in this
study was assessed by comparing its effect on the translation of mRNAs from
two plant viruses, TMV which has capped mRNA (Keith & Fraenkel-Conrat,
1975) and CPMV which has uncapped mRNA (Klootwijk et al. 1977). Both
contain mRNA components of similar molecular weight (1-3-2-2 x 10f> daltons)
and incorporate similar amounts of [35S]methionine into protein per /tg mRNA
under standard conditions in the message-dependent reticulocyte system (Table
1). The capped TMV RNA is markedly inhibited by m7GTP while the uncapped
CPMV mRNA is largely unaffected over the range of cap analogue concentrations utilized. In addition, the importance of the methyl group in the m7GTP
cap analogue was reconfirmed since GTP itself had no inhibitory effect on
translation (Fig. 1, Table 1).
The translation of mRNAs from mouse eggs, like the endogenous mRNAs
of non-nuclease treated reticulocyte lysate, is also reduced considerably in the
presence of the cap analogue m7GTP. All the polypeptide translation products
152
G. A. SCHULTZ, J. R. CLOUGH AND M. H. JOHNSON
resolved in one-dimensional polyacrylamide gels were reduced in quantity
suggesting that virtually all the translatable mRNA species are capped. Included
within this set would be the 'masked mRNAs' which code for the characteristic 35000 dalton molecular weight 2-cell polypeptides described by Braude
et al. (1979). Since most of the other polypeptides resolved on two-dimensional
gels of egg mRNA translates are also present in two-dimensional electropherograms of polypeptides synthesized by intact eggs (Braude et al. 1979), it appears
that nearly every template active mRNA (within the limits of sensitivity of
detection in these experiments) in unfertilized eggs is capped. The fact that cap
analogues do not reduce translation completely to background levels is a common observation in these types of experiments and likely reflects the fact that
initiation may occur by mechanisms not involving the cap structure but at
reduced efficiency (Kozak, 1978). The results of these experiments do not
allow us to conclude that every mRNA molecule in the unfertilized egg is
capped. Some messenger RNAs can be expected to be in too low a concentration to stimulate detectable amounts of protein synthesis. Also, if the egg
contains some uncapped mRNAs which do not translate (or translate with
low efficiency) in vitro, they would not be detected as being insensitive to
inhibition by m7GTP. For this reason, another set of experiments which do not
rely on the translatability of mRNA were undertaken to assess degree of
capping.
Poly(A)-containing RNA (putative message) was purified subsequent to
end-labelling procedures performed before and after removal of 5'-terminal
cap structures with tobacco and phosphatase. The total RNA content of 500
mouse eggs is about 275 ng (Olds, Stern & Biggers, 1973). On the basis of poly
(A) content of the mouse egg (Levey, Stull & Brinster 1978) and the assumption
that the average mRNA molecule in mouse eggs is about 1500 nucleotides long
(molecular weight of approximately 5 x 105 daltons) of which about one tenth
(150 nucleotides) is poly(A) tract, it can be calculated that 2-4% of the total
RNA is poly(A) + RNA or putative message. In absolute terms, this is 5-10 ng
(or on a molar basis, 10-20 fmoles) of poly(A) + RNA per 500 eggs. We extracted this RNA in the presence of 5 \L% of carrier tRNA from calf liver, which is
in 500 to 1000-fold excess over that of the poly(A) + RNA. It is therefore reasonable to expect that something less than 0-5 % of the resulting total RNA
preparation is represented by poly(A)-containing RNA.
The specific activity of the y-32P-ATP used in end-labelling was 6-5 x 103
cpm/fmole. Theoretically, if every 5'-end of poly(A) + RNA was labelled,
approximately 6-5-13-0 x 104 c.p.m. of 32P-label should be transferred to poly
(A) + RNA molecules. In the case of the experiment summarized in Table 2,
RNA was prepared from 616 eggs, and half was end-labelled after removal of
cap structures and half in the absence. In the former case, all poly(A)-containing
RNA molecules should have the potential to be end-labelled with y-32P-ATP
and poly-nucleotide kinase while in the latter, only non-capped species should
Caps on mouse egg mRNA
153
label. The RNA sample which was decapped with TAP (and represented RNA
extracted from 308 eggs) contained 18-14 x 103 c.p.m. of incorporated label in
poly(A) + RNA molecules (Table 2). On the basis of the calculations above,
as much as 40 to 80 x 103 c.p.m. was theoretically possible. The reduced level
obtained is not unreasonable, however, since recovery of RNA during extraction
must be assumed to be less than 100 % and since enzymatic decapping and endlabelling procedures do not go to total completion in the reaction times utilized
even when enzymes and ATP substrate are provided in excess (Efstratiadis et al.
