Vol. 7, 1415-1424, October 1996 Cell Growth & Differentiation 1415

Elements in the Murine c-mos Messenger RNA 5’-Untranslated Region Repress Translation ofDownstream Coding Sequences1

Laura F. Steel,2 Diane L Telly, Jackie Leonard,Bobbie A. Rice, Bob Monks, and Janet A. SawickiKimmel Cancer Institute, Thomas Jefferson University, Philadelphia,Pennsylvania 19107 [L F. S., J. L], and Lankenau Medical ResearchCenter, Wynnewood, Pennsylvania 19096 [0. L T., B. A. A., B. M.,J.A.S.]

Abstract

Munne c-mos transcripts isolated from testes have 5’-untranslated regions (5’UTRs) of -300 nucleotides witha series of four overlapping open reading frames(ORFs) upstream of the AUG codon that initiates theMos ORF. Ovarian c-mos transcripts have shorter5’UTRs (70-80 nucleotides) and contain only 1-2 of theupstream ORFs (uORFs). To test whether these 5’UTRsaffect translational efficiency, we have constructedplasmids for the expression of chimenc transcriptswith a mos-derived 5’UTR fused to the Escherichia coil

fi-galactosidase coding region. Translational efficiencyhas been evaluated by measuring f3-galactosidaseactivity in NIH3T3 cells transiently transfected withthese plasmids and with plasmids where variousmutations have been introduced into the 5’UTR. Weshow that the 5’UTR characteristic of testis-specificc-mos mRNA strongly represses translation relative tothe translation of transcripts that contain a 5’UTRderived from �-globin mRNA, and this is mainly due tothe four uORFs. Each of the four upstream AUG tripletscan be recognized as a start site for translation, andno single UAUG dominates the repressive effect. TheuORFs repress translation by a mechanism that is notaffected by the amino acid sequence in the COOH-terminal region of the uORF-encoded peptides. Thevery short uORF (AUGUGA) present in ovary-specifictranscripts does not repress translation. Staining oftestis sections from transgenic mice carrying chimericfi-galactosidase transgene constructs, which contain amos 5’UTR with or without the uATGs, suggests thatthe uORFs can dramatically change the pattern ofexpression in spermatogenic cells.

Received 6/27/96; revised 7/24/96; accepted 7/29/96.The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1 734 solely to mdi-cate this fact.1 This work was supported by Grant NP-859 from the American CancerSociety.2 To whom requests for reprints should be addressed, at Kimmel CancerInstitute, Thomas Jefferson University, 1 020 Locust Street, Philadelphia,PA 19107; Phone: (215) 503-7953; Fax: (215) 923-7144; E-mail:L_Steel@Iac.jci.tju.edu.

IntroductionThe c-mos proto-oncogene is the cellular homologue of thetransforming gene of the Moloney murine sarcoma virus. It

encodes a serine/threonine protein kinase (1) that partici-pates in the control of the meiotic cell cycle during vertebrate

oogenesis (2). Although low levels of mos transcripts havebeen reported in some somatic tissues, the highest levels ofaccumulation are detected in germ cells (3-5). The functionof Mos is best characterized during oogenesis where, inmice, its activity is required for the arrest of meiosis atmetaphase II prior to fertilization ofthe egg (2, 6, 7). Althoughmos transcripts have been found to accumulate in develop-ing spermatids, the role of Mos during spermatogenesis isless well understood. Mos function is apparently not essen-tial for either sperm or somatic cell development, sincemos� knockout mice are viable and males are fertile (6, 7).Nevertheless, inappropriate expression of mos in somaticcells has serious consequences and can result in cellulartransformation or death (8, 9). In addition, expression ofv-mos in premeiotic spermatogenic cells causes meiotic ar-

rest and male sterility (10).Regulation of mos expression has been demonstrated at

many levels, including tissue- and stage-specific transcrip-tion, and translation and stability of Mos mANA and protein.

In developing oocytes, mos is transcribed during the growthphase but remains translationally inactive until later, whenfully grown oocytes are hormonally stimulated to undergomeiotic maturation (4, 1 1 , 12). In oocytes of both Xenopusand mice, Mos mANA is translationally activated by cyto-plasmic polyadenylation of stored transcripts (1 3, 14). Once

activated, the mANA is quickly destabilized and is undetect-able by the two-cell stage of embryonic development (11,1 3). In developing sperm cells, Mos mRNA accumulation hasbeen detected in postmeiotic round spermatids (3-5, 15).The timing of Mos mRNA translation during spermatogenesishas not been studied, but an analysis of its distribution inpolysomes isolated from adult testes suggests that its overalltranslational efficiency is low (3).

