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Proc. Natl. Acad. Sci. USAVol. 87, pp. 7090-7094, September 1990Developmental Biology

Xenopus laevis a and .8 thyroid hormone receptors(alternative splicing/amphibian metamorphosis/5' untranslated region/translation control)

YOSHIO YAOITA, YUN-1o SHI, AND DONALD D. BROWNDepartment of Embryology, Carnegie Institution of Washington, 115 West University Parkway, Baltimore, MD 21210

Contributed by Donald D. Brown, June 18, 1990

ABSTRACT TheXenopus laevis genome encodes two genesfor the a (TRa) and two genes for the 18 (TRI) thyroid hormonereceptors. The two TRa genes closely resemble their rat,human, and chicken counterparts. No alternatively splicedTRa cDNA clones were found in the 5' untranslated region (5'UTR). In contrast, complex alternative splicing of TRl mRNAoccurs within the 5' UTR as well as possible alternativetranscriptional start sites. As many as eight exons encodingmainly the 5' UTR are alternatively spliced, giving rise to atleast two amino termini for each of the two TRf3 proteins. The5' UTR of transcripts from both TRa and TRj3 genes containmultiple AUG sequences with short open reading framessuggesting translational control mechanisms might play a rolein expression of TR genes.

We are studying amphibian metamorphosis as a cascade ofchanges in gene expression that occurs in every tissue of thetadpole as a response to thyroid hormone (TH). Only recentlyhas it become clear that the diverse responses of organismsto TH can be explained by the hormone's influence on geneexpression (see ref. 1). This idea has gained support from theidentification ofTH receptors (TRs) as members of a familyoftranscription factors termed "nuclear receptors" (2, 3). TRproteins activate or repress genes by binding to defined DNAelements called TH response elements. TH influences geneexpression by binding directly to the TR protein. Two closelyrelated families of TR called TRa and TRj3 were identifiedoriginally in chicken (4) and mammals (5) and shown to behighly conserved in evolution. Both families of proteins bindTH with high affinity (0.3-10x 10-1o M) in vitro (5, 6). ClonedTRs expressed in cells respond to TH by up-regulating thegrowth hormone gene (7) and down-regulating the gene forthyroid-stimulating hormone (8), mimicking the in vivo ef-fects (9-11). Furthermore, a patient requiring unusually highlevels of TH was found to have a mutant TR/3 gene (12).These discoveries constitute strong evidence that the TRproteins are true receptors for TH and are required interme-diates for many of the physiological effects of TH.By presuming that the first step of the metamorphosis

cascade is the binding of TH to one or more of its cognatereceptors, we have cloned and characterized the cDNAsencoding TRa and TR,8 from Xenopus laevis. In this paper wereport the sequences oftwo members for each of the TRa andthe TR,8 families in X. laevis.* The TRfl mRNAs are hetero-geneous due to alternative splicing of transcripts of as manyas eight exons encoding mainly the 5' untranslated region (5'UTR).

MATERIALS AND METHODSCloningTR Genes. Cloning X. laevis TR genes was initiated

by screening libraries with a cDNA encoding human TRB, agift ofR. Evans (5). Two cDNA libraries were prepared in the

A vector Zap II (Stratagene) from stage 49 X. laevis tadpolestreated with 5 nM 3,5,3'-L-triiodothyronine for 19 hr and fromstage 58 tadpoles. One genomic library in Charon 4A was thegift of I. Dawid (National Institutes of Health). A secondgenomic library was constructed by K. Joho from a partialSau3A digest of X. laevis homozygous diploid DNA (13)cloned into the Xho I half sites in LambdaGEM-11(Promega). The anchor polymerase chain reaction (PCR)method (14) was used to obtain the 5' ends ofthe cDNAs (Fig.1). The anchor primer for all PCRs was

