From the Department of Medicine and the Cancer Center, University
of Pennsylvania, Philadelphia, Pennsylvania 19104
Murine C2C12 myoblasts induced to
differentiate into multinucleated myotubes decrease their levels of
c-myc mRNA 3-10-fold through posttranscriptional
mechanisms that recognize regulatory elements contained in
protein-coding sequences in exons 2 and 3 of the mRNA. To determine
the mechanism by which these elements mediate c-myc
mRNA down-regulation, we examined the regulation of mutant
MYC and human
-globin-MYC fusion mRNAs.
Regulation of mRNAs containing MYC exon 2 or 3 is
abolished by insertion of an upstream termination codon indicating that
regulatory function depends on their translation. Exploiting this
translation dependence, we show that pharmacologic inhibition of
translation with cycloheximide abolishes the down-regulation of
regulated MYC and globin-MYC mRNAs and
induces their levels in differentiating C2C12 cells. We exclude the
possibility that this induction in mRNA levels results from
cycloheximide effects on transcription or processing of parts of the
RNA other than the regulatory elements, leading to the conclusion that
cycloheximide induction results from mRNA stabilization. We show
that the magnitude of cycloheximide induction can be used to estimate
turnover rates of mRNAs whose decay is translation-dependent. By using cycloheximide inducibility
to examine turnover rates of MYC and globin-MYC
mRNAs, we show that the MYC exon 2 and exon 3 regulatory elements, but not MYC 3'-untranslated region or
chloramphenicol acetyltransferase coding sequences, mediate accelerated
mRNA decay in differentiating, but not undifferentiated, C2C12
cells. We show that these regulatory elements must be translated to
confer accelerated mRNA decay and that increased turnover occurs in
the cytoplasm and not in the nucleus. Finally, using cycloheximide induction to examine mRNA half-lives, we show that mRNA
turnover is increased sufficiently by mechanisms targeting the exon 2 and 3 regulatory elements to account for the magnitude of
c-myc mRNA down-regulation during differentiation. We
conclude from these results that c-myc mRNA
down-regulation during myogenic differentiation is due to
translation-dependent mechanisms that target mRNAs
containing myc exon 2 and 3 regulatory elements for
accelerated decay.
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INTRODUCTION |
mRNA turnover rates play an important role in determining
levels of cellular gene expression, and regulated mRNA turnover has
been shown to regulate expression of a number of genes. mRNA stability has been shown to be regulated through a variety of mechanisms that target specific RNA sequences or motifs, RNA secondary structural elements, or the encoded peptide. For example, many labile
cytokine and proto-oncogene mRNAs (e.g. c-fos
and granulocyte macrophage-colony-stimulating factor) contain
AU-rich/AUUUA sequence elements in their 3'-untranslated regions
(UTRs)1 that target them for
rapid turnover and limit gene expression (1, 2), whereas
-globin
mRNA has a C-rich element in its 3'-UTR that allows formation of a
protein-mRNA complex that is thought to stabilize the mRNA and
allow prolonged gene expression (3). A secondary structural element in
the 3'-UTR of histone mRNA targets it for
translation-dependent turnover that is coupled to the
position of the cell in the cell cycle and DNA synthesis (4). Turnover
of transferrin receptor mRNA is regulated by stem-loop structures
in its 3'-UTR termed iron-responsive elements that are bound by iron
regulatory proteins in an iron-poor environment resulting in mRNA
stabilization. Conversely, in an iron-rich environment, the unprotected
mRNA is rapidly degraded. Autoregulation of
-tubulin mRNA
levels occurs through recognition of the first four amino acids
of the nascent peptide by excess free
-tubulin subunits, and
resulting dimerization targets the mRNA for accelerated turnover (5).
Regulated RNA stability plays a critical role in controlling expression
of c-myc, a proto-oncogene encoding a transcription factor
important in regulating cell proliferation and differentiation. The
short half-life of c-myc mRNA (15-30 min) (6, 7) allows cells to rapidly alter c-myc expression either through
transcriptional (8-12) or posttranscriptional (13-18) mechanisms. A
decrease in c-myc mRNA expression is seen when cells are
induced to differentiate (13-18), and a rapid increase is seen
following mitogen exposure (19). We have been characterizing
posttranscriptional mechanisms controlling c-myc mRNA
levels in C2C12 murine myoblasts (20, 21), and we have demonstrated
previously that sequences in the 3'-UTR determine the turnover rate and
steady-state levels of c-myc mRNA in proliferating C2C12
cells. However, these sequences are dispensable for the
posttranscriptional down-regulation of c-myc mRNA levels
when C2C12 cells are induced to differentiate into multinucleated
myotubes (17, 20, 21). Instead, coding elements in myc exons
2 and 3 target c-myc mRNA for down-regulation during
differentiation. In studies presented here, we characterize the
mechanism by which c-myc expression is posttranscriptionally down-regulated during C2C12 myogenic differentiation. We demonstrate that translation of exon 2 and exon 3 coding elements is necessary for
them to target mRNAs for down-regulation and that down-regulation occurs by translation-dependent accelerated mRNA decay in
the cytoplasm.
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EXPERIMENTAL PROCEDURES |
Cell Culture and DNA Transfection--
All experiments were
performed using C2C12 myoblasts (22) obtained from the ATCC (Rockville,
MD) and maintained in Dulbecco's minimum essential media (DMEM)
supplemented with 10% fetal calf serum, 5% CO2,
penicillin, and streptomycin. Plasmids containing test genes were
stably co-transfected into C2C12 cells with a plasmid containing a
neor gene using the calcium phosphate method. After
selection in 400 µg/ml G418 (Life Technologies, Inc.), pools of
25-50 surviving colonies were expanded for study. C2C12 cells were
induced to differentiate into multinucleated myotubes as described
previously (17). Briefly, undifferentiated C2C12 myoblasts were seeded at subconfluent density in DMEM supplemented with 10% fetal calf serum
and antibiotics. Cells were cultured to confluence and then induced to
differentiate by changing the media to differentiation media (DM), DMEM
containing 2% horse serum.
Plasmid Constructions--
Salient features of the plasmids used
in this article are diagrammed in Fig. 1.
The normal human c-MYC gene contains three exons. CM19 was
described previously (20) and is a pUC-based plasmid that contains all
of human c-MYC from the XhoI site (between the P1
and P2 promoters) to the EcoRI site (3' to exon 3) under the
transcriptional control of the Moloney murine leukemia virus (MLV) LTR.