Tissue-specific transcriptional start sites in the intron-freemurine c-mos gene result in testicular and ovarian mANAsthat differ in the length and structure of their 5’UTRs3 (3),suggesting that there may be different requirements for ac-tive translation of the testis- and ovary-derived Mos mANAs.The 5’UTR of mos transcripts isolated from testes containsfour AUG triplets that initiate a series of overlapping OAFsupstream of the AUG codon that initiates the Mos OAF, but

3 The abbreviations used are: UTR, untranslated region; OAF, open read-ing frame; u, upstream; CAT, chloramphenicol acetyltransferase; nt, nu-cleotide(s); wt, wild type.

a

-323 .253 -81 -53143

MOe ATG

�RF1

1416 Translational Repression by the c-mos mRNA 5’UTh

testis specific transcriptionalstart sites

ovary specific transcriptionalstart sites

_gI�., Ar � .� � /7� �

J�, �, .1

t�RF3

bMos ATG GGTGThATGC

-26 -21

t%DRF4

uATG].

uATG2

uATG3

UATG4

G�ACAGATGT

�CTGGGATGA

T�A�CIATGC

TGTCTQ�ATGT

Fig. 1. Structure of the murine c-mos 5’UTR. a, organization of transcriptional start sites and uORFs in the genomic region upstream of the murine Moscoding sequence. Hatched boxes with arrows, regions where testis- and ovary-specific transcriptional start sites have been identified, as discussed in thetext. The location of four uORFs, each initiated by a UATG and terminated prior the start of the Moe coding region, is depicted by striped boxes below thegene. The diagram is based on sequence data from Wood et a!. (50) and GenBank accession no. J00372. b, sequence context surrounding the ATGs.Nucleotides around each ATG that match the optimal context (24) are underiined, with matches at more important positions double underlined.

most of these uOAFs are absent from the shorter ovarianmos transcripts. Our current understanding of translationalinitiation predicts that the uORFs may seriously impedetranslation of testis-specific Mos mRNA (16), resulting in itsapparently inefficient translation. However, uORFs do notalways inhibit translation. For instance, both omithine decar-boxylase and c-sis mRNAs have 5’UTRs that strongly re-press translation (1 7-1 9), but uOAFs present in their 5’UTRscontribute little or nothing to the repression (1 7, 20).

In the work reported here, we have examined the effects ofthe uORFs present in murine c-mos transcripts on the trans-lation of downstream coding sequence to begin to defineboth whether and how they present a barrier to efficienttranslation. Our experiments show that the uORFs in the MosmRNA 5’UTR strongly repress translation in cultured fibro-blasts. Furthermore, evidence is presented suggesting thatthe uOAFs can dramatically alter the expression of a reportergene in spermatogenic cells.

ResultsStructure of the c-mos mRNA 5’UTR. Tissue-specific

transcriptional start sites lead to the production of Mos mA-NAs 1 .7 kb in size in spermatocytes and 1 .4 kb in oocytes (3,21). In both cases, the transcripts are intron free and termi-

nate at the same 3’ site (3). Testis-specific transcriptionalstart sites have been mapped by both nuclease protectionand primer extension to a region centered approximately 270bp upstream of the AUG codon that initiates translation ofthe Mos protein (3). We have obtained similar results (datanot shown) by sequencing several isolates of recombinantplasmids generated by 5’ rapid amplification of cDNA ends(22) to map testis-specific transcriptional start sites at posi-tions between -253 nt and -323 nt relative to the Mos AUGcodon. Ovary-specific transcriptional start sites have previ-ously been mapped to a region approximately 50-70 bpupstream of the Mos AUG (3, 23). Our sequence analysis offour independent plasmids generated by 5’ rapid amplifica-tion of cDNA ends indicated an ovary-specific start site atposition -81 (data not shown).

The longer 5’UTR of Mos mANA found in spermatocytesincludes several sequence elements that could have signif-icant effects on the translational efficiency of that mANA. Asshown in Fig. la, the Mos coding region is preceded by fouruAUGs that initiate a series of overlapping uOAFs in theregion from -143 to -8. The first three of these uOAFsencode peptides of 36, 22, and 21 amino acids, respectively,and the fourth is an AUG immediately followed by a UGAstop codon. The uAUGs, and in particular uAUG2 and

a

b

% �-gaI activity

3.6 ±0.7

AUG

I -2 3 -4 � n-gal tst-gal

frLx3

-4 n �-gaI ov-gal

� gb-gal

% mRNA level±SE.

41.3 ±5.9

372 ±62 98.9 ±21.7

100 100

100�

�,e0.

0� H

glo�gal 1st-gal ov.gal

Cell Growth & Differentiation 1417

Fig. 2. j3-Galactosidase activity andmRNA levels from chimeric mos-gal andgb-gal transcripts in transiently trans-fected cells. In a, chimeric transcripts areshown with a narrow line representing se-quence from the mos 5’UTR and numbersshowing the position of each of the fouruAUGs. A crookedllne represents (3-globin5’UTR sequence. The coding region isshown by a box, with the stippled orstriped area at the beginning representingthe first few codons encoding Mos or(3-globin, respectively. The remainder ofthe coding region is (3-galactosidase in allcases. f3-Galactosidase activity was meas-ured in transiently transfected cells andvalues (± SE) are given relative to thosemeasured in pglo-gal transfectants. Chi-meric mRNA levels were determined byNorthern blotting with quantitation using aPhosphorlmager, and results are pre-sented relative to mRNA levels in pglo-galtransfectants. The data are from four mdc-pendent series of transfections. In b, rela-tive (3-galactosidase activity levels havebeen normalized by dividing by the relativemRNA level and multiplying by 100 foreach set of transfected cells.

