Volume 13 Number 22 1985
Nucleic Acids Research
An analysis of the rate of metallothionein mRNA poly(A)-shortening using RNA blot
hybridization
Julian F.B.Mercer* and Samantha A.Wake
Births Defects Research Institute, Royal Children's Hospital, Flemington Road, Parkville,
Victoria 3052, Australia
Received 9 October 1985; Accepted 31 October 1985
ABSTRACT
A p r o g r e s s i v e r e d u c t i o n i n t h e s i z e of r a t m e t a l l o t h i o n e i n - 1 mRNA
following i n d u c t i o n by copper c h l o r i d e or dexamethasone was demonstrated on
RNA b l o t s , and was shown to be due to shortening of the p o l y ( A ) - t a i l . The
r a t e of poly(A) removal was t h e same i n r a t l i v e r and k i d n e y f o l l o w i n g
copper c h l o r i d e i n d u c t i o n , in r a t l i v e r following dexamethasone i n d u c t i o n ,
and i n mouse l i v e r f o l l o w i n g c o p p e r c h l o r i d e i n d u c t i o n .
I n mouse l i v e r
met a l l o t h i o n e i n - 1 a n d 2 mRNAs were s h o r t e n e d a t t h e same r a t e .
The
r e d u c t i o n of t h e p o l y ( A ) t a i l was more r a p i d i n t h e f i r s t 5 h o u r s
( a p p r o x i m a t e l y 20 n u c l e o t i d e s / h ) b u t much s l o w e r ( a p p r o x i m a t e l y 3
nucleotides/h) after the poly(A)-tail had been reduced to about 60
residues. Metallothionein mRNA molecules with poly(A) tail sizes less than
30-40 nucleotide* were not observed. Exonuclease digestion of the poly(A)t a i l is suggested, at least in the initial rapid phase. It is hypothesired
that poly(A)-tails longer than 30 are required for mRNA s t a b i l i t y and that
much longer poly(A) t a i l s may give newly synthesized mRNA molecules a
competitive advantage in protein synthesis.
INTRODUCTION
Host e u k a r y o t i c mRNA s p e c i e s have a sequence of a d e n y l i c a c i d
residues
adde4 to t h e i r 3 ' - e n d s f o l l o w i n g t r a n s c r i p t i o n (for reviews, see r e f s . 1
and 2). Despite extensive i n v e s t i g a t i o n the reason for the e x i s t e n c e of
the poly(A) t a i l i s s t i l l unclear. Newly synthesized mRNA molecules have
poly(A) t a i l s of about 200 n u c l e o t i d e s which a r e shortened r a p i d l y to
about 40-60 n u c l e o t i d e s ( 3 ) . This observation led to the suggestion that
the length of p o l y ( A ) may be r e g u l a t i n g mRNA s t a b i l i t y ( 3 , 4 ) and
a r t i f i c i a l l y d e a d e n y l a t e d mRNA i s l e s s stable when injected into Xenopus
oocytes ( 5 , 6 ) . A t a i l of 30 adenylate residues was found to be sufficient
to s t a b i l i z e globin mRNA ( 7 ) . However, newly s y n t h e s i z e d mRNA m o l e c u l e s ,
with long poly (A) t a i l s were shown to be equally likely to be degraded as
older mRNAs with short poly(A) t a i l s ( 8 ) .
Some evidence s u g g e s t s t h a t poly(A) may have a direct role in protein
s y n t h e s i s . For example, Doel and Carey ( 9 ) showed t h a t d e a d e n y l a t e d
chicken ovalbumin mRNA was t r a n s l a t e d poorly in rabbit r e t i c u l o c y t e lysates
© IRL Pros* Limited, Oxford, England.
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compared with the fully
adenylated molecule.
In addition, Jacobson and
Favreau (10) found that synthetic poly(A) had a specific inhibitory
effect
on translation of poly(A)+ mRHAs in reticulocyte lysates.
To examine hypotheses of poly(A) function,
s i r e analysis preferably
suitable methods of poly(A)
in specific mRNA species are required.
Early
procedures for analysis of poly(A) sire depended upon i t s resistance to
d i g e s t i o n by ribonucleases (11).