MYC(X/N) was described previously (20) and contains MYC sequences from the XhoI site to the
NsiI site 75 nucleotides 3' to the translation termination
codon fused to the simian virus 40 (SV40) early T antigen
polyadenylation signal (SVpA). The construction of plasmids
MYC(
41-178) and MYC(
265-433) in which
MYC codons 41-178 or 265-433 were deleted, respectively,
was described previously (23). MYC(T/N) was described
previously (21) and contains MYC cDNA sequences from the
ThaI site 5 nucleotides upstream of the exon 2 translation
initiation codon to the NsiI site and uses the SVpA.
MYC(Ex2Term3) was created by inserting an
oligonucleotide linker containing an in-frame termination codon into
the ClaI site at codon 262 in MYC(T/N). To create
MYC(Ex3/2), an XhoI site was inserted at codon 40 of MYC(T/N)(
265-433) (21) by substituting the
ThaI-PstI fragment of In40 (23). The
XhoI-EcoRI fragment of the resulting plasmid was
then substituted for the XhoI-EcoRI fragment of
MYC(T/N)(
41-178) (21) after insertion of an
XhoI site at codon 434 using the
ClaI-NsiI fragment of In434 (23). MYC(Ex3/2) contains MYC codons 1-40 fused
in-frame to codons 179-433, followed in-frame by MYC codons
40-265 and codons 434-439.

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Fig. 1.
Schematic diagram of recombinant genes used
in these studies. Construction of these genes is outlined under
"Experimental Procedures." Structural features of these genes and
the shading pattern of exonic sequences are depicted below the genes.
An NcoI restriction site was introduced by site-directed
mutagenesis into exon 3 of MLV- -Globin (20) to facilitate
construction of chimeric -globin genes.
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Construction of the
-globin-MYC plasmids,
Gm434SVpA,
Gm434MYCpA,
Gm263SVpA, and
Gm(40-262)SVpA, and
their nomenclature was described previously (20). These genes contain
an MLV-
-globin backbone encoding the first 140 globin amino acids
(out of 146) fused to variable MYC sequences. In
Gm434SVpA, MLV-
-globin is fused in-frame to the last six
MYC codons and the first 75 nucleotides of the
MYC 3'-UTR and uses the SVpA. The other genes were created from
Gm434SVpA with the following modifications: in
Gm434MYCpA, the entire MYC 3'-UTR and flanking
sequences to the EcoRI site was substituted for the SVpA; in
Gm263SVpA, almost all MYC exon 3 coding sequences (from
codon 263 to the NsiI site in MYC exon 3) are
fused in-frame to globin; and in
Gm(40-262)SVpA, MYC
cDNA sequences from codon 40-262 (most exon 2 coding sequences and the first 10 codons in exon 3) are fused in-frame to globin. To create
Gm263TermSVpA and
Gm(40-262)TermSVpA, a termination coding was inserted into the linker fusing globin and MYC domains
allowing globin sequences, but not MYC sequences, to be
translated.
Gm263SVpARI
and
Gm(40-262)SVpARI
were created by introducing a C to T
mutation by site-directed mutagenesis into
-globin codon 122 which
destroys an EcoRI site but preserves the encoded amino acid
(phenylalanine). In
G-CAT (20), the MLV-
-globin backbone is fused
to the last 181 codons (out of a total of 219) of the bacterial gene
encoding chloramphenicol acetyltransferase (CAT) and uses the SVpA.
Analysis of mRNA Levels in Stably Transfected Cells--
RNA
isolation for differentiation assays was described previously (21). To
examine induction in mRNA levels after inhibition of translation,
total cytoplasmic mRNA was isolated from duplicate tissue culture
plates of both undifferentiated and differentiating C2C12 cells, one
plate untreated and the other exposed to cycloheximide (Sigma) at a
concentration of 10 µg/ml for 3 h, unless otherwise stated.
Differentiating cells were used 36-48 h after exposure to DM, and
differentiation-induced c-myc mRNA down-regulation was
considered to have occurred when c-myc mRNA levels
decreased more than 3-fold compared with levels in undifferentiated
cells. mRNA levels were determined by Northern analysis using the
glyoxal method (24). RNA was electroblotted to Hybond N (Amersham
Pharmacia Biotech) and UV cross-linked. Hybridizations were carried out by modifications of the method of Church and Gilbert (25) using probes
labeled by random priming. Human MYC mRNAs were probed using a human c-MYC exon 1 probe (XhoI to
PvuII fragment) or a human c-MYC exon 2+3
cDNA probe from pSP65MYCIIA (23).
-Globin chimeric
mRNAs were probed using a full-length human
-globin cDNA
fragment from pSP
c (gift from Stephen Liebhaber). C2C12 c-myc mRNA was probed with a murine c-myc
exon 1 probe (BamHI to SacI fragment) or a human
c-MYC exon 2 + 3 cDNA probe from pSP65MYCIIA
(23). rpL32 mRNA was probed with a full-length cDNA probe (26).
Northern blots were analyzed on a Molecular Dynamics PhosphorImager
(Sunnyvale, CA) using ImageQuant software, and the relative levels of
MYC and globin-MYC fusion mRNAs during differentiation or after cycloheximide were determined by normalizing for RNA loading using the level of rpL32 mRNA. All experiments were
performed at least twice, and representative results are displayed in
the figures presented.
Derivation of mRNA Half-lives by Analysis of Induction of
mRNA Levels by Cycloheximide--
Induction in mRNA levels
3 h after inhibition of translation was used to deduce
MYC and globin-MYC mRNA half-lives. Assuming first order kinetics of mRNA decay, the steady-state level of a
given mRNA species, [mRNA]ss, is a function of
its rate of synthesis, ks, and its half-life,
t1/2, and can be calculated as shown in Equation 1.
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(Eq. 1)
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If mRNA decay is completely inhibited but the rate of
transcription continues at the same rate, the mRNA level at time
x, [mRNA]x, will be a function of the
steady-state mRNA level prior to inhibition of decay, the rate of
synthesis, and the time synthesis is allowed to continue,
tx, as shown in Equation 2.
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(Eq. 2)
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Under these conditions, the fold induction of a given mRNA
species, [mRNA]x
[mRNA]ss, is a
function of the length of time synthesis is allowed to continue and the
mRNA half-life prior to stabilization as shown in Equation 3.
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(Eq. 3)
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Therefore, the mRNA half-life can be calculated under these
conditions as shown in Equation 4.