5 '-GTCGACATCGATCTCGAGT18-3'.Sal I Cla I Xho I

The 18-mer antisense primers used for the PCR of TRacDNAs are identified by number in Fig. 1; their locations inthe cDNA sequence count the adenine of the translationinitiation codon as position 1. In some cases, extra nucleo-tides not present in the gene were added to introduce arestriction site at the 5' end of an oligonucleotide primer. Thisis shown by a vertical line in the sequence. They are asfollows: primer 1, 5'-CAGTTCGGTCTGATGACA998-3';primer 2, 5'-GGGGAITKCGTATCGTCAAGGTTA951-3'(underlined bases are a BamHI site); primer 3, 5'-TCCTC-AAAGACCTCAAGG1227-3'; primer 4, 5'-GGTGGAAA-GAGCTCGGTG1203-3' (underlined bases are a Sac I site);primer 5, 5'-AACAGCCTGCAACAGTGC973-3'. The an-tisense primers for PCR of TRB cDNAs are numbered bycounting the second guanine of the glycine codon that isadjacent (3') to the "changing point" in all TR,8 proteins asposition 1 (see Table 1). This is the first nucleotide of an exonthat encodes the first zinc finger (see Fig. 4). They are asfollows: primer 6, 5'-TGTAAACTGGCTGAAGGC519-3';primer 7, 5'-CCTCGGGCGCATTAACTA4'-3' (underlinedbases are an Ava I site); primer 8, 5'-GGGGIATCCA-AGTAGCTGGGTAT6-3' (underlined bases are a BamHIsite); primer 9, 5'-CCAAGiClTCCATAGTACACGGTT-18-3' (underlined bases are a HindIII site).A PCR product was cut by Xho I (located in the anchor

primer) and an appropriate restriction enzyme located in theother primer (Fig. 1); the doubly cleaved DNA was purifiedby gel electrophoresis and cloned into Bluescript KS(-)(Stratagene). Sequencing of cDNAs or subcloned genomicclones of TRaA and -B and TRPA was carried out for bothstrands by using the dideoxynucleotide method.

Isolation and Sequencing of Genomic TR, Clones. Individ-ual genomic clones in LambdaGEM-11 that contained mostof the TR,8A or TRBB coding regions (from the first finger tothe 3' UTR) were chosen for sequence analysis. The DNAsequences of exons for both TR/3A and TR,3B were deter-mined by direct sequencing of A DNA by using a series of

Abbreviations: TH, thyroid hormone; TR, TH receptor; UTR,untranslated region; ORF, open reading frame; PCR, polymerasechain reaction.*The sequences reported in this paper have been deposited in theGenBank data base (accession nos. M35343-M35362 for XenopusTRaA and -B cDNAs, TR,3A and -B cDNAs, and the 5' UTR exons,respectively).

7090

The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Proc. Natl. Acad. Sci. USA 87 (1990) 7091

I DNAI TH |

aA * 1Sam Hi

aB9

4 3* -i_

Sac II _

Hind III 5 3

8* Ir z*1 ===z Bar Hi

Hind III'3

7 6Ava I

4

FIG. 1. Strategy for cloning the TR cDNAs by anchor PCR. Thecoding region of TR mRNA is diagrammed at the top as a rectangle(DNA, DNA-binding domain; TH, TH-binding domain). Numbersabove arrows refer to PCR antisense primers. For their sequence andprecise location in the cDNA, see the text. Cloning of TRaA beganby sequencing a partial cDNA clone from the stage 49 tadpole cDNAlibrary. Primer 1 primed cDNA from 2 EAg of stage 58 tadpolepoly(A)+ RNA. The cDNA was tailed with poly(A) and the PCR wascarried out with the anchor primer (*) (with and then without thehomopolymer) and primer 2. Cloning ofTRaB began by sequencinga genomic clone. Primer 3 primed cDNA from tadpole poly(A)+RNA. The cDNA was tailed with poly(A) and the PCR was carriedout with the anchor primer (*) and primer 4. A partial cDNA clonewas obtained and sequenced. The PCR was repeated with the anchorprimer and primer 5. Cloning of the TRf3 cDNAs began with a partialcDNA cloned from a stage 58 tadpole library. Cloning the 5' ends wasaccomplished in two steps with the primers shown.

end-labeled primers corresponding to known TRO3A cDNAsequences. The method uses a repetitive unidirectional poly-merase reaction to increase the sequencing signal. The Ther-mus aquaticus polymerase sequencing kit (Pharmacia) wasused with the following modification. DNA (0.25 pg) wasdenatured with alkali and precipitated with ethanol. TheDNA was dissolved in 7 pl of H20, 8 1.l (-8 ng) of the labeledprimer, 2 pl of annealing buffer, and 4 pl of labeling mixture.The tube was cooled on ice and 1 Al of T. aquaticuspolymerase was added. The mixture (5 pl) was added to eachof the four tubes containing 3 pl of G, A, T, or C terminationmixture. The solutions were covered with a drop of paraffinoil, and the sequencing reaction was performed on a thermalcycler (Perkin-Elmer/Cetus) for 20 cycles. Each cycle con-sisted of 1.5 min at 94TC, 5 min at 55TC, and 3 min at 72TC.