uAUG3, are found within a sequence context that is at leastas strong for translational initiation (24) as the Mos AUG (Fig.lb). Although all four of the uOAFs are present on testis-

specific Mos mANAs, only uOAF4 and perhaps uORF3 arepresent on ovarian mos transcripts, depending on the startsite of transcription. Computer modeling (F0IdANA in theGCG Package; Ref. 25) indicates that the 5’UTA of testis-specific Mos mANA, with a G/C content of 48%, can befolded into a structure composed of several domains with anoverall free energy close to -90 kcaVmol. The shorter ovary-specific 5’UTR shows no significant secondary structure.Although both uORFs and secondary structure have thepotential to diminish the translational efficiency of mRNAs,we have focused our initial studies of the mos 5’UTR on theeffects of the uOAFs in modulating the translation of down-stream coding sequences.

Inhibition of Translation by the Mos mRNA 5’UTR. Wehave tested the effects of the uOAFs on translational effi-ciency by generating a series of plasmids in which the SV4Oearly promoter drives the expression of mos-gal chimerictranscripts, where the j3-galactosidase coding region is pre-ceded by 5’UTR sequence representative of either testis- orovary-specific mos transcripts (Fig. 2). In the plasmid ptst-gal, 354 bp of mos 5’UTR sequence has been placed up-stream of p-galactosidase coding sequence, whereas theplasmid pov-gal contains 65 bp of mos 5’UTA. The mos andfJ-galactosidase sequences have been joined after the fifthcodon of Mos coding sequence so that an extended MosAUG codon context is retained. The translational efficiency of

transcripts from these plasmids has been evaluated bymeasuring p-galactosidase activity in transiently transfectedNIH 3T3 cells. �3-gaIactosidase activity has been normalizedto CAT activity from cotransfected plasmids to control forvariations in transfection efficiency. An additional plasmid,pglo-gal, contains 5’UTR sequence from the human p-globingene and serves as a reference for comparison of f3-galac-tosidase activities measured in each series of transfections.

Reduced levels of �3-galactosidase activity were measuredin cells transiently transfected with plasmids containing ei-ther testicular or ovarian mos 5’UTR sequence relative tothose transfected with plasmids containing f3-globin 5’UTAsequence (Fig. 2). The longer testis-specific 5’UTR had themost dramatic effect, reducing activity essentially to back-ground levels, whereas the ovary-specific 5’UTR reducedactivity by -60%. RNA was isolated from transfected cells,and the level of chimeric �-galactosidase mANA present wasanalyzed by Northern blotting. Although there was a consist-ent reduction of 50-60% in the level of mANA accumulatedin ptst-gal transfectants relative to pglo-gal transfectants,this does not account for the 96% reduction in �-gaIacto-sidase activity. When the enzyme activity measurements arecorrected for the lower levels of mANA accumulation (Fig.

2b) it is evident that tst-gal transcripts are translated only-1 0% as efficiently as gb-gal transcripts. The level of chi-meric mANA detected in pov-gal transfectants was compa-rable to pglo-gal controls, and activity measurements nor-malized for mANA levels show that these transcripts aretranslated -40% as efficiently as gb-gal transcripts. We

AUG

-4 fl.��-gal ov.gal

AUG

_x�1 �-gaI ov/O-gal

b

37.8 ±6.7 87.3 ±16.9

Fig. 3. Effect of removing theuAUGs in chirneric tst-gal and

49.8 ±3.1 108.9 ±17.1 ov-gal transcripts. In a, chimerictranscripts are represented asdescribed for Fig. 2 with the po-sition of AUG to �4AG mutationsat each uAUG noted by an X.Data are from five independentseries of transfections for thetst-gal plasmids and four mdc-pendent transfections for theov-gal plasmids. In b, data arecorrected for relative mRNA 1ev-els, as for Fig. 2b.

r�HHH20�

gb-gal ‘ 1st-gal tSt4I-gal ov-gaI ov/0-gal

1418 TranslationalRepresskn by the c-mos rnRNA 5’UTR

aAUG

1 -2 3 -4 .b �.gaI

x-x x-x-� n-gal

100�

80�

J40.

t St . 9 a I

tSt/0 -gal

% n-gal activity±S.E.

2.5 ±0.6

18.0 ±3.8

% mRNA level±S.E.

40.6 ±7.8

53.1 ±6.1

conclude from these results that the testis-specific mos

5’UTR severely reduces translation of the chimeric mos-gal

transcript in transfected cells, whereas the shorter ovary-

specific 5’UTR is also inhibitory, but to a lesser extent. It is

possible that the sequence included as 5’UTR in the ptst-gal

plasmid also has some effect on the transcription of this

chimeric gene, although we have not yet ruled out transla-

tion-coupled effects on the stability of the transcript to cx-

plain the lower levels of RNA accumulation.