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(Eq. 4)
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RT-PCR+1 Assay of Comparative mRNA Abundance--
The
RT-PCR+1 assay for comparing the abundance of two
-globin-containing
mRNAs was performed as described previously (20) with minor
modifications. Total cytoplasmic RNA was extracted from stably
transfected C2C12 myoblasts by a reduced scale modification of the
method of Laski et al. (27). To isolate nuclear RNA, nuclei
were isolated from C2C12 cells after lysis in nuclear lysis buffer, NLB
(10 mM Tris-HCl (pH 7.4), 10 mM NaCl, 5 mM MgCl2, 0.5% Nonidet P-40). After washing
nuclei in NLB, nuclear RNA was isolated using Trizol reagent (Life
Technologies, Inc.) according to the manufacturer's instructions.
Sequence-specific reverse transcription of cytoplasmic and nuclear RNA
was performed by priming with oligonucleotide,
G3'Rev (see below),
using MLV reverse transcriptase (Life Technologies, Inc.) according to
manufacturer's instructions. PCR amplification of cDNA was
performed using Taq polymerase (Promega) in 50 mM Tris-HCl (pH 9.0), 20 mM
(NH4)2SO4, 1.5 mM
MgCl2, 200 mM dNTPs, and 1 mM
oligonucleotide primers on a PTC-100 thermal cycler (MJ Research,
Cambridge, MA). A 214-base pair sequence of
-globin cDNA was
amplified using oligonucleotide primers
GF2 and
G3'Rev (see
below) under the following conditions: 92 °C for 30 s, 62 °C
for 15 s, and 72 °C for 15 s for 30 cycles. A "+1"
cycle was conducted with a 32P-end-labeled nested primer,
GF1, and the products were restricted with EcoRI and
resolved by electrophoresis. A radiolabeled 329-base pair DNA fragment
was added to each EcoRI digestion reaction mixture to
indicate complete digestion of the RT-PCR+1 products. The relative abundance of the RT-PCR+1 products (and, hence, of the mRNAs) was
quantitated on a Molecular Dynamics PhosphorImager (Sunnyvale, CA) using ImageQuant software.
The primers and their sequences used in this study are listed as
follows: 1)
GF2, 5'AAGTGCTCGGTGCCTTTAGTGA3'; 2)
G3'Rev, 5'ACACCAGCCACCACTTTCTGA3'; 3)
GF1,
5'CAAGGGCACCTTTGCCACACT3'.
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RESULTS |
Murine C2C12 myoblasts are induced to differentiate into
multinucleated myotubes by mitogen deprivation. c-myc
expression is down-regulated early during differentiation, prior to the
up-regulation of muscle-specific genes (e.g. creatine
kinase) (17). This occurs through posttranscriptional mechanisms
demonstrated by nuclear run-on assays which showed that the rate of
c-myc transcription does not change significantly during
differentiation, whereas c-myc mRNA levels decrease
3-10-fold (17). We previously demonstrated that two protein coding
elements, one in exon 2 and the other in exon 3, are necessary for
targeting myc mRNA for down-regulation, whereas 5'- and
3'-UTR sequences and introns are dispensable.
Translation of Myc Exon 3 Is Required for Myc mRNA
Down-regulation during Differentiation--
The presence of regulatory
determinants within the protein coding region of c-myc
mRNA raises the possibility that their recognition may be coupled
to translation. To determine whether translation of the myc
exon 3 regulatory element is required for down-regulation, C2C12 cells
were stably transfected with MYC(T/N), a MYC
cDNA construct from which most 5'- and 3'-UTR sequences were
deleted, or with MYC(Ex2Term3), a construct identical to
MYC(T/N) except for the insertion of an in-frame nonsense
mutation at codon 262 that prevents translation of about 95% of exon 3 coding sequences, including the exon 3 regulatory element. Cytoplasmic
RNA was isolated on serial days from subconfluent cells, confluent
cells, and confluent cells induced to differentiate. Northern analysis
demonstrated that MYC(T/N) mRNA was maximally
down-regulated 3.7-fold during C2C12 differentiation, comparable to the
4.1-fold down-regulation in endogenous murine c-myc mRNA
(both normalized to rpL32 mRNA; Fig.
2A). (In figures showing
Northern blots from cells transfected with mutant human MYC
genes, transgene mRNA is labeled as Hu-MYC to
distinguish it from the endogenous murine c-myc
mRNA, labeled Mo-myc.) In contrast,
MYC(Ex2Term3) mRNA was not down-regulated and even
increased over 3-fold, whereas levels of endogenous c-myc mRNA in the same cells were down-regulated 6.7-fold (Fig.
2B). This showed that translation of the myc exon
3 regulatory element is required for myc mRNA
down-regulation.

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Fig. 2.
Down-regulation of myc mRNA during C2C12
differentiation is dependent on translation of exon 3 coding
sequences. C2C12 cells stably transfected with MYC(T/N)
(A), MYC(Ex2Term3) (B), or
MYC(Ex3/2) (C) were seeded at low density into
multiple culture plates, cultured to confluence, and induced to
differentiate using differentiation media (DM; DMEM containing 2%
horse serum). Northern analysis was performed on cytoplasmic RNA
isolated on serial days beginning the day after cells were plated.
Cells were subconfluent on day 1 and confluent on day 2. Medium was
changed to DM on day 2; therefore, days 3, 4, and 5 cells have been
exposed to DM for 1, 2, and 3 days, respectively. mRNA from the
transfected gene was detected using probe for the exon 2 + 3 coding
region of human (Hu) c-MYC(pSP65MYCIIA
(23)); the endogenous murine (Mo) c-myc mRNA
was detected using a murine c-myc exon 1 probe
(BamHI to SacI fragment; A and
B) or with a human MYC exon 2 + 3 probe
(C); and rpL32 mRNA was detected using a full-length
cDNA probe (26). RNA from untransfected C2C12 cells (U)
demonstrates probe specificity for transgene mRNA. The bar
graphs depict the levels of endogenous murine c-myc
mRNA (solid bars) and transgene mRNA (shaded
bars) relative to their mRNA levels in preconfluent,
undifferentiated cells after normalizing for RNA loading using the
level of rpL32 mRNA.
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The Order of Myc Exon 2 and 3 Regulatory Sequences Affects Myc
mRNA Down-regulation--
The translation dependence of exon 3 regulatory function made it impossible to determine whether function of
the myc exon 2 regulatory element is
translation-dependent in its normal context because insertion
of a stop codon upstream of exon 2 would prevent translation of both
elements. However, if the relative positions of the exon 2 and exon 3 elements are unimportant for myc regulation, the translation
dependence of exon 2 regulatory function could be tested in a mutant
mRNA in which the positions of the exon 2 and exon 3 coding
elements are reversed. To explore this possibility, C2C12 cells were
stably transfected with MYC(Ex3/2) in which the exon 3 regulatory element was placed 5' to the exon 2 regulatory element and
maintains the proper reading frame of both. Northern analysis showed
that MYC(Ex3/2) mRNA was not down-regulated during differentiation (Fig. 2C) demonstrating that the position of
the exon 2 and 3 regulatory elements affects their function, precluding testing of the translation dependence of exon 2 regulatory function in
the myc mRNA context.