a A a

E H E H

The reaction tubes were then cooled to 40C followed by theaddition of 3 Al of stop mixture. The samples were analyzedon an electrolyte gradient gel (15). Sequence of the genomicTRj3A exons from the first finger to the 3' UTR obtained fromthe genomic clone was identical to that determined from thecDNA clone with the exception of one silent polymorphicchange (T566 - G). The genomic TRI3B sequence was iden-tical to that determined for a partial TR,3B cDNA (the firstand part of the second zinc fingers). The exon sequenceswere confirmed by direct sequencing of the A clone from bothdirections by using primers homologous to adjacent introns.

Labeling DNA Fragments. A convenient method for label-ing short DNA fragments uses a unidirectional repetitivepolymerase reaction with one primer. The 20-1.l reactionmixture contained lx PCR buffer (Perkin-Elmer/Cetus),10-20 ng of an exon-specific DNA fragment, 0.2 mM dATP,0.2 mM dGTP, 0.2 mM dTTP, 30 ng of a specific primer, 30

OCi ofdCTP (3000 Ci/mmol; 1 Ci = 37 GBq; Amersham), and0.3 unit of Thermus aquaticus DNA polymerase. The firstcycle (950C, 3 min/550C, 2 min/720C, 30 min) was followedby four cycles of a step program (950C, 40 sec/550C, 2min/720C, 30 min). The labeled DNA was purified by passagethrough a Sephadex G-50 spin column.

RESULTSThere Are Two a and Two fi TR Genes. Representative

Southern blots of homozygous diploid DNA (Fig. 2) showthat there are two genomic copies of each family of TR perhaploid complement of Xenopus DNA. Each of the TRaprobes hybridized to its own gene strongly and also hybrid-ized weakly to the other TRa gene. Each TR46 probe hybrid-ized specifically with its own gene at high stringency.The amino acid sequences of TRaA and TRaB (Fig. 3)

differed in only 7 out of 418 amino acids (98% homology) and35 nucleotides in the entire coding region (97% homology).Another TRaA clone had glycine-137 replaced by serine, anda TRaB genomic clone had arginine at residue 392.The sequence of one X. Iaevis TRa gene has been pub-

lished (18). It is identical to our sequence ofTRaA except thattyrosine replaced histidine-54 and glycine replaced serine-137(the same as one of our identified polymorphisms).DNA- and the TH-binding domains of the two Xenopus

TRa proteins are highly conserved with those of otherspecies, and in vitro-synthesized proteins bind 3,5,3'-L-[1251]triiodothyronine in a filter binding assay (A. Kanamoriand D.D.B., unpublished data). The sequences for thechicken (4) and rat TRa (16) are included in Fig. 3 forcomparison. The most divergent regions are the amino ter-

PA A BB E H B E H

1 8 --

2 --

6.:2 _-IN 6.2 -_. _

5 53-_15-0

-.) n _-Mb -2. 02 -4..

_

5.0 -_

0.7 -_

FIG. 2. Southern blot hybridization analysis of genomic DNA from homozygous diploid X. laevis using TR probes, as indicated. Highmolecular mass homozygous diploid DNA was digested with Bgl II (lanes B), EcoRI (lanes E), or HindIll (lanes H) and, after gel electrophoresis,blotted onto a Nytran filter. Probes forTRaA and TRaB were nick-translated cloned Xenopus genomic DNA fragments of 2.2 and 2.0 kilobases,respectively, encoding part of the hormone-binding domain and part of the 3' UTR. The former is a HindIII-EcoRI fragment; the latter is aHindIII fragment that does not have an EcoRI site. The TRf3A and TRI3B probes encoded just the first zinc finger of the DNA-binding domainthat had been cloned in a vector. These 100-base-pair fragments were labeled by a linear polymerase reaction. The filters were washed understringent conditions. Band sizes are indicated in kilobase pairs.

Developmental Biology: Yaoita et al.