Elimination of All Four uAUGs Partially Restores Trans-lational Activity. To test whether the uORFs present in the

chimeric mos-gal transcripts are responsible for translational

repression, we used site-directed mutagenesis to eliminate

all of the uATGs (ATG to AAG), generating the plasmids

ptst/O-gal and pov/O-gal. The results in Fig. 3 indicate that

cells transiently transfected with ptst/0-gal showed a sub-

stantial increase in �3-galactosidase activity (-7x) relative to

ptst-gal transfectants. However, activity was still less than

20% of that detected in pglo-gal transfectants. Correcting for

the lower mRNA levels found in ptst/0-gal transfectants,

f3-galactosidase activity was -35% of that measured in

pgbo-gal transfectants (Fig. 3b). Cells transfected with the

pov/O-gal plasmid showed a slight increase in �-galactosid-

ase activity relative to pov-gal transfectants, but this wasparalleled by a similar increase in mRNA levels. Both pov-gal

and pov/O-gal transfectants showed reduced f3-galactosid-ase activity when compared to pglo-gal transfectants (Fig. 3).

These results indicate that uORFs 1-3 do repress translation,

but other features of the 5’UTR could also play a role. The

sequence AUGUGA that constitutes uORF4 does not affect

the translational efficiency of ov-gal transcripts.

In all experiments, relative translational efficiencies of dif-

ferent transcripts have been determined by comparison to

transcripts with a 5’UTR derived from human �-gbobin. It is

important to note that in the pglo-gal reference plasmids, the

ATG that initiates the p-gal coding sequence is derived from

the p-globin sequence and is in a very strong context for

initiation (CACCATGG). The Mos AUG context, on the other

hand, contains a purine (G) at the important -3 position butis otherwise slightly suboptimal. We have tested whether this

difference accounts for our inability to regain full translational

activity when uAUGs are removed from mos-gal transcripts.We have found that improving the context surrounding the

Mos AUG increases the translational activity of all transcripts

(with or without uAUG5) by approximately 2-fold. Therefore,

ov-gal, ov/0-gal, and gb-gal transcripts are translated withequal efficiency when the Mos AUG context is improved.However, tst/O-gal transcripts are translated approximately

2-fold less efficiently than gb-gal transcripts, even with an

improved Mos AUG context (data not shown).

uAUG1 , uAUG2, and uAUG3 Each Contribute to Trans-lational Repression. A series of plasmids was derived from

ptst-gal to test the ability of each individual uAUG to repress

translation in the absence of other uAUGs. In each plasmid,

three of the uATGs were eliminated by site-directed mu-tagenesis, while one was left intact. Cells transiently trans-fected with these plasmids were analyzed as above. Results

presented in Fig. 4 indicate that transcripts that retain only

x-x X-4 �jJ�gal

Fig. 4. Inhibition of translationby individual uAUGs in tst-galtranscripts. In a, chimenc tran-scripts are represented as de-scribed in Figs. 2 and 3. [3-Ga-lactosidase activity wasmeasured in three sets of trans-fections, and mRNA levels weredetermined for one of thosesets. In b, (3-galactosidase activ-ity is normalized using enzymeactivity and mRNA levels deter-mined from the same transfec-tion experiment.

b

54.1

58.1

64.3tst(+ATG4)gal 15.0 ±2.2

100#{149}

�

- �HHHH�‘ �t � �‘ .�‘ �� � eg C’)

� �2 �2 P‘C ‘C < <

+ + + +�. �. �. �.2� � � Mi

Cell Growth & Differentiation 1419

a

1 -X x-x31 0-gal

X-2 XXfjJ�gal

x-x 3 -X�fi��al

uAUG1 , uAUG2, or uAUG3 are translated at levels interme-

diate between tst-gal and tst/0-gal transcripts. In three sep-

arate experiments, p-galactosidase activity levels were con-

sistently slightly higher than the background levels detected

in ptst-gal transfectants but not as high as those observed in

ptst/O-gal transfectants. In cells transfected with

ptst(+ATG4)-gal, however, j3-galactosidase activity levels

were similar to those in ptst/O-gal transfectants. This is con-

sistent with the results of Fig. 3, where uAUG4 in ov-gal

transcripts had little effect on translational activity.

It appears from these results that the overall translational

repression observed with the testis-specific mos 5’UTR se-

quence is a consequence of smaller inhibitory effects con-

tributed by each of the first three uAUGs individually. Furtherevidence that a single uAUG does not dominate the trans-

lational repression comes from experiments where each

uAUG was removed individually, leaving the others in place.

In these experiments, all �-gaIactosidase activities were at

background levels (data not shown), so removal of any single

uAUG does not release the translational repression.