Myc Regulatory Elements Must Be Translated to Confer
Down-regulation onto Globin mRNA--
To determine the importance
of translation for exon 2 function, we examined regulation of chimeric
mRNAs in which MYC sequences were fused in-frame to
human
-globin mRNA. We previously showed that human
-globin
mRNA was down-regulated during differentiation when fused to either
the exon 2 or 3 regulatory element but not when fused to MYC
3'-UTR sequences or to CAT or rpL32 coding sequences (Ref. 21 and see
Fig. 4). These globin-MYC chimeric genes allowed us to test
the translation dependence of function of each of the myc
mRNA regulatory domains independent of the other element. To that
end, C2C12 cells were stably transfected with
Gm(40-262)TermSVpA and
Gm263TermSVpA, constructs which contain a stop codon 5' to the
MYC exon 2 or 3 regulatory element, respectively (ribosomes translate globin codons but not MYC codons). When C2C12
transfectants were induced to differentiate, levels of
Gm(40-262)TermSVpA mRNA decreased only 1.3-fold and levels of
Gm263TermSVpA mRNA were essentially unchanged, whereas levels of
endogenous c-myc mRNA decreased 4.8- and over 10-fold in
these respective stable transfectants (Fig.
3, A and B). Thus,
the regulatory elements in myc exons 2 and 3 must be
translated to confer down-regulation on globin mRNA.

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Fig. 3.
Regulatory elements in MYC exons
2 and 3 must be translated to confer down-regulation on -globin
mRNA. Differentiation assays were conducted on C2C12 cells
stably transfected with Gm(40-262)TermSVpA or Gm263TermSVpA.
(Refer to legend in Fig. 2 for details of the assay.) Autoradiographs
of Northern blots display mRNA levels in cells transfected with
Gm(40-262)TermSVpA (A) and Gm263TermSVpA
(B). mRNA from the transfected gene was detected using a
full-length human -globin cDNA probe (labeled Globin)
pSP c; endogenous murine c-myc (labeled
c-myc) and rpL32 mRNAs were detected using
myc exons 2/3 and full-length rpL32 probes, respectively.
RNA from untransfected C2C12 cells (U) was used to
demonstrate specificity of the human -globin cDNA probe. The
bar graphs depict the levels of endogenous murine
c-myc mRNA (solid bars) and transgene
mRNA (shaded bars) relative to their mRNA levels in
preconfluent, undifferentiated cells after normalizing for RNA loading
using the level of rpL32 mRNA.
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Cycloheximide Studies Reveal That Myc Exons 2 and 3 Contain
Translation-dependent Instability
Determinants--
Whereas coding sequences in myc exons 2 and 3 serve as translation-dependent, conditional mRNA
regulatory elements, the mechanism by which they down-regulate mRNA
is unclear. If the regulatory elements mediate down-regulation by
accelerating mRNA decay in differentiating C2C12 cells, inhibiting
their function should result in stabilization and increased levels of
their mRNAs. Since their function is
translation-dependent, we tested whether cycloheximide might produce these effects in differentiating C2C12 cells stably transfected with
Gm(40-262)SVpA or
Gm263SVpA, in which the
regulatory element from MYC exon 2 or exon 3 was fused to
globin, respectively. Following 3 h of cycloheximide treatment,
levels of
Gm(40-262)SVpA mRNA were 6-fold higher, and
levels of
Gm263SVpA mRNA were 8.7-fold higher than in
untreated cells (Fig. 4, A and
B). These results are consistent with the idea that
myc exon 2 and exon 3 elements down-regulate their mRNAs
by accelerating mRNA decay in a translation-dependent manner. Cycloheximide induced levels of
Gm(40-262)SVpA and
Gm263SVpA mRNAs an insignificant 2.2- and 1.5-fold,
respectively, in subconfluent, undifferentiated cells (Fig. 4,
A and B). This contrasted with a 7- and 17-fold
induction in the endogenous c-myc mRNA, respectively, demonstrating that the insignificant induction in
Gm(40-262)SVpA and
Gm263SVpA mRNA levels in undifferentiated cells was not
due to lack of cycloheximide effect (in all our cycloheximide studies, induction of endogenous c-myc mRNA levels was used as a
positive control for cycloheximide effect). These results indicate that the destabilizing function of the myc exon 2 and exon 3 regulatory elements requires differentiation.

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Fig. 4.
MYC regulatory elements confer
regulated cycloheximide inducibility (CHX Ind) on
-globin mRNA during myoblast differentiation. Induction in
levels of globin-MYC fusion mRNAs was examined in C2C12
cells stably transfected with Gm(40-262)SVpA, Gm263SVpA,
Gm434MYCpA, or G-CAT. Cytoplasmic RNA was isolated
from preconfluent cells (lanes 1 and 2) and cells
induced to differentiate for 36 h in differentiation media
(lanes 3 and 4) that were untreated ( ) or
exposed to cycloheximide (CHX) for 3 h (+).
Autoradiographs of Northern blots display mRNA levels in cells
transfected with Gm(40-262)SVpA (A), Gm263SVpA
(B), Gm434MYCpA (C), and G-CAT
(D). mRNA from the transfected gene was detected using a
full-length human -globin cDNA probe, pSP c (A,
B, and C), or with a probe for the CAT coding
region (D); the endogenous murine c-myc mRNA
was detected using a probe for the Hu-MYC exon 2 + 3 coding
region, and rpL32 mRNA was detected using a full-length cDNA
probe (26). RNA from untransfected C2C12 cells (lane 5)
demonstrates probe specificity for transgene mRNA. The fold
induction in transgene mRNA levels after cycloheximide when
normalized for RNA loading using the level of rpL32 mRNA is
displayed. Fold induction = (c-myc mRNA level in
cycloheximide-treated cells c-myc mRNA level in
untreated cells) (rpL32 mRNA level in cycloheximide-treated
cells rpL32 mRNA level in untreated cells).