7092 Developmental Biology: Yaoita et al.

X. aAX.aBC. alR. mlX.PAX.PBR. 1

MDQNLSGLDCLSEPDEKRWPEDGKRKRKSQCMGKSGMSGDSLVSLPS GY-- I---- -I --- P ---

-E-KP-T--P----EDT- ------- S--- LV--S-- _-E-KP-KVE-G-D-E- __------- --PL--S-- _

S *MP-S--_*MP-S--_

89 amino acids -

IPSYLDKDEPCVVCSDKATGYHYRCITCEGCKGFFRRTIQKNLHPSYSCK

-Q--------G---------------------- Q-- G----------------------------- T-----------Q----G------------------------------T-------------L----G--------------------------------------------L----G----------------------------------------L----G----------- ------ ---------S--------

YDGCCIIDKITRNQCQLCRFKKCIAVGMAMCLVLD RRK IEENR-------------------------------D---D--- ----___---D-SV-S __- ----- _____

SV----i-Z---------------__ ------__ Q

-E-K-V---V------E--------- T-- __ --L-----------E-K-V---V ------E------KT----T --L-----------E-K-----V------E-------Y----T --L----------

50

100

QRRRKEEMIKTLQQRPEPSSEEWELIRIVTEAHRSTNAQGSHWKQRRKFL 200V-C-E---------S--H--S--A------HV-----------------K----E--------RS-------TP---D--HVA --------------------EK---D-IQ-S-V-K---TQ------QV-----VA----------K----EK---D-IQ-SIV-----TQ------QV--- VA----------K----EK--R--LQ-SIGHK---TD------KT-----VA----------K----

PEDIGQSPMASMPDGDKVDLEAFSEFTKIITPAITRVVDFAKKLPMFSEL 250

-D------IV----------------------------------------

[-----A-IVNA-E-GQ--------Q----------------------C-------A-IVNA-E-G-------Q----------------------C-------A-IVNA-E-GQ------Q-----------------------C--

TCEDQIILLKGCCMEIMSLRAAVRYDPDSETLTLSGEMAVKREQLKNGGL 300

I-- 6 V-- -A - ---

P--------------------------E------N-----T-G------P---------------------------E------N-----T-G-------P---------------------------------N-----NT-G-------

GVVSDAIFDLGRSLAAFNLDDTEVALLQAVLLMSSDRTGLICTDKIEKCQ 350

-----------K--S---------------------------V-------------- E--K--S-------------------T--S--L-V ----S-

-----------V--SS-S-------------------.P--ASVER----------------V--SS-S--------P-----------P--SSVER----------------M--SS----P-----------------P--A-VER---Y-

ETYLLAFEHYINHRKHNIPHFWPKLLMKVTDLRMIGACIASRFLHMKVEC 400_- _-_-_-_-_--- --_ --- C-

PTELFPPLFLEVFEDQEVFIG.3. Amino acid sequee c o o Ts X

FIG. 3. Amino acid sequence comparison of TRs. XenopusTRaA and TRaB, chicken TRal (4), rat TRal (16), Xenopus TR.8Aand TR,8B, and rat TRP1 (17) are compared. Amino acids identicalto Xenopus TRaA, taken as the standard, are shown as dashes. Twoadditional amino acids (senine and alanine) are present in the amino-terminal domain of rat TRal as indicated. Vertical lines (from aminoto carboxyl terminus) separate the amino-terminal variable region(called the "changing point" in TRB) from the DNA-binding domain,then the junction region, and finally the TH-binding domain. Aconserved heptapeptide in all TRa proteins located in the amino-terminal hypervariable region is boxed as is a nonapeptide region justafter the DNA-binding domain. These are possible nuclear localiza-tion signals. No sequence similarity was detected between theamino-terminal 89 amino acids of rat TRB1 and Xenopus or chickenTRJ3. The star (*) next to the amino termini of Xenopus TRBA and-B means that these are just one of two amino-terminal sequencesencoded by each gene (see Table 1).

mini and a region between the DNA- and TH-binding do-mains. We note seven conserved amino acids in the former(boxed in Fig. 3) that are reminiscent ofa nuclear localizationsignal (19, 20) in addition to the well-conserved nuclear-localization peptide between the DNA-binding and hormone-binding domain (also boxed in Fig. 3). We analyzed 24 clonesprepared by the anchor PCR method and found that they