Messenger RNA molecules containing
different poly(A)-tail lengths have been separated using thermal
elution
from poly(A)-binding materials such as oligo (dT)-cellulose (12), but often
this analysis has proved unsuitable for mRNA species with poly(A) below
about 20 residues since these bind poorly (12).
cloned probes bound to s o l i d
More recently the use of
supports has f a c i l i t a t e d
poly(A)-sizes in some individual mRNAs (13).
analysis
of
Despite the large number of
studies, most conclusions about poly(A)-function have been based upon
analysis of globin mRNA or of total mRNA in cultured cells.
poly(A)-shortening rates in mRNAs in different
No analysis of
organs of an animal have
been presented.
In t h i s
paper we use RNA blots to analyze the shortening of
the
poly(A)-tails on metallothionein mRNA in rat liver and kidney following the
induction by copper chloride or dexamethasone.
MATERIALS AND METHODS
Materials
Oligo(dT)
Biochemicals.
(12-18) and RNAase H were purchased from Pharmacia-PL
a - 3 2p-deoxycytidine triphosphate
supplied by Amersham.
(3000 Ci/mmole) was
Dexamethasone phosphate was obtained from David Bull
Laboratories, Melbourne, Victoria, Australia.
RHA Isolation
Total
RHA was i s o l a t e d from the l i v e r s or kidneys of adult male
Sprague-Dawley r a t s (200g) or C57 black mice at various times
treatment
following
with CuCl2 (3mg CuCl2/kg in 0.9Z NaCl, i n t r a p e r i t o n e a l )
dexamethasone (4 mg/kg intraperitoneal).
or
The RNA i s o l a t i o n procedure has
been described previously (14).
RNA Blots
Gel electrophoresis of RNA was performed as described by Dobner et a l .
(15).
Total RNA (5 ug per lane) was heated for 5-10 min at 65°C in a mix
containing 61 Wv formaldehyde, 50Z formamide, 0.02Z bromophenol blue and
20 mM MOPS, 1 mH EDTA, 5 mM sodium a c e t a t e pH 7 . 0 .
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performed
using
2.2Z W v
horizontal
formaldehyde, 20 mM MOPS, 1 mM EDTA, 5 mM sodium a c e t a t e pH 7 . 0 ,
hours a t 30-40 mA c o n s t a n t c u r r e n t .
w
agarose g e l s c o n t a i n i n g 6Z
for
/v
16
The electrophoresis buffer was 20 mM
MOPS, 1 mM EDTA, 5 mM NaAc pH 7 . 0 .
The t o t a l RNA was v i s u a l i z e d
using
ethidium bromide to ensure i n t e g r i t y ,
then transferred to n i t r o c e l l u l o s e
e s s e n t i a l l y as described by Thomas (16).
The f i l t e r s
80°C in vacuo,
then p r e h y b r i d i z e d
were baked 2 h r .
SSCE (0.75 sodium chloride, 0.075M sodium c i t r a t e , 25 mM EDTA pH 8 . 0 ) ,
Denhardts
reagent
(0.02Z
at
16 hours at 25°C in 50Z formamide, 5 x
bovine
serum albumin,
0.02Z F i c o l l ,
polyvinylpyrrdidone) and 250 ug/ml h e r r i n g sperm DNA.
1 x
0.02Z
The f i l t e r s
were
then hybridized 24 hours a t 25°C in the above solution which also contained
100 ug/ml of both poly(O) and poly(C) together with approx. 100 ng of mouse
metallothionein-1
cDNA (17) approx. 400 bp or a mouse metallothionein-2
to a specific a c t i v i t y of >10 8
probe approx. 200 bp (18) n i c k - t r a n s l a t e d
cpm/ug.
min
After hybridization, f i l t e r s were washed in 2 x SSC, 0.1Z SDS (30
room
temp.)
then
in
0.5
x SSC,
0.1Z
SDS ( 6 0
under
55°C).
Cross-hybridization
between m e t a l l o t h i o n e i n - 1
c o n d i t i o n s was <5Z.
Autoradiography was carried out using Kodak X-AR5 film
together with Dupont intensifying
and - 2
min,
these
screens.
RHAase H analysis of t o t a l RMA samples
T o t a l RNA i s o l a t e d from the l i v e r s of copper-injected r a t s (5 and 8 h
p o s t - i n j e c t i o n ) was used for the i n i t i a l study.
similar
to t h a t d e s c r i b e d by Sippel
made 1 mM in EDTA, and heated a t
ice,
oligo(dT)
( 0 . 3 ug) added,
et a l .
The RNAase H treatment was
(19).