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While the foregoing results support a translation-dependent
destabilizing function of myc exon 2 and 3 regulatory
elements, cycloheximide could also be affecting transcription from the
MLV LTR or regulation from non-myc portions of the
mRNAs. To exclude these possibilities, the effect of cycloheximide
was examined in C2C12 cells stably transfected with (i)
G-CAT in
which CAT sequences are fused in-frame to globin in place of
myc sequences, or (ii)
Gm434MYCpA which
contains the last six MYC codons fused in-frame to globin
followed by the entire MYC 3'-UTR. Levels of mRNAs
encoded by these genes were induced no more than 2-fold by
cycloheximide in either preconfluent or differentiating C2C12 cells
(Fig. 4, C and D). Cycloheximide inducibility
depends on mRNA instability being translation-dependent, but the
magnitude of induction depends on the turnover rate of the mRNA
prior to stabilization, i.e. a given increase in the amount
of a stable mRNA is fractionally smaller and more difficult to
appreciate than the same increase in the amount of an unstable
mRNA.
Gm434MYCpA mRNA has a decay rate comparable
to
Gm263SVpA mRNA under growth conditions (20) and should be
comparably induced if cycloheximide affected its metabolism,
e.g. through increased transcription from the MLV LTR. These
results, therefore, exclude the possibility that cycloheximide
induction of
Gm(40-262)SVpA and
Gm263SVpA mRNAs resulted
from increased MLV LTR transcription or from cycloheximide effects on
globin or SV40 polyadenylation sequences during differentiation. Additionally, they show that MYC sequences that do not
confer down-regulation during C2C12 differentiation (i.e.
the 3'-UTR) do not confer cycloheximide inducibility. Thus, sequences
from either MYC exons 2 or 3 specifically confer
cycloheximide inducibility on
-globin and function as
translation-dependent mRNA instability determinants during
C2C12 differentiation. If transcription and mRNA processing rates
are unchanged by cycloheximide, the 3-6-fold difference in
cycloheximide inducibility between undifferentiated and differentiating
cells suggests that the decay rates of these mRNAs are accelerated
3-6-fold in differentiating cells (see "Discussion").
Coding Sequences in Both Exons 2 and 3 and Their Translatability
Are Necessary for Cycloheximide-induction of myc mRNA during
Differentiation--
If accelerated myc mRNA decay
accounts for its down-regulation during C2C12 differentiation,
mutations that disrupt myc mRNA down-regulation should
prevent its accelerated decay. To test this hypothesis, we examined the
effect of cycloheximide on the following: (a)
MYC(
41-178) mRNA, a mutant human MYC
mRNA from which most of exon 2 was deleted; (b)
MYC(
265-433) mRNA, a MYC mutant from
which most exon 3 coding sequences were deleted; (c) MYC(X/N) mRNA, a MYC mutant from which most
3'-UTR sequences were deleted; or (d) MYC(T/N)
mRNA, a MYC mutant from which most 5'- and 3'-UTR
sequences were deleted. Previously, we showed that MYC(
41-178) and MYC(
265-433) mRNAs
are not down-regulated and that MYC(X/N) and
MYC(T/N) mRNAs are down-regulated during differentiation (21). MYC(
41-178) and MYC(
265-433)
mRNAs were very modestly induced by cycloheximide treatment in
preconfluent cells but were not induced in differentiating C2C12 cells,
whereas levels of endogenous c-myc mRNA were induced
(Fig. 5, A and B).
In contrast, MYC(X/N) and MYC(T/N) mRNAs,
which were poorly induced by cycloheximide in preconfluent cells, were
induced 3.2- and 5.5-fold, respectively, in differentiating cells (Fig.
5, C and D). Thus, mutant MYC
mRNAs that are down-regulated during C2C12 differentiation are
cycloheximide-inducible, whereas those that are not down-regulated
during differentiation are not cycloheximide-inducible. These results
demonstrate that coding sequences in both myc exons 2 and 3 are necessary for cycloheximide induction of myc mRNA
and support the hypothesis that they, but not 5' or 3'-UTR sequences,
are necessary for translation-dependent accelerated
c-myc mRNA decay during C2C12 differentiation.

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Fig. 5.
Coding sequences from MYC exons 2 and 3 are necessary for regulated cycloheximide inducibility (CHX
Ind) of myc mRNA during myoblast differentiation.
Induction in myc mRNA levels was examined in C2C12 cells
stably transfected with MYC( 41-178),
MYC( 265-433), MYC(X/N), or
MYC(T/N). Cytoplasmic RNA was isolated from preconfluent
cells (lanes 1 and 2) and cells induced to
differentiate for 36 h in differentiation media (lanes
3 and 4) that were untreated ( ) or exposed to
cycloheximide for 3 h (+). Autoradiographs of Northern blots
display mRNA levels in cells transfected with
MYC( 41-178) (A), MYC( 265-433)
(B), MYC(X/N) (C), or
MYC(T/N) (D). mRNA from the transfected gene
was detected using a human (Hu) c-MYC exon 1 probe (XhoI to PvuII fragment) (A,
B, and C) or with a probe for the exon 2 + 3 coding region of human c-MYC (D); the endogenous
murine (Mo) c-myc mRNA was detected using a
murine c-myc exon 1 probe (A, B, and
C) or with a probe for the Hu-MYC exon 2 + 3 coding region (D); and rpL32 mRNA was detected using a
full-length cDNA probe (26). RNA from untransfected C2C12 cells
(lane 5) demonstrates probe specificity for transgene
mRNA. The fold induction in transgene mRNA levels after
cycloheximide when normalized for RNA loading using the level of rpL32
mRNA is displayed. Fold induction = (c-myc mRNA
level in cycloheximide-treated cells c-myc mRNA
level in untreated cells) (rpL32 mRNA level in cycloheximide
treated cells rpL32 mRNA level in untreated cells).
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If accelerated mRNA decay during differentiation is dependent on
translatability of the myc exon 2 and 3 regulatory elements, we would predict that cycloheximide inducibility should also depend on
the translatability of these elements. As expected if myc
exon 3 sequences must be translatable to confer cycloheximide
inducibility, levels of MYC(Ex2Term3) mRNA were not
induced by cycloheximide in either preconfluent or differentiating
cells (Fig. 6A). Since the
translation dependence of exon 2 sequences cannot be examined in
myc mRNA without perturbing translation of exon 3 sequences, we examined the effect of their translatability on
cycloheximide inducibility using
Gm(40-262)TermSVpA. Levels of
Gm(40-262)TermSVpA mRNA were not induced after
cycloheximide in either preconfluent or differentiating cells (Fig.
6B) showing that coding elements in both myc
exons 2 and 3 must be translatable to confer regulated cycloheximide
inducibility, i.e. accelerated mRNA decay during C2C12
differentiation.

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Fig. 6.