Table 1. Amino termini of X. Iaevis TRB3 proteinsTRj3 protein Amino acid sequence Variant(s)

,8A-I MPSSMSIGYIP-- Al, 2, 3, 483A-II MEIGYIP-- A5, 6PB-I MPSSMSIGYIP-- Bi, 3f3B-II MPSSMSVRLFTASAAQRKKIQ B2

EGDCCVVLAGKTQGRFILIGAVARVSIGYIP--

Vertical lines delineate the changing point. See Fig. 4 for splicingpattern of variants.

differed only in the length of the 5' UTR. The longest 5' UTRof TRa mRNA among these clones was approximately 600base pairs.As for the two TRa genes, the coding regions of the two

TRA genes are very similar to each other and to theirmammalian counterpart (5, 7, 17). We identified two aminotermini for each TR,3 protein (Table 1). These are producedby alternative splicing at the same position, as has beendescribed for a rat pituitary-specific variant of TR,82 (6). Wecall this site just before the DNA-binding domain the "chang-ing point." It represents an exon-intron boundary that sep-arates an upstream region that encodes mainly the variable 5'UTR from a downstream constant region that begins with thefirst zinc finger and encodes most of the protein. However,alternative splicing at the 5' end of the TRP mRNAs is muchmore complex than suggested by just the protein differencesthat it causes.

Heterogeneous 5' Ends of the TRP mRNAs. Fig. 4 diagramsnine 5' ends from 31 TR,8 cDNA clones isolated from ananchor PCR mixture of tadpole mRNA. They fall into sixgroups associated with TRf3A and three groups associatedwith TR,3B. Twenty of the other 22 clones were identical tothese nine variants except for changes attributable to poly-morphisms (see Fig. 5) or to truncated 5' ends. The other twoclones were incomplete splicing products containing intronsequences (data not shown). The sequences of the exons aregiven in Fig. 5. No clone has been found that combines anexon of the TR,8A gene with one from the TRP3B gene, andthe order of the exons is consistent with that shown in Fig.4. No single clone has been found containing both exons a andb, so their order in the genome is unknown. Most clones beginwith either exon a or b. This means that there may be at leasttwo transcription start sites for each TRf3 gene beginning atthe 5' end ofexons a and b. Exons a, b, c, f, and g are commonto both TRP3A and TR/B genes and are 61-86% identicalexcept for exon g, which is more highly conserved. IndividualcDNA clones with the same set of exons occasionally dif-fered by one or more nucleotides as shown in Fig. 5. Since thepoly(A)+ RNA used for PCR was pooled from many tadpoles,this heterogeneity is probably due to polymorphism. Thearrangement of the sequences in Fig. 4 is evidence enoughthat they are on separate exons except for exons f and g,which are spliced together in all nine variants. Characteriza-tion of genomic clones confirm the separation by introns ofexons a, b, f, g, h, and i (unpublished data). Southern blotsprobed with cloned individual exons, exons a and h fromTR,8A and exons a and i from TRp8B, conclusively showedthat each is represented by a single genomic sequence inhomozygous diploid (data not shown).

DISCUSSIONAlternative Splicing and Developmental Regulation of TRI3.

In the rat, TRj31 and TRfi2 mRNAs are generated by alter-native splicing at the site where the isoforms ofXenopus TR,8differ; we call this site the changing point. Rat TR,82 isexpressed only in the pituitary (6), suggesting by analogy thatalternative splicing of TRI3 in Xenopus might be tissue-

Proc. Natl. Acad. Sci. USA 87 (1990)

-GF-

Developmental Biology: Yaoita et al. Proc. Natl. Acad. Sci. USA 87 (1990) 7093

~AIIIIZiLILZZDLIZIPA |El H E g C

1 < - v -

' -- -~"' ' 't,'T ';s

3 * -. ---- --

Fin

I I: I,"I , --

%I v III\ S ,v

d --l' I y-% I '/--

<q

II IIs,,o

+4-a b c

I- - I, [~f g i

41, , _I"