Total RNA (30 ug) was
100°C for 5 min.
Tubes were c h i l l e d on
and allowed
to hybridize 10 min a t 25°C.
Reactions were then made 50 mM in KC1, and hybridization was c o n t i n u e d
a further
KC1,
10 min a t 25°C.
for
The f i n a l r e a c t i o n mix (40 ul) contained 25 mM
2 8 mM Mg C l 2 , 20 oM Tris pH 8.0, 0.5 mM EDTA, 0.3 ug oligo(dT) and 30
ug t o t a l tRNA.
An aliquot was removed a t t = 0 min before
RNAase H (0.5U,
min a t 37°C).
oligo(dT).
1 U catalyses
the a d d i t i o n of
t h e production of 1 nmole nucleotide in 20
Reactions were incubated at 37OC.
Control reactions lacked
Aliquots of r e a c t i o n were removed at 7.5 min and 15 min from
experimental
tubes and a t 15 min from control tubes.
These a l i q u o t s were
e x t r a c t e d with phenol/CHCl3 (1:1) buffered to pH 8.0 with 10 mM T r i s , 1 mM
EDTA.
The t o t a l RNA was then p r e c i p i t a t e d ,
washed two times w i t h 70Z
e t h a n o l , dried and dissolved in H2O. Sample preparation for
was as d e s c r i b e d above.
Transfer,
prehybridization
and
electrophoresis
hybridization
conditions were also as described above.
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12
15
20
TIME AFTER Cu (hr)
F i g u r e 1,
S i r e changes in metal l o t h i o n e i n - 1 mRNA in rat l i v e r following
copper chloride administration.
RNA was i s o l a t e d from rat l i v e r at various
times following i n j e c t i o n (IP) of copper c h l o r i d e .
Total RNA (5 ug) was
e l e c t r o p h o r e s e d on 2 . 2 1 agarose g e l s c o n t a i n i n g 62 formaldehyde ( 1 5 ) .
After transfer to n i t r o c e l l u l o s e ( 1 6 ) t h e f i l t e r was probed w i t h a n i c k
t r a n s l a t e d mouse metallothionein-1 cDNA ( 1 7 ) . After washing to remove the
n o n - h y b r i d i z e d p r o b e , the m e t a l l o t h i o n e i n - 1 mRNA was d e t e c t e d by
autoradiography. The s i z e markers are pBR322 digested with Taq 1.
RESULTS
The s i z e change of m e t a l l o t h i o n e i n nRNA i n r a t l i v e r f o l l o w i n g
copper
chloride i n j e c t i o n i s due to polv(A) removal
We have previously reported t h a t m e t a l l o t h i o n e i n mRNA i n r a t
rapidly
increases
(16 f o l d i n 7 h o u r s )
thereafter the mRNA concentration i n the l i v e r d e c r e a s e s w i t h a
of 3 to 4 hours ( 1 4 ) .
following
When t o t a l
half-life
l i v e r RNA i s o l a t e d at d i f f e r e n t
times
CuCl2 i n j e c t i o n was a n a l y z e d by RNA blot hybridization using a
mouse metallothionein-1 cDNA probe,
the RNA was seen to decrease r a p i d l y
s i z e from about 600 n u c l e o t i d e s when f i r s t
about 440 n u c l e o t i d e s
tines
liver
f o l l o w i n g an i n j e c t i o n of CuCl2;
(e.g.
nucleotides.
12 to 15 hours a f t e r
5 or 8 hours) mRNA s i z e s
in
d e t e c t e d ( F i g . 1 1 hour) to
injection.
At
intermediate
ranged between about 600 and 470
At 20 hours a small amount of mRNA, larger than that
present
a t 15 h o u r s , was d e t e c t e d which probably resulted from a s t r e s s induction
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5
8
Omin(-)
5
8
5
8
5 8
Omin(+) 7.5min(+)
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15min(-)
Figure 2, RNAase H treatment of metallothionein mRNA. Total RHA isolated
5 or 8 hours following copper administration was incubated for 0, 7.5 or 15
min. with ribonuclease H in the presence (+) or absence (-) of oligo(dT) as
described in the text. The RHA was then analysed by the RNA blot procedure
as described for Figure 1. Only the relevant portion of the blot is shown.
rather than the CuCl2 injection.
Shortening of the poly(A) tail (23) was the most likely explanation of
these changes.
and oligo(dT).