1977).
The finding that much more 32P is incorporated into poly(A)-containing
RNA during end-labelling procedures on molecules subjected to enzymatic
decapping than in those which were sham treated (Tables 2 and 3) supports the
previous results which demonstrated that the majority of translatable mRNAs
in mouse eggs is capped. Further, there is no observable difference between
unfertilized and fertilized egg RNA. The validity of these conclusions depends
upon the fact that the enzymatic decapping and end-labelling procedures do not
result in fragmentation of RNA molecules to yield other 5'-termini which can be
labelled. It is imperative that the enzymes utilized are free of contaminating
nucleases. The TAP and polynucleotide kinase preparations utilized here have
been previously used to end-label /?-globin (Efstratiadis et al. 1977) and protamine (Gedamu & Dixon, 1978) mRNA without degradation. In this study,
mRNA components from TRV have been enzymically decapped and endlabelled without observable degradation when compared to starting material
on polyacrylamide gels (Fig. 5). Because the TRV RNA molecules resolved are
very large, generation of smaller molecules because of nuclease cleavage should
have been readily detected as a number of labelled bands in the lower part of the
gel. In addition, affinity chromatography on oligo-dT cellulose does not appear
to lead to degradation since poly(A)-containing mRNA purified from mouse
eggs by this procedure still has translational activity (Johnson, Schultz &
Braude, unpublished results). Qualitative analysis of poly(A) + RNA molecules
on polyacrylamide gels (Fig. 5) also reveals a marked increase in the intensity
and number of RNA species end-labelled in the presence of TAP compared
to those end-labelled in the absence of TAP treatment. These are clearly distinguishable from the background of control carrier tRNA preparations. Finally,
if extensive fragmentation of RNA molecules occurred during the reactions,
many additional 5'-termini available for end-labelling would be generated, a
result which is not consistent with the amount of 32P-incorporation we observed.
That a small amount of RNA cleavage has occurred during end-labelling procedures cannot be ruled out. The consequence of such events would be the
generation of 5'-termini which would behave as uncapped molecules. Therefore,
the observation that about 80% of the poly(A) + RNA molecules are capped
must be regarded as a minimal estimate and conceivably all of the molecules
could be capped.
154
G. A. SCHULTZ, J. R. CLOUGH AND M. H. JOHNSON
In summary, virtually all the mRNA molecules extracted from unfertilized
mouse eggs that lead to detectable polypeptide products during in vitro translation are sensitive to inhibition by m 7 GTP. In experiments in which poly(A)
+ RNA (putative message) was analysed after end-labelling procedures with
and without treatment with TAP, a minimum of 80 % of the molecules were
observed to contain cap structures in each case. Taken together, the results
suggest that the majority of mRNAs in mouse eggs contain cap structures. These
findings are not unexpected as caps provide protection from 5'-exonuclease
degradation and appear to augment translational efficiency in eukaryotic cells.
The mRNA of unfertilized sea-urchin eggs has also been shown to be predominantly capped (Hickey et al. 1916b). The methodology utilized in this study
is not sufficiently sensitive to rule out the existence of some uncapped mRNA
species in mouse eggs or minor alterations in capping which might accompany
fertilization events, and may well be compatible with the small amount of
[3H]guanosine incorporated into cap structures in newly fertilized mouse eggs
observed by Young (1977). However, no quantitative data were presented in the
latter study making it difficult to assess the significance of this observation in
respect to what percentage of mRNA molecules might be affected. Our results
indicate that a considerable proportion of mRNA molecules in the mouse egg
contains cap structures and argue against addition of cap structures to pre-existent
mRNA molecules of maternal origin as a major mechanism of post-transcriptional regulation in early post-fertilization events.