I< I-_ _ _

< I , I==-- ==, -I

%I/ I,

w I L~-- vI .,, JIt v EV

FIG. 4. Alternative splicing in 5' regions ofTRBA and TR3B mRNA. Rectangles outlined by solid or stippled lines represent exons ofTRBAor TRBB, respectively. The order of exons a and b is not known. The exons are predicted from the cDNA sequences and for exons f and g bysequencing of genomic clones (unpublished data). Dashed lines indicate spliced-out introns; their sizes are unknown. Each large arrow marksthe 5' end of the longest cDNA insert found for each group. Only a single clone was found for variants A4, A5, A6, and B3. Small arrows pointingto the right mark the translation initiation site of each mRNA.

specific. TRA in chicken has a truncated amino terminussimilar to Xenopus TR3 (21). The conservation of the chang-

CAGCG CTTTATCCTGACTGA

AGCTTCATTATCCTGACTGA

ACTGAGGCAGGGGTAACGCTGGGAGTGACTGGCATAGCAGGGGCTGCGGGGAGGCACTTC

ACACAAGCAGGGATAACGCTGGGAGTGACTGGCATAGCAGGGGCTGCAGGGAGGCACTTC

AAG TCCGTGCCAAGTCCAACATTGTAGCTAGTGACGAGAATCGTACTACAGTGCGGG

ATAATCCGTGCCAAATCCAACGTTGTAGCGAGTGACGAGAATCGTAG AGTGCGCGGA

C TCTCACTAAGTGACGCTCGAATTCGGGAAGAACGACGCGGCAGCTGTTGCATT,

ACAGTCTCAC G GACGCTGGGGTTTGGGAAGGACGACGCGGCAGCTGTTGCACTACG

GTGCGTCTG TAGGTCGGAGAGCCGGCG 259

TTACGTCTAACTCTATAGGTTGGAGAGCTGACG 226G

Ab ATTTCAGGACAGCCCAGCGCCCTGGTGCACGATCAGCTGTAGATCTCCCTGTCTGTGTCG

Bb AGCTGTAGATCTCC TGTCTGTGTTGT

CTGC TGCCGCTGCTACTTCAGTTCCTCTGACTGTCAG 97

CTGCCACTGCTGTTGCTGCTCCAGTTCCTCTGACTGTCAG 65

ing point (at the amino acid level) suggests that TR/3s ofchicken (21, 22), rat, and Xenopus have the same exon-intron

TAf GTTCCTCTCAAGC CCAGGAACAAAAACCGGAAA TTTTTC

Bf CTACAGGTTTCCCTCAAGCACCAAGAACGAAAACCAGAAAGAATTTGCAGAGAATTTTTCG C

AA=;AG 4 6

AAAIAG 67

Ag GCTATuGTGATTCTTAGAAGAA=AGCGGACCTTCCAATCCATAZCAAGCAGTATGBg GTTATaMTGLCTTAGAAGA=AGCAGACCTTCCAATCCATACAAGCAGTATG

TCAG 64

TCAG 64T

G

Ah GCAGAGTATGGTTTAGAAGAACTAACACAGAAGTTTTTTGTTGGACACTACTCTCCATCTAG

G A C

AIACAAGATTTCCATTGTAACATCCTAATTGTA4CCAGTAATCAG ,/,CTGCTT

TGGACAGTGCTTACAGCTTTTTTAPAGAGATTTT TATT-TTGCTTTGCATC(P-ACCGTGTAC7MAAG 191

Ac .TTGAAGACTGATTGGGGTTAAGCAGGCACATAC AAGAA (AAG) 44

Bc ZATTGAAGAGTGATTGGGGTTAAGCAGGCACATACTGTACAAGAAAAAG 50

A T

Ad ACAGAAGCCGTGAACCAACAGAATTACAGGAAAGGACGAGGATTGAAACATCTGTACaZ3:AGAAGGAATTTCTG (AAG) 79

Ae

Bi TTCGGCTTTTCACTGCATCTGCCGCACAAAGAAAGAAGATACAGGAAGGGGATTGCTGTGTGGTGCTCGCTGGAAAAACCCAGGGCCGGTTTATATTGATAGGAGCAGTGGCCCGGGTATCAG 123

TTAAAGTTGAAGTATTTCTGGTCAGGTGATCTCTGAGGCAGCGCACAGGCCCTCACAAAATGGTGGCTC(AAG) 72

FIG. 5. Sequences of the exons found in the 5' UTR ofTR3A and TRBB. Paired dots mean identical nucleotides between TR/3A and TRAB.Differences attributable to polymorphism including nucleotide exchanges, insertions, and deletions are indicated above or below the sequence.The ATG codons within the 5' UTRs are underlined. The true translation initiation codons are boxed. The exact boundaries of some exons weredetermined by sequencing genomic clones. It is not known whether a part of the AAG in parentheses belongs to the end of exons c, d, and e,as shown in the figure, or to the beginning of exons d, e, and f.