To test this we incubated RNA in the presence of RNAase H
RNAase H has the property of degrading the RNA strand of a
DNA/RNA hybrid, and has been used to remove the poly(A)-tail from mRNA
(12). Incubation of RNA isolated 5 or 8 hours after CuCl2 injection with
RNAase H and oligo(dT) for 7.5 min. reduced both RNA samples to the same
size (about 400 nucleotides, Fig. 2) indicating that both the heterogeneity
and sire differences in the two samples was due to differing poly(A) tail
lengths. Incubation for 15 min. in the absence of oligo(dT) did not cause
any size alteration, showing that the size reduction was indeed due to
RNAase U and not a contaminating nuclease.
Significantly, the RNAase B
treated sample (presumably completely deadenylated) was smaller than the
smallest size (440 nucleotides) seen in 15 hours following CuCl2 injection
(Fig. 1).
Poly(A) removal from kidney metallothionein RNA following copper induction
The simplicity of this method of analysis suggested that we could
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2
-
5
5
8
12
15
20
TIME AFTER Cu (hr)
Figure 3.
Site changes in metallothionein-1 mRNA in rat kidney following
copper chloride administration. Total RNA was isolated from rat kidneys at
various times following injection (IP) of copper chloride and analyzed by
RNA blot hybridization as described in Figure 1.
study the rate of poly(A) removal and mBNA stability for a defined mRNA in
different
tissues and with different inducers.
Figure 3 shows an RNA blot
analysis of metallothionein-1 RNA from the kidneys of rats following copper
chloride i n j e c t i o n .
The metallothionein-1 mRNA was found to shorten at a
similar r a t e to that in the l i v e r .
The rapid decrease in mRNA levels
between 5 and 8 hours suggested that the b a l f - l i f e of metallothionein-1
mRNA in the kidney may be shorter than in the l i v e r ,
more animals failed
however, analysis of
to demonstrate a consistent difference between mRNA
half-lives in the two tissues (results not shown).
Poly(A) removal from liver metallothionein RNA following dexamethasone
treatment
The RNA blot shown in Fig. 4. shovs that a similar shortening of
poly(A) occurs with r a t liver metallothionein-1 mRNA induced following
dexamethasone i n j e c t i o n .
I t is of interest that the 4 hour time point
shows a fairly discrete band of reduced size, indicating that the poly(A)
shortening is due to an exonuclease rather than a random endonuclease
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4
0
6
8
10 Cu(5h)
i
TIME AFTER DEX (h)
Figure 4.
Size changes in hepatic m e t a l l o t h i o n e i n - 1 mRNA following
dezamethasone administration. RNA was isolated from the livers of rats at
various times following treatment with dexamethasone.
Metallothionein-1
mRNA was detected by RNA blot hybridization as described in Figure 1. Cu
(5h) refers to a liver RNA sample 5 hours a f t e r i n j e c t i o n of a r a t with
CuCl2.
cleavage which would have generated a wider range of molecular s i z e s .
6 and
8 hour
samples
show t h a t
molecules) are r e a p p e a r i n g ,
probably
dexamethasone from the i n j e c t i o n
mRNA has largely disappeared.
hours a f t e r
injection,
some new t r a n s c r i p t s
site.
The
(long poly(A)
due to c o n t i n u i n g
release
of
By 10 hours the metallothionein
Lane 8 shows a copper induced RNA from 5
illustrating
that the size range was identical to
the hormonal ly induced sample, but that the degree of induction achieved by
copper was much greater than with dexamethasone at the doses used.
Comparison of the rate of shortening of poly(A) in metallothionein mRMA in
liver and kidney with different
inducers
To more d i r e c t l y analyze the r a t e of shortening of poly(A) under
different physiological conditions, results from a number of separate RNA
b l o t s were combined for the data shown in Fig. 5.
We plotted the smallest
size of mRNA present at any given time because average or maximum mRNA size
would be influenced by the appearance of new mRHAs over an extended period,
particularly
in the liver following CuCl2 injection (where new transcripts
appeared for up to 5 hours), or when absorption was delayed. The curves for
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Dexamethasone liver
Copper kidney
Copper liver
600
200
<n
a>
3
•5
•D
o
O
3
O
•3
C
X
1—
500
100
o
LU
rr
E
RNAaseH treated
400
2
4
6
8
10
12
2
14
TIME AFTER INDUCTION (hours)
Figure 5 .
