c- myc mRNA Is Down-regulated during Myogenic Differentiation by Accelerated Decay That Depends on Translation of Regulatory Coding Elements*

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 b -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-reg-ulation 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

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 3Ј-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 3Ј-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 3Ј-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 stemloop structures in its 3Ј-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)(14)(15)(16)(17)(18) mechanisms. A decrease in c-myc mRNA expression is seen when cells are induced to differentiate (13)(14)(15)(16)(17)(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 3Ј-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 downregulation during differentiation. In studies presented here, we characterize the mechanism by which c-myc expression is post-transcriptionally 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.

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% CO 2 , penicillin, and streptomycin. Plasmids containing test genes were stably co-transfected into C2C12 cells with a plasmid containing a neo r gene using the calcium phosphate method. After selection in 400 g/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 (3Ј 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 3Ј 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.
Construction of the ␤-globin-MYC plasmids, ␤Gm434SVpA, ␤Gm434-MYCpA, ␤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 3Ј-UTR and uses the SVpA. The other genes were created from ␤Gm434SVpA with the following modifications: in ␤Gm434MYCpA, the entire MYC 3Ј-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. ␤Gm-263SVpARI Ϫ and ␤Gm(40 -262)SVpARI Ϫ 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 g/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 2ϩ3 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 ϩ 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 halflives. 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, k s , and its half-life, t1 ⁄2 , and can be calculated as shown in Equation 1.
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, t x , as shown in Equation 2.
Under these conditions, the fold induction of a given mRNA species, [mRNA] x Ϭ [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.
RT-PCRϩ1 Assay of Comparative mRNA Abundance-The RT-PCRϩ1 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 MgCl 2 , 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, ␤G3ЈRev (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 (NH 4 ) 2 SO 4 , 1.5 mM MgCl 2 , 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 ␤G3ЈRev (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 "ϩ1" cycle was conducted with a 32 P-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-PCRϩ1 products. The relative abundance of the RT-PCRϩ1 products (and, hence, of the mRNAs) was quantitated on a Molecular Dynamics PhosphorImager (Sunnyvale, CA) using ImageQuant software.

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 5Ј-and 3Ј-UTR sequences and introns are dispensable.
Translation of Myc Exon 3 Is Required for Myc mRNA Downregulation 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 5Ј-and 3Ј-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 downregulation 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.
The Order of Myc Exon 2 and 3 Regulatory Sequences Affects , 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 ϩ 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 ϩ 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.
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 5Ј 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 3Ј-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 5Ј 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.

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 ␤Gm263-SVpA 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 . 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.  1 and 2) and cells induced to differentiate for 36 h in differentiation media (lanes 3 and 4) that were untreated (Ϫ) or exposed to cycloheximide (CHX) for 3 h (ϩ). 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 ϩ 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 ϭ (c-myc mRNA level in cycloheximide-treated cells Ϭ c-myc mRNA level in untreated cells) Ϭ (rpL32 mRNA level in cycloheximide-treated cells Ϭ rpL32 mRNA level in untreated cells). 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 ␤Gm263-SVpA 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.
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 3Ј-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 3Ј-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:  (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,butnot5Јor3Ј-UTRsequences,arenecessaryfortranslationdependent accelerated c-myc mRNA decay during C2C12 differentiation.
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)Term-SVpA 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.
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.
Accelerated mRNA Decay during Differentiation Affects Cytoplasmic but Not Nuclear mRNA Levels-Previous studies demonstrated that myc exon 2-or 3-mediated mRNA downregulation 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 Ϫ , which encodes an mRNA that is induced by cycloheximide in differentiating cells, and ␤Gm434SVpA, which encodes a noninducible mRNA. ␤Gm(40 -262)SVpARI Ϫ 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-PCRϩ1 analysis, the comparative level of spliced ␤Gm434SVpA:␤Gm(40 -262)-SVpARI Ϫ 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 Ϫ 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 Ϫ 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 Ϫ 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).  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 (Ϫ) or exposed to cycloheximide for 3 h (ϩ). 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 ϩ 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 ϩ 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 ϭ (c-myc mRNA level in cycloheximide-treated cells Ϭ c-myc mRNA level in untreated cells) Ϭ (rpL32 mRNA level in cycloheximide-treated cells Ϭ rpL32 mRNA level in untreated cells).

FIG. 7. Cycloheximide (CHX) induction of c-myc mRNA in-
creases 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 ϭ (c-myc mRNA level in cycloheximide-treated cells Ϭ c-myc mRNA level in untreated cells) Ϭ (rpL32 mRNA level in cycloheximide-treated cells Ϭ rpL32 mRNA level in untreated cells).

DISCUSSION
Regulation of expression of the c-myc proto-oncogene occurs through posttranscriptional mechanisms in many cell lines (13)(14)(15)(16)(17)(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 3Ј-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 5Ј-and/or 3Ј-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)(36)(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)(36)(37)(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 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 Ϫ 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-PCRϩ1, and the products were resolved on a 6% denaturing polyacrylamide gel. Autoradiographs display the following RT-PCRϩ1 products: EcoRI-digested RT-PCRϩ1 products, lanes 1-8; undigested RT-PCRϩ1 products, lanes 9 -16 and 19 -22. EcoRI-digested RT-PCRϩ1 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 PCRϩ1 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-PCRϩ1 cut (Ct) and uncut (U) products are labeled. Treated and untreated are designated as (ϩ) and (Ϫ), respectively. 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 cmyc 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 downregulation, 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 3Ј-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 3Ј-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 downregulation 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 3Ј-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 3Ј-UTR sequences does not affect down-regulation of c-myc mRNA during C2C12 differentiation. Their effects are superimposable, however, so that 3Ј-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 3Ј-UTR-containing mRNAs reach lower steadystate levels in differentiating C2C12 cells than mRNAs without myc 3Ј-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.