MYC exon 2 and 3 regulatory elements
must be translatable to confer regulated cycloheximide inducibility
(CHX Ind) during myoblast differentiation. Induction
in levels of MYC or globin-MYC fusion mRNAs
was examined in C2C12 cells stably transfected with
MYC(Ex2Term3) or Gm(40-262)TermSVpA. Cytoplasmic RNA was
isolated from preconfluent cells (lanes 1 and 2)
and cells induced to differentiate for 36 h in differentiation
media (lanes 3 and 4) that were untreated ( ) or
exposed to cycloheximide for 3 h (+). Autoradiographs of Northern
blots display mRNA levels in cells transfected with
MYC(Ex2Term3) (A) and Gm(40-262)TermSVpA
(B). mRNA from the transfected gene was detected with a
probe for the exon 2 + 3 coding region of human (Hu)
c-MYC (A) or a full-length human -globin
cDNA probe, pSP c (B); the endogenous murine
(Mo) c-myc mRNA was detected using a probe
for the human MYC exon 2 + 3 coding region; and rpL32
mRNA was detected using a full-length cDNA probe (26). RNA from
untransfected C2C12 cells (lane 5) demonstrates probe
specificity for transgene mRNA. The fold induction in transgene
mRNA levels after cycloheximide when normalized for RNA loading
using the level of rpL32 mRNA is displayed. Fold induction = (c-myc mRNA level in cycloheximide-treated cells c-myc mRNA level in untreated cells) (rpL32 mRNA
level in cycloheximide-treated cells rpL32 mRNA level in
untreated cells).
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Induction in Levels of c-myc mRNA by Cycloheximide Increases
when C2C12 Myoblasts Differentiate--
Measurements of
c-myc mRNA decay after actinomycin D show only a modest
increase in its turnover rate in differentiating cells compared with
undifferentiated cells (19). It is likely that these results
overestimate the half-life of c-myc mRNA in
differentiating cells because actinomycin D interferes with the
destabilizing function of myc exon 3 coding sequences (19),
and its effects on myc exon 2 regulatory function are
unknown. To determine whether c-myc mRNA turnover is
accelerated during differentiation, we examined cycloheximide induction
of c-myc mRNA levels in undifferentiated and
differentiating C2C12 myoblasts. After 3 h of cycloheximide treatment, levels of c-myc mRNA were induced 11-14-fold
in preconfluent and confluent cells, respectively (Fig.
7, lanes 1-4), whereas levels
were induced 63-fold in differentiating cells (Fig. 7, lanes
5 and 6). This greater level of induction resulted more from the lower c-myc mRNA levels in differentiating
cells not exposed to cycloheximide (Fig. 7, compare lanes 1, 3, and 5) than from higher levels of c-myc
mRNA after cycloheximide (Fig. 7, compare lanes 2, 4,
and 6). This 4-6-fold increase in cycloheximide inducibility at 3 h suggests that the turnover of c-myc
mRNA is 4-6-fold faster in differentiating C2C12 cells which is
consistent with the magnitude of down-regulation seen during
differentiation and with estimates of the degree of destabilization
imposed on globin mRNAs by the myc regulatory
elements.

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Fig. 7.
Cycloheximide (CHX) induction of
c-myc mRNA increases during myoblast
differentiation. C2C12 cells were seeded at low density into
multiple culture plates, cultured to confluence, and induced to
differentiate for 36 h using differentiation media. Cytoplasmic
RNA was isolated from preconfluent (lanes 1 and
2), confluent (lanes 3 and 4), and
differentiating myoblasts (lanes 5 and 6).
Displayed is an autoradiograph of a Northern blot probed for
c-myc mRNA using a murine c-myc exon 1 probe
(BamHI to SacI fragment) and for rpL32 mRNA
using a full-length cDNA probe (26). The autoradiograph
demonstrates mRNA levels in untreated cells (lanes 1, 3,
and 5) and in cells exposed to cycloheximide for 3 h
(lanes 2, 4, and 6). The fold induction in
c-myc mRNA levels after cycloheximide when normalized
for RNA loading using the level of rpL32 mRNA is displayed. Fold
induction = (c-myc mRNA level in
cycloheximide-treated cells c-myc mRNA level in
untreated cells) (rpL32 mRNA level in cycloheximide-treated
cells rpL32 mRNA level in untreated cells).
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Accelerated mRNA Decay during Differentiation Affects
Cytoplasmic but Not Nuclear mRNA Levels--
Previous studies
demonstrated that myc exon 2- or 3-mediated mRNA
down-regulation during C2C12 differentiation affects cytoplasmic but
not spliced nuclear mRNA levels (21). If accelerated mRNA decay
is responsible for down-regulation, one would predict that it too
should be a cytoplasmic event. To examine whether cytoplasmic RNA
turnover is accelerated during differentiation, we examined the effect
of cycloheximide on nuclear and cytoplasmic mRNA levels using an
RT-PCR-based assay for comparing the relative abundance of two
globin-MYC fusion mRNAs (20). To examine the effect of myc exon 2 coding sequences on cytoplasmic mRNA
turnover, C2C12 cells were stably co-transfected with
Gm(40-262)SVpARI
, which encodes an mRNA that is
induced by cycloheximide in differentiating cells, and
Gm434SVpA,
which encodes a noninducible mRNA.
Gm(40-262)SVpARI
is identical to
Gm(40-262)SVpA except for a silent C to T mutation introduced
at globin codon 122 that destroys an EcoRI site but does not
affect mRNA processing or stability (20). By RT-PCR+1 analysis, the
comparative level of spliced
Gm434SVpA:
Gm(40-262)SVpARI
mRNAs in a
nuclear RNA preparation from undifferentiated C2C12 cells was 1:1 (Fig.
8, lane 5). Their comparative
levels did not change when cells were induced to differentiate (Fig. 8,
lane 7) or when undifferentiated or differentiating cells
were exposed to cycloheximide (Fig. 8, lanes 6 and
8), demonstrating that nuclear mRNA levels are not
affected by exon 2 sequences during differentiation or after
cycloheximide. The relative abundance of these mRNAs in a
cytoplasmic RNA preparation from undifferentiated cells was 1.3:1 (Fig.