C o m p a r i s o n of t h e r a t e s of p o l y ( A ) s h o r t e n i n g i n
metal l o t h i o n e i n - 1 mRNA following CuCl2 or dezamethasone injection. Data
from 3 RNA blots were combined to produce the l i v e r r e s u l t s and one b l o t
for the kidney. The smallest mRNA detected at any one time was plotted
against the time following treatment. The points are shown mean _+ 2 SEH.
The s i z e of mRNA deadenylated with RNAase H is shown on the right. Liver
RNA from dexamethasone treated animals •
• ; liver RHA from CuC12 treated
rats *
*
; kidney RHA from CuCl2 treated rats •
•.
l i v e r mRNA a f t e r dexamethasone and copper are an average s i z e _+ 2SE,
estimated from 3 separate RNA b l o t s . The kidney data shows results of a
single b l o t . There i s no s i g n i f i c a n t d i f f e r e n c e between the r a t e s of
s h o r t e n i n g of p o l y ( A ) in the l i v e r and kidney or between copper and
dexamethasone as inducers. Comparison of the dexamethasone and copper
induced mRNA on one blot showed identity of s i z e s , (Fig. 4 and unpublished
data). It i s apparent that the poly(A) shortening was more rapid in the
f i r s t 6 hours following s y n t h e s i s (approx. 20 nucleotides per hour), at
which point the poly(A) t a i l length i s approximately 60 r e s i d u e s . Over the
f o l l o w i n g 8 hours the mRNA decreased in s i z e more slowly (approx. 3
nucleotides/hour). No RNA molecules were observed that had a poly(A) t a i l
smaller than 30-40 nucleotides.
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B
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2.5
5
10
20
30
TIME AFTER Cu (hr)
Figure 6. A comparison of the metallothionein-1 and -2 mRNA sire changes
in mouse l i v e r following CuCl2 administration. RNA blots of total liver
RNA from mice at various times a f t e r i n j e c t i o n were probed with e i t h e r
metallothionein-1 (A) or metallothionein-2 (B) cDNAs.
A comparison
of m e t a l l o t h i o n e i n - 1 and -2 mRNA s i z e s in mouse l i v e r s
following copper chloride injection
In the mouse there are two metallotbionein genes which are apparently
induced co-ordinately (18).
Since the molecules d i f f e r
significantly
in
t h e i r nucleotide sequences i t seemed possible that differences in poly(A)
removal rate or degradation of the mRNA might be detectable.
Fig. 6A shows
an RNA blot analysis of mouse liver RNA following copper chloride injection
using a m e t a l l o t h i o n e i n - 1 probe.
I t i s immediately apparent
that
the
p a t t e r n of poly(A) shortening was not as consistent as that shown for the
rat (Fig. 1). This wa6 a consequence of the higher level of mRNA found
some u n t r e a t e d
animals
(e.g.
Fig.
6 zero
in
t i m e ) . In t h i s sample an
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intermediate aired mRHA was p r e s e n t . The 1 hour sample showed t h e f u l l
range of s i z e s , presumably t h e r e s u l t of induction of new mRNA ( l a r g e ) ,
superimposed upon o l d e r mRNA ( s m a l l e r ) . The 2.5 hour sample probably
c o n s i s t e d of a l l new mRNA and t h e 5 hour sample showed evidence of
shortening of the induced RNA. We generally observe much lower endogenous
m e t a l l o t h i o n e i n mRNA levels in the r a t compared with the mouse. Durnam and
Palmiter (21) r e p o r t e d t h a t mice have q u i t e v a r i a b l e h e p a t i c
m e t a l l o t h i o n e i n mRHA levels and t h i s was presumed to be due to s t r e s s . The
d i s c r e t e sited mRHA in lane 1 suggests that the induction i n t h e s e animals
i s not a continuous event but occurs in discrete pulses, since continuous
induction would have y i e l d e d mRNAs c o n t a i n i n g a wider range of poly(A)
lengths.
F i g . 6B s h o w s t h e same RHA b l o t r e p r o b e d w i t h a m o u s e
m e t a l l o t h i o n e i n - 2 cDNA, under c o n d i t i o n s t h a t p r e v e n t s i g n i f i c a n t
cross-hybridization.
C l e a r l y the metallothionein-2 mRHA has a v i r t u a l l y
i d e n t i c a l s i z e d i s t r i b u t i o n to m e t a l l o t h i o n e i n - 1 mRNA in a l l t r a c k s ,
s u g g e s t i n g t h a t the r a t e of poly(A)-removal i s most probably the same for
each.