Each uAUG Is Recognized as a Translational Start Site.To confirm that the uAUGs of the testis-specific mos 5’UTR

can be recognized by scanning ribosomes as translational

start sites, plasmids were constructed where f3-galactosid-

ase coding sequence was fused to the 5’UTR at the position

of each uATG, in turn. In each case, the remaining 5’UTR was

tst(+ATG1 )gal

% 0-gal activity

±5-a

7.6 ±0.4

tst(+ATG2)gal 9.7 ±1.6

tst(+ATG3)gal 6.1 ±1.6

% mRNA level

40.5

devoid of additional uATGs. �-Galactosidase activitiesmeasured in cells transfected with these plasmids showedthat a relatively high level of translational initiation is possibleat each of the uAUGs (Fig. 5). That is, nothing such asstructure or context precludes the scanning ribosome from

recognizing the uAUGs as authentic translation initiation

sites. The differences observed in activities derived fromeach of the plasmids are generally consistent with predic-

tions based on the sequence context preceding each of the

uAUGs (Ref. 24; Fig. ib). For instance, fusion of 13-galacto-sidase coding sequence to uATG4 results in the lowest ac-tivity, and uATG4 lies in the most unfavorable context. Fusionat uATG3, on the other hand, yields the highest levels of

13-galactosidase activity, and uATG3 lies in the best context.The relative strength of translation from each of the uAUGs is

also consistent with the slight differences noted (Fig. 4)among their abilities to repress translation when presentindividually in the 5’UTR. These results support the idea that,

in the presence of the mos 5’UTR, initiation occurs by a

conventional scanning mechanism, and each uAUG is eval-

uated by ribosomes as a potential start site.

Changes in the Amino Acid Sequence of the PeptidesEncoded by the uORFs Do Not Relieve Translational Re-pression. In some cases, uORFs present in the 5’UTR of

mammalian and viral mRNAs have been shown to mediate

translational repression by a mechanism that is sensitive to

1420 Translational Repression by the c-mos mRNA 5’UTR

aMos AUG

x-x x-x-� n-gal

uAUG1

-Ei�iIIIuA1�G2

x-I 0-gal

uA1�G3

x-x�1 �-gaI

uA1�G4

x-xxH �.gal

.�

I

b

ATGM-gal

uATGI -gal

uATG2-gal

uATG3-gal

uATG4-gal

% �-gaI activity

18.5

41.1

44.2

62.5

13.6

% mRNA level

48.9

53.8

46.2

56.1

56.1

C

0000

C) � :� � �

�, � �e � �

Fig. 5. Recognition of theuAUGs as translational startsites. In a, transcripts with [3-ga-lactosidase coding sequencejoined to the testis-specific MosmRNA 5’UTR at the position ofeach of the uAUGs or the MosAUG are depicted as in Figs. 2and 3. [3-Galactosidase activitywas measured in two sets oftransfections, and mRNA levelswere determined for one ofthose sets. In b, data are nor-malized using f3-galactosidaseactivities and mRNA levels fromthe same transfection experi-ment. c, Northern blot analysisof chimeric f3-galactosidasetranscript levels.

changes in the amino acid sequence of the uORF-encoded

peptide, particularly at the COOH terminus of the peptide

(26). We have used site-directed mutagenesis to introduce

changes in the COOH-terminal residues of both uORF1/2

and uORF3 to test two separate mutations at each of two

positions for each of these uORFs (Fig. 6a). �3-GaIactosidase

activity was measured after transient transfection of cultured

cells and normalized as described above. For these experi-

ments, the SV4O promoter of our previous reporter plasmids

was replaced with the stronger CMV/IE promoter and testis-specific 5’UTR sequence beginning at -325 (rather than

-354) was used (see “Materials and Methods”). Together,

these changes resulted in somewhat higher overall expres-

sion from the mos-gal transcripts. Nevertheless, as shown in

Fig. 6b, none of the uORF peptide coding mutations tested

led to a relief of the translational inhibition. That is, mutations

introduced into the COOH terminus of uORF1/2 did not result

in higher activity than that obtained from transcripts with the

wild-type uORF1/2 present in the 5’UTR. Similarly, mutations

that changed the amino acid sequence at the COOH termi-nus of uORF3 did not increase activity from levels obtained

from transcripts with the wild-type uORF3 in the 5’UTR. It is

unlikely, therefore, that the sequence of the uORF-encoded

peptides plays a role in the translational repression mediated

by these uORFs.

uORFs Can Alter the Pattern of Expression of a Re-porter Transgene in Developing Spermatogenic Cells.Although it is evident that the uORFs present in the 5’UTR of

testis-specific mos transcripts are potent inhibitors of trans-

lation in cultured fibroblasts, it is ultimately our interest to

discover if these elements act to regulate the timing or extent

of Mos mRNA translation during spermatogenesis. As part of

these investigations, we have constructed mouse strains

carrying chimeric mos-gal transgenes. Two different trans-

genes have been constructed, where the �-galactosidasecoding region is preceded by 325 nt of wild-type mos 5’UTR

sequence (wt-mos/gal) or by 325 nt of 5’UTR, where all

uATGs have been removed by point mutations (OATG-mos/

gal). In both of the transgenes, expression is driven by a

CMV/lE promoter to minimize cell- and tissue-specific dif-

ferences in transcription. Testes were removed from mice

expressing these transgenes, stained to detect f3-galacto-

sidase activity, and sectioned. Results shown in Fig. 7 mdi-cate that there are dramatic differences in the spermatogenic

cell types that express the two different transgenes. In OATG-

mos/gal testis sections, expression is detected in a broad

a

b

+ATG1/2:

-1 MWLVLRfl(EQGKGTGMKMIFSHAPKLPWLFLLISP I I �-pai

II-��i -

I I_,. A - p(+ATGI/2)B.gai

F-� T -

L�+ A - p(+ATGI/2)D-gai.ATG3:

I-..F � p(+ATG3)1-oaiI_.#{248}.L - p(+ATG3)2.gai

-� A - p(+ATG3)3-gai

T - p(+ATG3)4-gaI

100

C

In..H.H.nH.H

�ri

:ELj�Q5� (� � � � 0� � - c� c� � ,.,

&

‘i..