8, lane 1) but changed to 3.9:1 when cells were induced to
differentiate (Fig. 8, lane 3), demonstrating that
Gm(40-262)SVpARI
mRNA was down-regulated 3-fold
compared with
Gm434SVpA mRNA. In undifferentiated cells exposed
to cycloheximide, the comparative cytoplasmic mRNA ratio was 1.1:1,
an insignificant change from the ratio in untreated cells (Fig. 8,
compare lanes 1 and 2). However, in
differentiating cells exposed to cycloheximide, the comparative
mRNA ratio changed from the 3.9:1 ratio seen in untreated cells to
1:1.1, a 3.5-fold relative increase in cytoplasmic levels of
Gm(40-262)SVpARI
mRNA compared with
Gm434SVpA
mRNA (Fig. 8, compare lanes 3 and 4). These
results are consistent with previous results demonstrating that fusion
of coding elements from MYC exon 2 confers down-regulation on a globin-MYC fusion mRNA, and they validate results
shown here demonstrating that exon 2 confers cycloheximide inducibility
under conditions of differentiation. Furthermore, they demonstrate that cycloheximide affects cytoplasmic but not nuclear mRNA levels, suggesting that cytoplasmic but not nuclear turnover of myc
mRNA is accelerated during differentiation. Analyses of
Gm263SVpARI
demonstrated that MYC exon 3 affected the cycloheximide inducibility of cytoplasmic, but not
nuclear, mRNA under conditions of differentiation suggesting that
its regulatory element also affects cytoplasmic mRNA turnover
during differentiation (data not shown).

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Fig. 8.
myc exon 2-mediated cycloheximide
inducibility during C2C12 differentiation results in increased
cytoplasmic, but not nuclear, mRNA levels. C2C12 cells were
stably co-transfected with Gm(40-262)SVpARI and
Gm434SVpA. Cytoplasmic (C) and nuclear (N) RNA
were extracted from preconfluent cells (P) and cells induced
to differentiate for 36 h in differentiation media (D)
that were untreated or exposed to cycloheximide for 3 h.
Comparative levels of mRNAs from the -globin-MYC
genes were determined by RT-PCR+1, and the products were resolved on a
6% denaturing polyacrylamide gel. Autoradiographs display the
following RT-PCR+1 products: EcoRI-digested RT-PCR+1
products, lanes 1-8; undigested RT-PCR+1 products,
lanes 9-16 and 19-22. EcoRI-digested
RT-PCR+1 products were spiked with an EcoRI cutting control
prior to digestion. Digested and undigested EcoRI cutting
control are shown in lanes 17 and 18,
respectively. Results of PCR+1 amplification of RNA obtained from both
untreated cells and cells treated with cycloheximide (B)
(i.e. not reverse-transcribed) are shown in lanes
19-22. EcoRI and RT-PCR+1 cut (Ct) and
uncut (U) products are labeled. Treated and untreated are
designated as (+) and ( ), respectively.
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DISCUSSION |
Regulation of expression of the c-myc proto-oncogene
occurs through posttranscriptional mechanisms in many cell lines
(13-18), and its down-regulation is thought to be a critical
determinant in cell differentiation (15, 16, 28-32). We previously
demonstrated that coding elements in myc exons 2 and 3 are
necessary for myc mRNA down-regulation during
differentiation of C2C12 myoblasts and sufficient to confer
down-regulation on globin mRNA (21). Studies presented here examine
the mechanism by which this occurs. The results show that function of
these regulatory elements depends on their translation. Because
function of the myc regulatory elements is dependent on
their own translation, we reasoned that a pharmacologic inhibitor of
translation, like cycloheximide, should also block the function of
translation-dependent regulatory elements, resulting in
increased mRNA levels in differentiating cells. This was confirmed when levels of globin-MYC fusion mRNAs containing coding
sequences from either MYC exon 2 or 3 were induced by
cycloheximide in differentiating C2C12 cells but not in
undifferentiated myoblasts. Cycloheximide inducibility was not seen in
globin fusion mRNAs containing MYC 3'-UTR or CAT coding
sequences and, therefore, could not have resulted from cycloheximide
effects on transcription, globin mRNA metabolism, or SV40
polyadenylation function. The importance of myc exon 2 and 3 regulatory sequences in conferring cycloheximide inducibility in
differentiating C2C12 cells was confirmed by demonstration that
deletion of exon 2 or 3 regulatory sequences from MYC
mRNA abolished inducibility, whereas inducibility was unaffected in MYC mRNAs in which 5'- and/or 3'-UTR sequences were
deleted. Thus, using two independent approaches, the function of the
elements in myc exons 2 and 3 that mediate mRNA
down-regulation during differentiation was shown to be dependent on
translation. Although translation in cis is clearly
necessary for down-regulation, our results do not rule out the
possibility that short-lived trans-acting factors are also
involved in the regulation of myc mRNA.
The translation dependence of exon 2 in mediating down-regulation
during C2C12 differentiation contrasts with the translation independence of another regulatory function ascribed to exon 2. Morello
and co-workers (33) have suggested that myc exons 2 and 3 contain independent elements that posttranscriptionally modulate
c-myc mRNA levels in transgenic mice based on
tissue-specific expression levels and mRNA inducibility in liver
regeneration and after inhibition of protein synthesis with
cycloheximide. In their model, regulation conferred by exon 2 sequences
was independent of its own translation (34). A different mechanism
targeting exon 2 sequences for regulation is suggested by these studies which is difficult to reconcile with that suggested by our studies except that there are obvious differences in experimental systems. Thus, exon 2 sequences may play multiple roles in posttranscriptionally regulating myc mRNA levels, one under conditions of cell
differentiation and another in hepatic regeneration and determining
tissue-specific mRNA levels.
The mechanism by which c-myc mRNA is down-regulated
during C2C12 differentiation involves accelerated mRNA decay as
demonstrated by analyses of cycloheximide inducibility of
MYC and globin-MYC fusion mRNAs. Treatment of
cells with cycloheximide or other protein synthesis inhibitors has long
been known to stabilize mRNAs encoded by early response genes
resulting in induction in their levels (35-37). It has been presumed
that stabilization of these mRNAs results from inhibition of
translation, thus implying that decay of these mRNAs is coupled to
translation, either directly or indirectly by the requirement of a
short-lived trans-acting factor involved in mRNA decay.
However, no studies have directly examined the mechanism by which these
agents stabilize mRNAs, and translation inhibitors can have
pleiotropic effects on cell metabolism (35-38). Studies here show that
increased transcription does not account for mRNA cycloheximide
inducibility (see above and Ref. 19); therefore, the increase in levels
of mRNAs containing myc exon 2 and 3 sequences after
cycloheximide is best explained by stabilization of previously unstable
mRNAs. Kinetic considerations indicate that the rapidity of and
fold increase in the level of an mRNA following its stabilization
are a function of its turnover rate prior to stabilization (see
"Experimental Procedures"). This suggests that mRNAs containing
myc exon 2 or 3 sequences are very rapidly turned over in a
translation-dependent manner in differentiating C2C12
cells. Coupled with the observation that those mRNAs containing MYC exon 2 or 3 sequences that are cycloheximide-inducible
are also down-regulated during C2C12 differentiation, it is reasonable to think of these regulatory elements as conditional (i.e.
differentiation-associated), translation-dependent,
mRNA instability determinants. Analyses of nuclear and cytoplasmic
mRNA levels demonstrated these elements mediate accelerated
mRNA decay in the cytoplasm.