This work was supported by grants from the Ford Foundation and the Medical Research
Council to Dr M. H. Johnson, Department of Anatomy, University of Cambridge, and in
part by an operating grant from the Medical Research Council of Canada to G.A. S. The
authors are grateful to all individuals who provided enzymes and viral mRNAs as gifts.
Valuable discussion and helpful criticism from Dr P. R. Braude and Dr H. R. B. Pelham is
also noted.
REFERENCES
BERGMANN, J. E., TRACHSEL, H., SONENBURG, N., SHATKIN, A. & LODISH, H. F. (1979).
Characterization of rabbit reticulocyte factor(s) that stimulates the translation of mRNAs
lacking 5'-terminal 7-methylguanosine. / . biol. Chem. 254, 1440-1443.
BOTH, G. W., BANERJEE, A. K. & SHATKIN, A. J. (.1975). Methylation-dependent translation
of viral messenger RNAs in vitro. Proc. natn. Acad. Sci. U.S.A. 72,1189-1193.
BRAUDE, P. R. & PELHAM, H. R. B. (1979). A microsystem for the extraction and in vitro
translation of mouse embryo mRNA. /. Reprod. Fert. 56,153-158.
BRAUDE, P. R., PELHAM, H. R. B., FLACH, G. & LOBATTO, R. (1979). Post-transcriptional
control in the early mouse embryo. Nature, Lond. 282, 102-105.
CANAANI, D., REVEL, M. & GRONER, Y. (1976). Translational discrimination of 'capped'
and 'non-capped' mRNAs: Inhibition by a series of chemical analogues of 7mCpppX.
FEBSLett. 64, 326-331.
CHU, L. Y. & RHOADS, F. E. (1978). Translational recognition of the 5'-terminal methylguanosine of globin messenger RNA as a function of ionic strength. Biochemistry 17,
2450-2454.
Caps on mouse egg mRNA
155
EFSTRATIADIS, A., VOURNAKIS, J., DORIS-KELLER, H., CHACONAS, G., DOUGALL, D. &
KAFATOS, F. C. (1977). End-labelling of enzymatically decapped mRNA. Nucleic Acid
Res. 4, 4165-4172.
L. & DIXON, G. H. (1978). Effect of enzymatic 'decapping' on protamine translation in wheat germ S-30. Biochem. biophys. Res. Comm. 85, 114-124.
HELD, W. A., WEST, K. & GALLAGHER, J. F. (1977). Importance of initiation factor preparations in the translation of reovirus and globin mRNAs lacking a 5'-terminal 7-methylguanosine. /. biol. Chem. 252, 8489-8497.
HICKEY, E. D., WEBER, L. A. & BAGLIONI, C. 0976a). Inhibition of initiation of protein synthesis by 7-methylguanosine-5'-monophosphate. Proc. natn Acad. Sci. U.S.A. 73, 1923.
HICKEY, E. D., WEBER, L. A. & BAGLIONI, C. (1976/?). Translation of RNA from unfertilized
sea urchin eggs does not require methylation and is inhibited by 7-methylguanosine-5'phosphate. Nature, Loud. 261, 71-73.
JOHNSON, M. H. (1979). Intrinsic and extrinsic factors in preimplantation development.
J. Reprod. Fert. 55, 255-265.
KEITH, J. & FRAENKEL-CONRAT, H. (1975). Tobacco mosaic virus RNA carries 5'-terminal
triphosphorylated guanosine blocked by 5'-linked 7-methyl-guanosine. FEBS Lett. 57,
31-33.
KEMPER, B. & STOLARSKY, L. (1977). Dependence on potassium concentration of the translation of messenger ribonucleic acid by 7-methylguanosine-5'-phosphate. Biochemistry 16,
5676-5680.
KLOOTWIJK, J., KLEIN, I., ZABEL, P. & VAN KAMMEN, A. (1977). Cowpea mosaic virus RNAs
have neither m 7 GppN... nor mono-, di-, or triphosphates at their 5'-ends. Cell 11,
73-82.