_4� �

:��‘ �� .4

.&‘ �., �, �3

C, < �-

a,.

.. .�. �a

.� -�c, .

:� �, � ,.�,- �a �1’

Cell Growth & Differentiation 1421

Fig. 6. Effects of changes inthe amino acid sequence ofuORF-encoded peptides. In a,the amino acid sequence of thepeptide encoded by uORF1/2and uORF3 is shown in theboxed region of separate dia-grams of the structure of the5’UTRs of (+ATG1/2)-gal and(+ATG3)-gal transcripts. For themutant plasmids, the sequencechange and position are shownbelow each diagram. In b and c,(3-galactosidase activities andmRNA levels were measured incells transfected with the mdi-cated plasmids and normalizedas in previous experiments.

�8O

�6O

� 40

� 20

�.

r�

Fig. 7. Testis sections from adult transgenic mice and control mouse stained for f3-galactosidase activity. a, wt-mos/gal transgenic. b, OATG-mos/galtransgenic. C, nontransgenic littermate of a wt-mos/gal mouse. All photographs are shown at the same scale, and the line in c signifies 100 pm.

range of spermatogenic cell types, with some concentration

of activity in the pre-meiotic spermatocytes located toward

the periphery of the tubules. In wt-mos/gal testis sections,

there is a very pronounced shift in staining to the more

luminal, postmeiotic spermatids. This pattern of expression

has been consistently observed in transgenic lines derived

from three different founders for each of the two transgenes.

No j3-galactosidase staining is observed in the germ cells of

testes from nontransgenic mice (Fig. 7c) although endoge-

nous f3-galactosidase activity is observed in Leydig cells of

somatic origin in both transgenic and nontransgenic mice.

Further analysis will be necessary to determine whether

these expression patterns reflect differences in translational

activity of transcripts from the transgenes.

DiscussionWe have shown that the 5’UTR associated with murine tes-

tis-specific c-mos transcripts severely inhibits the translation

of downstream coding sequences in transiently transfected

fibroblasts. This is due, in part, to a set of four overlapping

OAFs that are present in the region from -143 to - 8 nt in the

5’UTR. Removal of all four uAUGs improved translational

activity -7-fold, and activity could be increased a further

1422 Translational Repression by the c-mos mRNA 5’UTR

2-fold by improving the sequence context of the Mos AUG.

The shorter 5’UTR characteristic of ovarian Mos mRNAscontains only a single uAUG that is in a poor context and isimmediately followed by a UGA stop codon. This “uOAF”apparently has no effect on translational activity.

The strong translational repression imposed by the testis-

specific Mos mRNA 5’UTR raises the question of how trans-lation of c-mos transcripts, or similarly uOAF-burdened tran-scripts, is allowed when needed. In the simplest case, thiscould be explained by “leaky scanning” where ribosomespass uAUGs that are in a weak context, initiating at the firstAUG they find in a strong context. However, some of theuAUGs in the Mos mRNA 5’UTA are not in a weaker contextthan the Mos AUG, and our results show that each of the fouruAUGs can be recognized as a start site for translation.Initiation by leaky scanning, then, would require ribosomes tobypass four potential start sites to finally initiate at a site thatis not significantly different from the ones it passed. It ap-pears more likely that ribosomes do initiate at one of theuAUGs, and translation of Mos requires termination at theend of a uORF with subsequent reinitiation at the start of theMos OAF. This interpretation is consistent with a number ofearlier studies that have established that reinitiation is pos-

sible in eukaryotic cells if translation of a short uOAF isterminated prior to the downstream AUG start codon (27-30).

The efficiency of reinitiation of translation of eukaryoticmANAs can be affected by both the distance between auOAF and the downstream start site and by the nature of thesequence following the upstream termination codon (31 , 32).In the testis-specific Mos mANA, the close proximity of theuORFs to the Mos OAF leads to the prediction that reinitia-tion will be very inefficient, and that is supported by our data.It is also possible for the frequency of reinitiation to beaffected by the sequence of the peptide encoded by theuOAF (26). For instance, in both AdoMetDC mANA (33) andcytomegalovirus gp48 mANA (34), uOAFs located relatively

far upstream of the major OAF repress translation in mam-malian cells, but that repression can be abolished if changesare made in the amino acid sequence of the uORF-encodedpeptide, particularly at the COOH-terminal end. Our resultsshow that repression by the Mos mANA 5’UTA is not relievedby changing the sequence at the COOH terminus of thepeptide encoded by either uORF1/2 or uOAF3. In addition,

the overall level of repression results from contributions from

uOAFs 1 , 2, and 3 and is not dominated by a single uORF.We think it is unlikely that a specific uORF peptide sequenceplays a role in the translational repression.