4

5

6

OB

2

3

Finger 1

Aa

Ba

7094 Developmental Biology: Yaoita et al.

boundary. It remains to be seen if expression of the TRf3 genein chicken and rat also involves complex alternative splicingof exons in the 5' UTR. In chicken, expression of TRa isdescribed as constitutive and ubiquitous, not correlating withdevelopmental changes. In contrast, TRj3 mRNA accumula-tion in the chicken either correlates with development orshows tissue specificity (21). In studies on the developmentalexpression ofXenopus TRa and TR,8 mRNAs to be publishedelsewhere, we found a close correlation of expression ofTR,8mRNA with metamorphosis. Both TR,8 genes are up-regulated by TH, reaching maximal levels at the climax ofmetamorphosis when endogenous TH is maximal. TRamRNA levels are only minimally influenced by TH (Y.Y. andD.D.B., unpublished data).

Heterogeneity in the 5' UTR has been reported for mouseand human retinoic acid receptor y (23, 24). These variationsinvolve both alternative promoters and alternative splicing of5' UTRs to a constant exon that has the first AUG of thecoding region (23). Heterogeneous alternatively spliced 5'UTRs ofmRNAs are probably more common than previouslysuspected since variants are likely to be overlooked bytraditional cDNA cloning methods.

Possible Function of Diverse 5' UTRs in TRfi mRNA. TR,8and TRa mRNAs are both approximately 10 kilobases long(Y.Y. and D.D.B., unpublished data), and about 8 kilobasesare in the 3' UTR, a region that we have not yet analyzed. Inthis paper we have focused on the 5' UTR. The two TRamRNAs have 5' UTRs of about 600 nucleotides with noevidence for alternative splicing. However, the 5' UTR ofTRB mRNAs has multiple variants formed possibly by twostart sites and complex alternative splicing of as many as 8exons resulting in two proteins for each TR,8 gene (Table 1).

Striking features of the 5' UTRs of the TRI3 mRNAs aretheir length (200-600 base pair), their complexity, and thepresence ofmany small open reading frames (ORFs) (Fig. 5).It has been shown that upstream ORFs can regulate trans-lation by reducing initiation events at the bona fide transla-tion start site. The 5' leader sequence of GCN4 mRNA inyeast contains four small ORFs, each of which has a repres-sive effect on translation (25). The closer an AUG is to thetrue initiation codon the more inhibitory it is. The mostconserved 5' exon between TR/3A and TRBB is exon g. Exong is adjacent to the translation start site in many variants, andit contains multiple AUG codons with in-frame terminators(Fig. 5). The TR genes are related to erbA, and manyoncogenes not only have unusually long 5' UTRs but alsoshort ORFs preceding the true translation initiation codon(26). The Xenopus TRa mRNAs also have short ORFspreceding their translation start codons, as do the genes forchicken (22) and mammalian (5, 7) TRs. Therefore, onepossible function of these alternately spliced exons might beto regulate the rate of TR protein synthesis by control oftranslation.However, it does not explain a functional need, should

there be one, for the various isoforms of TR,3 mRNA.Perhaps the heterogeneity reflects specificity of splicing in

various tissues or is in some way related to up-regulation ofXenopus TRP mRNA by TH that occurs in tadpoles atmetamorphosis (Y.Y. and D.D.B., unpublished data).

We thank Ronald Evans for a gift of the human TR,8cDNA and ourcolleagues for helpful criticisms. This research was supported in partby grants from the National Institutes of Health and the Lucille P.Markey Charitable Trust.

1. Oppenheimer, J. H. & Samuels, H. H. (1983) Molecular Basisof Thyroid Hormone Action (Academic, New York).

2. Evans, R. M. (1988) Science 240, 889-895.3. Green, S. & Chambon, P. (1988) Trends Genet. 4, 309-314.4. Sap, J., Mufioz, A., Damm, K., Goldberg, Y., Ghysdael, J.,

Leutz, A., Beug, H. & Vennstrom, B. (1986) Nature (London)324, 635-640.

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