KOZAK, M. (1978). How do eucaryotic ribosomes select initiation regions in messenger RNA?
Celin, 1109-1123.
3
14
LASKEY, R. A. & MILLS, A. D. (1975). Quantitative film detection of H and C in polyacrylamide gels by fluorography. Eur. J. Biochem. 56, 335-341.
LEVEY, I. L., STULL, G. B. & BRINSTER, R. L. (1978). Poly(A) and synthesis of polyadenylated
RNA in the preimplantation mouse embryo. Devi Biol. 64, 140-148.
LOCKARD, R. E. & LANE, C. (1978). Requirement for 7-methyl-guanosine in translation of
globin mRNA in vivo. Nucleic Acid Res. 5, 3237-3248.
LODISH, H. F. & ROSE, J. K. (1977). Relative importance of 7-methylguanosine in ribosome
binding and translation of vesicular stomatitis virus mRNA in wheat-germ and reticulocyte
cell-free systems. /. biol. Chem. 252, 1181-1188.
MUTHUKRISHNAN, S., FURUCHi, Y., BOTH, G. W. & SHATKIN, A. J. (1975). Effect of 5'terminal structures on the binding of ribopolymers to eukaryotic ribosomes. Frog. Nucl.
Acid Res. Mol. Biol. 19, 473-476.
OLDS, P. J., STERN, S. & BIGGERS, J. D. (1973). Chemical estimates of the RNA and DNA
contents of the early mouse embryo. /. exp. Zool. 186, 39-46.
PEACOCK, A. C. & DINGMAN, C. W. (1967). Resolution of multiple ribonucleic acid species
by polyacrylamide gel electrophoresis. Biochemistry 6, 1818-1827.
PELHAM, H. R. B. & JACKSON, R. J. (1976). An efficient mRNA-dependent translation system
from reticulocyte lysates. Eur. J. Biochem. 67, 247-256.
REVEL, M. & GRONER, Y. (1978). Post-transcriptional and translational controls of gene
expression in eukaryotes. Ann. Rev. Biochem. 47, 1079-1126.
SANGER, F. & COULSON, A. R. (1975). A rapid method for determining sequences in DNA
by primed synthesis with DNA polymerase. / . molec. Biol. 94, 441-448.
SHATKIN, A. J. (1976). Capping of eukaryotic mRNAs. Cell 9, 645-653.
VAN BLERKOM, J. (1977). Molecular approaches to the study of oocyte maturation and embryonic development. In Lmmunobiology of Gametes (ed. M. Edidin & M. H. Johnson),
pp. 184-206. Cambridge University Press.
WEBER, L. A., FENMAN, E. R., HrocEY, E. D., WILLIAMS, M. C. & BAGLIONI, C. (1976).
Inhibition of HeLa cell messenger RNA translation by 7-methylguanosine-5'-monophosphate. /. biol. Chem. 251, 5657-5662.
GEDAMU,
156
G. A. S C H U L T Z , J. R. C L O U G H AND M. H. JOHNSON
L. A., HICKEY, E. D. & BAGLIONI, C. (1978). Influence of potassium salt concentration and temperature on inhibition of mRNA translation by 7-methylguanosine-5'monophosphate. J. biol. Chem. 253, 178-183.
WHITTINGHAM, D. G. & WALES, R. G. (1969). Storage of two-cell mouse embryos in vitro.
Aust. J. biol. Sci. 22, 1065-1068.
YOUNG, R. J. (1977). Appearance of 7-methylguanosine-5'-monophosphate in the RNA of
mouse 1-cell embryos three hours after fertilization. Biochem. Biophys. Res. Commun.
76, 32-39.
WEBER,
ZAN-KOWALCZEWSKA, M., BRETNER, M., SIERAKOWSKA, H., SZCZESNA, E., FILIPOWICZ, W.
& SHATKIN, A. J. (1977). Removal of 5'-terminal m7G from eukaryotic mRNAs by potato
nucleotide pyrophosphatase and its effect on translation. Nucleic Acid Res. 4, 3065-3081.
{Received 14 August 1979, revised 2 November 1979)