A second question raised by the strongly repressiveuORFs is whether that repression is constitutive or regulated.For over 90% of mANAs of higher eukaryotes, the scanningmodel of translation initiation correctly predicts that proteinsynthesis will start at the 5’ proximal AUG triplet (35). Theobservation that many of the mANAs that initiate down-stream of one or more AUG triplets encode regulatory pro-teins or proto-oncogenes has led to the suggestion thatuAUGs or uORFs constitutively down-regulate the transla-tion of proteins whose overproduction could be deleteriousto cells (36). Support for this idea is found in experiments

showing that the activation of the oncogenic potential ofsome genes, including c-mos (8, 37-40) and c-Ick (41), can

be greatly enhanced by rearrangements that remove theuOAF-containing sequence upstream of the coding region.There is also evidence, however, that the repressive effectsof a 5’UTA can be overcome to allow expression of the geneproduct in specific cell types. For instance, elements in the5’UTR of c-sis mANA can strongly inhibit its translation, butthat repression is transiently relieved in differentiatingmegakaryocytes (42). In addition, there appears to be varia-tion in the ability of different cell types to translate someuOAF-containing mRNAs, including AdoMetDC mRNA (43,44) and retinoic acid receptor /32 mRNA (45).

Our results directly demonstrate that the uAUGs present inmurine testis-specific c-mos transcripts can serve to reducetranslational initiation at the Mos AUG in fibroblasts. It will be

of interest to learn whether these uOAFs act primarily toprovide protection from the inappropriate expression of c-mos in somatic tissues or whether they also mediate regu-latory events that control the timing and nature of translationof Mos protein in the testis. To begin an analysis of the rolethese elements play in regulating mos expression in devel-oping spermatocytes, we have generated transgenic mousestrains where chimeric 13-galactosidase transcripts carry theuOAF-burdened wt mos 5’UTR or the corresponding uORF-free “OATG” 5’UTA. These strains show dramatic differencesin the spermatogenic cell populations that accumulate frga-

lactosidase activity. Interestingly, in the wt-mos/gal mice,significant levels of f3-galactosidase activity are not detecteduntil cells have reached postmeiotic stages of development,where there is strong f3-galactosidase expression. TheCMV/lE promoter used in the transgene constructs can beactive at earlier spermatogenic stages, as demonstrated by

the accumulation of f3-galactosidase activity in premeioticcells in the testes of OATG-mos/gal mice. The wt-mos/galand OATG-mos/gal transgenes are identical, except for thepresence of the four uATGs in the mos 5’UTR, suggestingthat the differences could be due to changes in the ability of

cells to translate uORF-burdened mRNAs. However, other

explanations are clearly possible, and current experimentsare aimed at understanding the basis of the differential ex-pression of these transgenes during spermatogenesis.

Materials and MethodsCell Lines and MedIa. NIH 3T3 cells, purchased from American TypeCulture Collection, were maintained at low cell densities in 5% CO2.balance air, at 37*C in DMEM (4.5 g/liter glucose) and 1 0% newborn calf

serum.

Plasmid Constructions. All plasmids are derived from the vectorpGL2-Control (Promega), which contains the SV4O early promoter and

5V40 3’UTR, intron, and polyadenylation signal. PCR-based molecularcloning techniques were used to replace the luciferase coding region of

pGL2-Control with the coding region for E. coli (3-galactosidase, engi-neered to contain an XbaI site just upstream of the eighth codon. Se-quences to be tested as 5’UTRs were then inserted as HindIIl-Xbal frag-ments between the HindlIl site that lies just downstream of the SV4Opromoter in the vector and the Xbal site upstream of the (3-galactosidase

coding sequence. For the plasmid ptst-gal, PCR was used to copy se-quence from position -354 to +15 (the A of the major ORF ATG is ±1)

from the murine c-mos genomic plasmid pMSTX, bounded by Hindlll andXbal sites (pMSTX was the gift of Dr. G. Vande Woude, NCI-Frederick

Cancer Center, Frederick, MD). The plasmid pov-gal contains c-mos 5’

Cell Growth & Differentiation 1423

sequence from -65 to +15. Similariy, PCR was used to amplify 5’sequencefrom a human (3-globmn cDNA plasmid from position -50 to +8,

bounded by Hindlll and Xbal sites, for insertion between the SV4O pro-moter and (3-galactosidase coding sequence to generate the plasmidpglo-gal. In all of these plasmids, fusion with f3-galactosidase coding

sequence is just downstream of the major OAF ATG associated with the5’UTR sequence, so that ATG retains its original context.

The Hindlll-Xbal fragment containing testis-specific Mos mRNA 5’UTR

sequence from ptst-gal was subcloned into the vector pGEM7Zf(+) (Pro-mega) for subsequent mutagenesis at each of the uATGs. Oligonucleoti-

dc-directed mutagenesis (Amersham kit, version 2.1) was used to intro-duce ATG to MG mutations at each uATG, both individually and incombinations, to generate plasmids that retained any single uATG, lacked

any single uATG, or lacked all four uATGs. After confirmation by DNAsequence analysis, the mutated Hindlll-Xbal fragments were reinsertedinto the [3-galactosidase expression vector to generate the plasmid ptstl

0-gal (which lacked all four uATGs), the plasmid series ptst(+ATGX)gal,where X = 1, 2, 3, or 4 and only uATG1, 2, 3, or 4 was left intact, and the

series ptst(-ATGX)gal, where only uATG1 , 2, 3, or 4 was removed. AHindIll-XbaI fragment encoding the 5’UTR of the plasmid pov/0-gal wasderived from ptstlo-gal by PCR using the same primer set as for con-struction of the pov-gal plasmid.