DISCUSSION
This study d e m o n s t r a t e s t h e u s e f u l n e s s of an RHA blot analysis for
studying the metabolism of poly(A) in a m e t a l l o t h i o n e i n mRNA. The method
i s r a p i d and has t h e advantage of demonstrating poly(A) size by detection
of the mRNA to which i t i s attached rather than by l a b e l l i n g of the poly(A)
moiety i t s e l f .
In t h e l a t t e r method one must a d j u s t t h e measured
d i s t r i b u t i o n , s i n c e the c o n t r i b u t i o n of r a d i o a c t i v i t y i a i n d i r e c t
p r o p o r t i o n to t h e l e n g t h of the molecule and a f a i l u r e to appreciate t h i s
has l e d t o an i n c o r r e c t c o n c l u s i o n t h a t the synthesis i n h i b i t o r emeteine
did not a f f e c t t h e r a t e of poly(A) s h o r t e n i n g ( 2 1 ) . The a n a l y s i s i s
a p p l i c a b l e to metallothionein mRHA since i t i s small enough for changes in
poly(A) length to have an appreciable effect upon gel migration. A s i m i l a r
use of RNA b l o t s and RNAaee H was r e p o r t e d by Rosenthal et a l . (22) in
their study of a l t e r a t i o n in patterns of p o l y a d e n y l a t i o n following
f e r t i l i z a t i o n of clam eggs. Some changes in size of molecules as large as
6,000 nucleotides were detected, but detailed analysis was not possible,
since the change in mobility caused by loss of the poly(A) was very small.
To extend the method to larger mRNA molecules, these could be hybridized to
a synthetic oligonucleotide complementary to a region about 200 nucleotides
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5 1 - from the poly(A) addition s i t e . Digestion with RNAase-H p r i o r to the
gel e l e c t r o p h o r e s i s would s p l i t the mRNA in two, with the poly(A) on a
piece long enough to allow s p e c i f i c probe d e t e c t i o n but s h o r t enough to
permit detailed analysis of poly(A) s i r e changes.
Since poly(A) molecules have been shown to have an anomalous m i g r a t i o n
r e l a t i v e t o r i b o s o m a l RNA markers in some gel systems ( 2 3 ) , we were
concerned t h a t our mRNA s i z e e s t i m a t e s may be in e r r o r .
The g e n e r a l
agreement of our poly(A) length estimates, i . e . newly synthesized molecules
with p o l y ( A ) - t a i l s of 150-200 n u c l e o t i d e s t h a t s h o r t e n to about 30-40
r e s i d u e s , with o t h e r l i t e r a t u r e r e p o r t s ( 1 , 2 , 2 1 ) , convinced us that the
denaturing gel system we used gave reasonably accurate s i r e estimates.
We analyred the changes in poly(A) s i r e in metallothionein mRNA in the
l i v e r and kidney of r a t s and mice f o l l o w i n g i n d u c t i o n by c o p p e r or
d e x a m e t h a s o n e , u s i n g a m e t a l 1 o t h i o n e i n - 1 p r o b e w i t h t h e r a t and
metallothionein -1 and -2 with the mouse RNA samples. Metallothionein mRNA
which f i r s t appeared following induction contained a long poly(A)-tail of
about 150-220 nucleotides, similar in size to most newly s y n t h e s i z e d mRNA
molecules ( 3 ) . The poly(A) underwent a rapid shortening, such that by 5
hours after induction some of the poly(A) molecules reached a l e n g t h of
about 60 n u c l e o t i d e s . The s h o r t e n i n g a f t e r t h i s time seemed to be much
slower. The pattern of s i r e reduction was not r e a l l y c o n s i s t e n t with the
random e n d o n u c l e a s e c l e a v a g e favoured by Sheiness e t a l . ( 2 1 ) .
In
contrast, some samples demonstrate that the poly(A) becomes p r o g r e s s i v e l y
s h o r t e r following an i n d u c t i o n event (e.g. Fig. 4A, 4 hr. sample) and we
conclude th»t the pattern of s h o r t e n i n g i s s u g g e s t i v e of an e x o n u c l e a s e
removal of poly(A) from m e t a l l o t h i o n e i n mRNA at l e a s t during the i n i t i a l
rapid shortening period. The data of Sheiness et a l . (21) was, however,
c o n s i s t e n t w i t h an e x o n u c l e a s e a c t i o n which became slower as the
p o l y ( A ) - t a i l s shortened and t h i s i s e x a c t l y t h e p a t t e r n we f i n d i n
metallothionein mRNA (Fig. 5 ) .
