Download Expression of two aldolase A mRNA species in different human and

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BIOCHEMICAL SOCIETY TRANSACTIONS
Hunter, I. & Skerrow, D. (1982) Biochim. Biophys. Acia 714, 164169
Matoltsy, A. G. (1964) Nature (London) 201, 113&1131
McGuire, J., Osber, M. & Lightfoot, L. (1984) Br. J . Dermaiol. 111
(Suppl. 27), 27-37
Expression of two aldolase A mRNA species in different human and rat tissues
F. SCHWEIGHOFFER, F. MENNECIER, P. MAIRE,
L. BACHNER, and A. KAHN
Inserm U 129, 24 rue du Fg St Jacques, 75674 Paris
Cedex 14, France
The development stage- and tissue-specific expression of
multiple mRNAs encoding a same protein is now a welldocumented phenomenon (Schibler et al., 1983; MacGrogan et al., 1985). Recently, it has been shown in rat
(Mukai et al., 1984; Tsutsumi et al., 1984) that aldolase A is
translated from two mRNAs, of 1650 and 1550 bases; the
latter was detected in adult muscle, whereas the former was
present in brain and re-expressed in hepatomas.
Fig. 1 shows a ‘Northern’ blot analysis of the two aldolase
A mRNAs in different rat and human tissues.
In rat, the heavier species is the only one detected in
non-muscular organs, while the lighter species is the single
detectable form in adult gastrocnemius. In foetus, by contrast, only the heavier form was seen before the 19th day of
foetal life; then the 1550 base-long mRNA progressively
takes over the 1650 one. The lighter aldolase A mRNA
species appears to be specific to fast twitch muscle since, by
contrast, adult soleus and heart muscle exhibit only the
heavier mRNA species, like the non-muscular tissues.
In man, the lighter mRNA form is already present in a
3-month-old foetus muscle, in an equal amount to the
heavier species. Also in contrast to the rat, this 1650 base
mRNA is still detectable as a minor species in the adult
muscle. The peculiar occurrence of slow twitch fibres in
human skeletal muscle compared with rat muscle might
explain this result.
The whole content of aldolase A mRNA is roughly the
same in human adult and foetal skeletal muscle, and this is
in contrast with the aldolase A protein level, which increases
during muscle development.
Fig. 2 shows primer extension experiments performed
with rat brain, heart and gastrocnemius muscle mRNAs as
templates. Adult gastrocnemius allowed a 370 base extension of our primer, while heart and brain mRNA used as
templates gave longer extensions, of 420,490 and 560 bases.
Therefore, the aldolase A mRNAs present in these tissues
differ by their 5’ ends. An heterogeneity of the heavier
species can be observed in brain and heart.
Discussion
The products of extension using the same primer upon the
two types of aldolase A mRNA were different in size; this
size difference is of the same range as the size difference
between the two mRNAs. Therefore, the 5’ end differences
can in themselves account for the total difference between
those two messengers.
It has been previously demonstrated that a single aldolase
A isoenzyme exists in different tissues (Gracy et al., 1970;
Ikehara et a]., 1970) and that both heavier and lighter
mRNA species have a very homologous, probably identical,
3’ non-coding extension (Mukai e t al., 1984).
Fig. 1. Northern blot experimenl
Total mRNA was deposited on to each slot. Man: (1) adult liver, 1Opg of mRNA; (2)
3-month-old foetus liver, 10 pg; (3) adult lung, 5 pg; (4) adult muscle, 2 pg; (5) 3-monthold foetus muscle, 4pg; (6) testis, 5 pg; (7) pineal gland, 6pg; (8) adult muscle, 2pg. Rat:
(1) 17-day-old foetus muscle, 30 pg; (2) and (10) adult brain, 20pg; (3) 19-day-old foetus
muscle, 30pg; (4) and (8) adult gastrocnemius muscle, 3pg; (5) newborn rat muscle,
25 pg; (6) adult soleus muscle, 15 pg; (7) 3-day-old muscle, 18 pg; (9) adult heart, 20 pg;
(1 1) 6-day-old muscle, 15 pg.