The 3-10-fold decrease in c-myc mRNA levels observed
during C2C12 differentiation would predict a 3-10-fold increase in the rate of its decay if accelerated mRNA turnover is entirely
responsible for its down-regulation. The difference in cycloheximide
inducibility of MYC and globin-MYC mRNAs seen
in undifferentiated cells compared with differentiating cells strongly
suggests that accelerated mRNA decay does account for the
3-10-fold myc mRNA down-regulation. If one assumes no
change in transcription rates and complete mRNA stabilization after
cycloheximide, the magnitude of increase in mRNA levels over time
can be used to calculate mRNA half-lives (see "Experimental
Procedures"). Although these assumptions demand caution in applying
this calculation, we believe it likely that all of the constructs
tested would be affected equally by any changes in transcription (since
they are all transcribed from the same promoter/enhancer elements), and
there is little reason to think that cycloheximide would stabilize
their mRNAs to differing extents. Therefore, the magnitude of
change in cycloheximide inducibility seen with different states of cell
differentiation should accurately reflect the magnitude of change in
mRNA half-life. The 3-6-fold greater cycloheximide inducibility
conferred by MYC exon 2 and 3 regulatory elements in
differentiating cells compared with undifferentiated cells suggests
that they confer approximately a 3-6-fold increase in mRNA
turnover rates, and thus account for the magnitude of mRNA
down-regulation. Confirming this suggestion, the magnitude of
cycloheximide inducibility of c-myc mRNA was found to
increase 4-6-fold when undifferentiated cells were compared with
differentiating cells.
The suggestion that accelerated mRNA decay mediates
c-myc mRNA down-regulation contrasts with studies of the
decay rate of c-myc mRNA using actinomycin D. Results of
these studies suggest that myc mRNA decay is only
modestly accelerated in differentiated C2C12 cells and not to an extent
that would explain the 3-10-fold decrease in steady-state mRNA
levels (17). Actinomycin D studies and analyses of cycloheximide
inducibility of c-myc mRNA predict similar half-lives
for c-myc mRNA (approximately 15-30 min) in undifferentiated C2C12 cells. However, they yield different results in
differentiating C2C12 cells with actinomycin D studies predicting a
longer half-life of c-myc mRNA than do cycloheximide
studies. The myc mRNA half-life predicted by actinomycin
D studies in differentiating cells is likely artifactually long since
actinomycin D interferes with the destabilizing function of
myc exon 3 coding sequences (19) which is necessary for
myc mRNA down-regulation, and its effects on
myc exon 2 regulatory function are unknown. Studies presented here analyzing induction in mRNA levels after
cycloheximide are unlikely to yield artifactual results because they
reflect mRNA turnover rates prior to the addition of pharmacologic
agents, and the mechanism of stabilization will not affect the kinetics of induction.
The mechanism by which exon 2 and 3 coding elements target
c-myc mRNA for translation-dependent
accelerated turnover during differentiation remains to be elucidated. A
number of other mRNAs have been shown to be targeted for decay by
protein coding region instability determinants. Like c-myc
mRNA, c-fos (2), and
-interferon (39) mRNAs have
been shown to contain independent instability determinants in both
their protein coding domains and their 3'-UTRs. It remains to be
determined whether the c-fos and
-interferon elements are
simply redundant destabilizing elements or whether, like the
c-myc coding region and 3'-UTR elements, they destabilize the mRNA under different conditions. Yeast MAT
1 (40) and
mammalian
-tubulin (5) mRNAs have also been shown to contain
instability elements in their coding sequences. Of these various coding
region instability determinants, the mechanism of recognition has been determined only for
-tubulin mRNA in which auto-regulation of
-tubulin mRNA levels depends on translation of its first four codons. Excess free
-tubulin monomers target
-tubulin mRNA
for accelerated turnover by recognition of the encoded amino acids rather than the RNA sequence or structure (5). Our results have not
excluded primary or secondary structure of the myc
regulatory elements or the amino acid sequence encoded as the feature
targeting c-myc mRNA for down-regulation. The stability
of these and many labile mRNAs has been shown to be coupled to
translation (for review, see Ref. 41). With mRNAs other than
-tubulin mRNA, the mechanism coupling translation to mRNA
decay is unclear. Several models have been suggested including the
following: (i) association of nucleases or other proteins involved in
RNA decay with the translation machinery; (ii) disruption by transiting
ribosomes of RNA secondary structural elements important in RNA
stability; (iii) dislocation of proteins involved in RNA stability by
transiting ribosomes; or (iv) localization of the RNA to the
subcellular region involved in RNA decay by the translation machinery
(41). Our results do not exclude any of these possibilities as the
mechanism by which translation targets c-myc mRNA for
accelerated decay during differentiation.
In conclusion, we demonstrated that function of the exon 2 and exon 3 elements that mediate posttranscriptional down-regulation of
c-myc mRNA during C2C12 differentiation is dependent on
their translation. We have demonstrated that these elements mediate down-regulation through accelerated turnover of cytoplasmic, but not
nuclear, mRNA. Furthermore, the translation-dependent
destabilizing function of these elements is conditional, destabilizing
the mRNA in differentiating C2C12 cells but not in undifferentiated
myoblasts. Thus, c-myc mRNA is regulated
posttranscriptionally by a variety of mechanisms. Under normal growth
conditions, c-myc mRNA is maintained at low steady-state
levels by translation-independent instability elements within the
3'-UTR (20). When C2C12 cells differentiate into multinucleated
myotubes, c-myc mRNA is down-regulated through a
translation-dependent mechanism targeting elements in the
coding regions of exons 2 and 3 that function to destabilize the
mRNA during differentiation but not in undifferentiated cells.
These two mRNA regulatory pathways do not appear to interact and
seem to function independently of each other. Thus, deletion of coding sequences in exons 2 and 3 does not affect steady-state myc
mRNA levels under growth conditions, and deletion of myc
3'-UTR sequences does not affect down-regulation of c-myc
mRNA during C2C12 differentiation. Their effects are
superimposable, however, so that 3'-UTR sequences contribute to low
steady-state myc mRNA levels during differentiation even
though they do not confer regulation. Moreover, we have not ruled out
the possibility that myc 3'-UTR-containing mRNAs reach lower steady-state levels in differentiating C2C12 cells than mRNAs
without myc 3'-UTR sequences since their steady-state levels are lower prior to differentiation. Future studies will attempt to
localize and define the elements in exons 2 and 3 targeting myc mRNA for down-regulation and identify the
trans-acting factors that mediate its down-regulation.