The f a c t t h a t the newly s y n t h e s i z e d molecules are longer and become
progressively shorter with time allows an assessment of the r e c e n t h i s t o r y
of i n d u c t i o n i n t h e a n i m a l . This was i l l u s t r a t e d in Fig. 6, in which two
sire classes of metallothionein mRNA were superimposed in the mouse RNAs
examined. From the s i z e s of the mRNA molecules, we could deduce that two
different induction events were involved, one being u n c o n t r o l l e d , p o s s i b l y
due to s t r e s s and r e s t r i c t e d in time since the mRNA d e r i v i n g from t h i s
appeared as a r e l a t i v e l y d i s c r e t e band, and the o t h e r i n d u c t i o n being due
7939
Nucleic Acids Research
to the copper chloride injection.
Without an appreciation of the nature of
the size distribution, r e s u l t s as shown in Fig. 6 would be d i f f i c u l t
interpret.
We h a v e
found
this
analysis
useful
for
to
analyzing
metallothionein mRNA in neonatal rats and in tissue culture ( J . F . B . Mercer
and T. Stevenson, unpublished data).
We found no evidence for differential
two tissues, or with the two inducers.
rates of poly(A)-removal in the
The r a t e of poly(A)-removal
also
appeared to be the same with both metallothionein-1 and -2 mRNAs.
This
suggests that poly(A) removal rates may well be constant for many messages,
however,
mRNAs.
t h i s w i l l need to be confirmed by further analysis of a range of
Under the conditions examined,
relatively
the mRNA h a l f - l i v e s
also seemed
constant, and both metallothionein-1 and metallothionein-2 mRNA
species also appeared coordinately regulated and degraded (Fig. 4 ) .
It
would be informative to examine the kinetics of poly(A) removal using mRNAa
vith d i f f e r e n t
half-lives
to further
explore the r e l a t i o n s h i p
between
poly(A) lengths and mRMA s t a b i l i t y .
Others have reported t h a t the i n i t i a l phase of poly(A)-shortening is
more rapid, and also a steady state population of apparently more s t a b l e
mRNA speciea with poly(A)-tails of 40-65 nucleotides has been found (3,4).
Our data and these r e s u l t s are c o n s i s t e n t v i t h a model that
restriction
places a
on t h e r a t e of poly(A)-decay below a length of about 60
nucleotides, possibly due to a p r o t e i n t i g h t l y
binding to t h i s
region,
perhaps one t h a t overlaps the 3* untranslated region as has been proposed
by L i t t a u e r and Soreq ( 1 ) .
sections
The p r o t e i n s a s s o c i a t e d
of poly(A) may be leas e f f e c t i v e
against nuclease attack.
with poly(A) t a i l s
with
the
longer
in p r o t e c t i n g the molecule
The absence of m e t a l l o t h i o n e i n mRNA molecules
shorter than about 30 r e s i d u e s i s in agreement with
s t u d i e s with other tnRNAs (24,25) and i t is likely that the molecules become
metabolically l a b i l e as soon as these residues are removed.
Nudel et a l . (26) reported that the s t a b i l i t y of globin mRNA molecules
with a poly(A)-tail length of only 34 residues was i n d i s t i n g u i s h a b l e
t h o s e molecules with longer p o l y ( A ) - t a i l s vhen i n j e c t e d
oocytes.
This fact,
from
i n t o Xenopus
taken together with the observed equal probability of
decay of nev or old mRHAs (with long or short p o l y ( A ) - t a i l s r e s p e c t i v e l y )
(8),
suggests t h a t above a length of 30 nucleotides the poly(A)-tail does
not have a role in s t a b i l i z i n g mRNA.
Why then should mRNA molecules be
synthesized with such long poly(A) t a i l a ?
One possible explanation is that
the longer poly(A) t r a c t s confer a competitive a d v a n t a g e
7940
in
protein
Nucleic Acids Research
synthesis,
for example by increasing the r a t e of i n i t i a t i o n of protein
synthesis.