1986
616th MEETING, LONDON
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Fig. 2. Primer extension experiment
Templates used: (1) rat brain poly (A’) RNA, 28 pg; (2) rat
heart poly (A’) RNA, 22pg; (3) rat gastrocnemius poly (A’)
RNA, 5pg; (4) rat liver poly (A’) RNA, 5pg; ( 5 ) short
exposure of lane (3). The primer was a 80 base-pair fragment
Hinfl-Hue111 of the aldolase A cDNA, labelled with [a-”P]dATP by filling the 3’ recessed end at the Hinfl site.
The differences between the aldolase A mRNA forms
seem therefore to occur through their 5‘ non-coding stretches.
Several possible mechanisms leading to the expression of
such different messengers could then be proposed. Two
promoters, one ubiquitous and the other specific to adult
fast twitch muscle, could exist. Otherwise, the same pre-
mRNA transcript might be differentially spliced, or even
both mechanisms might occur, as has been demonstrated
for myosin light chains (Periasmy et al., 1984).
Nevertheless, one cannot totally exclude that the different
aldolase A mRNAs could be specified by different genes. It
has been shown that in man, at least two genes carried by
different chromosomes (Hagenauer et a[., 1985), and in rat
at least five genes (P.Maire, unpublished work) are present.
There is no information until now on how many of these
genes are transcribed.
In fast twitch fibres, the rise of the aldolase A mRNA
lighter species level is parallel to that of other specific muscular protein mRNAs such as glycogen phosphorylase M,
creatine kinase M (F. Schweighhoffer, unpublished work)
cr-actin (Minty et al., 1982) and adult myosin heavy chain
(Wild et al., 1984).
In conclusion, the expression of aldolase A mRNAs is
qualitatively and quantitatively related to muscle development. The lighter mRNA species is absolutely specific to the
differentiated fast twitch muscle fibres, while the heavier
species accounts for foetal, ubiquitous aldolase A expression.
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Expression of brain-specific ID sequence is not restricted to the brain, and a novel
complementary cID sequence is found in L-type pyruvate kinase mRNA (a liver-specific
messenger)
YU-CHUN LONE, MARIE-PIERRE SIMON,
AXEL KAHN and JOBLLE MARIE
Inserm U 129, Chu Cochin. 24 rue du Fg St Jacques,
75014 Paris Cedex 14, France
We have recently shown that the pyruvate kinase (PK) gene
is transcribed in the liver in three distinct translatable
mRNA species of 3.2 2.2 and 2 k b (Simon et al., 1983).
These three different pyruvate kinase mRNAs only differ by
their 3’ untranslated extension (J. Marie, M. P. Simon,
Y . C. Lone, M. Cognet & A. Kahn, unpublished work).
In order to study the role of these messengers and the
regulation of their expression in the liver, we have sequenced
both the whole coding region and the 3’ untranslated region
of the 3.2 kb PK mRNA.
Abbreviations used: kb, kilobases; PK, pyruvate kinase.
Vol. 14
Fig. 1 shows the nucleotide sequence of 3PK cDNA
clones (llC6, 12H2, 2B8) which covers the whole 3’ noncoding region of the 3.2 kb PK mRNA. We have found that
the 3.2 kb mRNA possesses in its 3’ non-coding region a
sequence complementary to the brain-specific identifier
sequence (ID) described by Sutcliffe and co-workers (Sutcliffe et al., 1982, 1984a, b; Milner et al., 1984); however, this
sequence which is surrounded by two A h family elements is
absent in the two other PK mRNAs (Fig. I).
This cID sequence present in the PK mRNA was subcloned in both orientations in M 13 single-stranded phage,
both strands being used as probes to analysis the expression
of transcripts containing I D and cID sequences in various
tissues.
Fig. 2 shows the Northern blot and dot blot analysis in
various tissues, ID sequence probe hybridized, as expected,
with the two small brain-specific RNAs, BCI and BC2