In the plasmid series ptst(uATGX)gal, where X = 1 , 2, 3, or 4, the

(3-galactosidase coding region was joined to testis-specific 5’UTR

sequence at the position of each uATG, or at the Mos ATG forptst(ATGM)gal. PCR was used to amplify sequence from the Hindlll-Xbal fragment of ptst/0-gal using a 3’ primer designed in each case to

reinsert a uATG (or the Mos ATG) at its original position, followed by an

Xbal site to allow joining of the f3-galactosidase coding region. In these

plasmids, the uATG that now initiates the major ORF is the only ATG inthe 5’UTR, and sequence directly following the ATG is identical for the

plasmids of this series (�J�AGTCTAGAAGTC). The structure of allplasmids was confirmed by DNA sequence analysis.

Plasmids used to test the importance of the amino acid sequenceencoded by the uORFs are based on pGL-Basic (Promega). For these

plasmids, the luciferase coding region has been replaced with the (3-ga-lactosidase coding region, as above, and the SV4O promoter has been

replaced with the stronger CMV/IE promoter. The transcription start site is

3 bp upstream of a Hindlll site used for insertion of 5’UTR sequence.Testis-specific 5’UTR sequence from -325 to + 1 5, bounded by HindIll

and Xbal sites, was amplified by PCR from plasmids that retained either

uATG1 and 2 or uATG3, described above, and cloned into the vectorpGEM7Zf(+). Site-directed mutations were introduced using mutagenicoligonucleotides with degenerate sequence at the first position of eitherthe last or second to last codon of uORF1/2 or uORF3. After DNA se-

quence analysis, plasmids were chosen that carried mutations creatingdifferent amino acid ceding changes at each position, respectively. The

mutated 5’UTR sequence was excised with HindlIl and XbaI and insertedinto the CMV/(3-galactosidase expression plasmid.

Transfectlon, Enzyme Assays, and RNA Analysis. For transfection,7.5 x 106 NIH 3T3 cells were electroporated in 0.4 ml of PBS containing

1 0 �g of the chimeric (3-galactosidase “test construct” DNA and 1 0 �g ofpCAT-Control DNA (Promega). Following electroporation, cells were re-suspended in DMEM containing 10% newborn calf serum, and 3.75 x 106

cells were seeded into each of two 1 00-mm culture dishes. After 24 hincubation, the DNA-containing medium was replaced with fresh medium.Twenty-four h later, cell lysates were prepared for protein determination,

enzyme assays, and RNA isolation. Cell lysates from one dish were

prepared using 0.9 mI/100-mm dish Reporter Lysis Buffer (Promega) andaliquoted into two tubes. One aliquot, for CAT determination, was heated

at 60CC for 10 mm to inactivate endogenous deacetylase activity, and thenboth tubes were stored at -80”C until use. The second aliquot was usedto determine protein concentration using the BCA Protein Assay Kit(Pierce) and j3-galactosidase enzyme activity by a spectrophotometric

assay (46). CAT enzyme activity was determined using a liquid scintillationcounting assay according to the manufacturer’s directions (Promega).RNA was prepared from the second dish of transfected cells by the

guanidinium isothiocyanate procedure (47). RNA was treated with glyoxal

(48) and transferred to Zeta-Probe membrane (Bio-Rad) after electro-phoresis in agarose gels. Agarose gels were loaded with RNA correspond-

ing to an equivalent amount of CAT activity from each group of transfectedcells. The Northern blots were probed with a radiolabeled DNA fragment

from the E. coli lacZ gene, using standard procedures, and hybridizationwas quantitated on a Molecular Dynamics Phosphorlrnager.

Transgenic Mice and �-Galactosidase Hlstochemlcal Staining.Transgenic mice were generated by DNA microinjection of B6C3F2 fer-tilized eggs using standard techniques (49). The presence of the transgene

in pups was identified by PCR amplification ofgenomic DNA isolated from

tails. To test for f3-galactosidase activity in the testes of 7-week-oldtransgenic mice, the testis was fixed in 4% paraformaldehyde for 1 h at

4”C, washed twice in PBS (pH 7.3) for 30 minutes each, and then incu-bated overnight at 30”C in staining solution (PBS containing 5 m� potassium

ferrocyanide, 5 mM potassium ferricyanide, 0.5% NP4O, 2 m�.i MgCl2, and 1mg/mI 5-bromo-4-chloro-3-indot�d-(3-r-galactopyranoside). Stained tissues

were embedded in paraffin, and 15-20-pm sections were prepared.

AcknowledgmentsWe acknowledge the technical assistance of Klara Peto and GwenGilliard.

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