A role for poly(A) in protein synthesis has been suggested by
Jacobson and Favreau (10) who found that translation of poly(A)+ mfiNAs in
rabbit reticulocyte lysates could be selectively
i n h i b i t e d by addition of
free poly(A).
a 2u globulin mRNA species
Other studies have shown that
with long poly(A)-tails more rapidly reached maximum translation rates
poly(A)-poor mRNA when injected into Xenopus oocvtes (27);
than
and Geoghegen
et a l . (28) found that actin mRNA molecules with short poly(A)-tails tended
to be concentrated
suggest
the
rate
in the smaller polysomes.
of
initiation
poly(A)-deficient mRNA molecules.
al.
of
Both of these
findings
t r a n s l a t i o n was slower
In contrast, an earlier study by Bard et
(29) had concluded that species of mRNA with different
poly(A)-tail
lengths could r e i n i t i a t e with equal frequencies in mouse L-cells after
shock.
with
heat
Their data did show, however, a 14Z increase in newly synthesized
mRHAs in the polysomes following recovery from heat shock, but t h i s was
dismissed
as a l a b e l l i n g
artefact.
I t could be, however,
i n i t i a t i o n rate enhancement by poly(A) d i f f e r s
that
the
between mRNA species and
a n a l y s i s of bulk mRHA could obscure this effect.
complete a n a l y s i s of t h i s question using s p e c i f i c
This argues for a more
probes and a simple
analysis system such as we have described would be useful for this.
One can envisage that i t could be advantageous for a c e l l to have a
mechanism for
metallothionein.
enhancement of
the t r a n s l a t i o n of inducible mRNAs such as
Such mRHAs are produced rapidly in response to inducers
such as heavy metals and hormones, but enter a cytoplasm already full of
active mRHAs most of which will have shorter poly ( A ) - t a i l s.
A mechanism
for promoting the t r a n s l a t i o n of the newly synthesized mRHAs would a s s i s t
in achieving a more effective response to an inducing signal.
fairly
In addition,
rapid removal of the p o l y ( A ) - t a i l would reduce t h i s
ensuring the mRNA did not out-compete the longer
extended
period.
Of c o u r s e ,
other
lived
advantage,
mRNAs for
poly(A)-independent
an
mechanisms
controlling the half-life of the mRNAs are presumably involved, for example
sequences in the 3 ' - u n t r a n s l a t e d region (30).
The apparent link between
protein synthesis and poly(A)-shortening (21) i s not i n c o n s i s t e n t with a
r o l e of poly(A) in protein synthesis.
If the removal of the poly(A) during
the rapid phase (to 60 residues) were linked to protein s y n t h e s i s ,
then
t h i s would control the number of times that an mRHA could be translated and'
s t i l l maintain a competitive advantage.
This is a variant of the t i c k e t i n g
hypothesis of Sussmann (31), but we suggest i t is notraRHAstability
that
7941
Nucleic Acids Research
i s being r e g u l a t e d by the removal of
poly(A)
but
its
efficiency
of
translation.
In
conclusion,
our d a t a s u g g e s t s t h a t
the p o l y ( A ) - s h o r t e n i n g of
metallothionein mRHA occurs by an exonuclease a c t i v i t y , and is c o n s i d e r a b l y
slower
once
the
po ly ( A ) - t a i 1 s have s h o r t e n e d below 60 n u c l e o t i d e s ,
metallothionein mRNA molecules with poly(A)-lengths of l e s s than 30 a r e
found.
These r e s u l t s and e v i d e n c e in
that poly(A) may have two r o l e s :
(i)
s y n t h e s i z e d mRNA molecules and ( i i )
which
is
needed
to
maintain
not
the l i t e r a t u r e lead us to suggest
to f a c i l i t a t e
t r a n s l a t i o n of
newly
a c r i t i c a l length of about 30 residues
mRNA s t a b i l i t y .
An a n a l y s i s
of
p o l y ( A ) - s h o r t e n i n g and s i r e d i s t r i b u t i o n i n polysomes from a number of
d i f f e r e n t mRNAs could now be examined u s i n g a modified b l o t p r o c e d u r e
to
t e s t these hypotheses.
ACKNOWLEDGEMENTS
We a r e g r a t e f u l
t o Professor David Danks for h i s advice,
and c r i t i c i s m of the manuscript.
encouragement
This work was supported in p a r t by g r a n t s
from the National Health and Medical Research Council of A u s t r a l i a .
*To whom correspondence should be addressed
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