Alternative Splicing Generates Variants in Important Functional ...

T H EJOURNAL OF BIOLOGICAL CHEMISTRY

Vol. 262, No. 33, Issue of November 25, pp. 16122-16126,1987 Printed in U.S.A .

Alternative Splicing Generates Variants in Important Functional Domains of Human Slow Skeletal Troponin T* (Received for publication, April 24, 1987)

Reinhold GahlmannS, AnthonyB. TrouttQ,Robert P. Wadell, Peter Gunning11, and Larry Kedes** From the MEDIGEN Project, Department of Medicine. StanfordMedical School and Veterans Administration Medical Center, Palo Alto, California 94305

We provide the first nucleotide sequence information for the slow isoform of troponin T (TnT). Sequence and hybridization analyses revealedthat a single slow TnT gene present in the human genome gives riseto at least two different slow TnT variants by alternative splicing. The observed variations in slow TnT splicing generated major structural differences between the two corresponding slow TnT proteins in a domain that is likely to be involved in critical interactions with troponin C, troponin I, and tropomyosin in the thin filament. Corresponding variations have not been found for fast or for cardiac TnT. The comparison of splicing patterns for fast, cardiac, and slow TnT reveals that the splicing pattern for each isoform is unique. These features raise important questions of why and howall the individual members of the closely related TnT gene family developed such complex but different schemes of alternative splicing to create sets of variant proteins. This unusual familial trait is not known in any other muscle or nonmuscle multigene family.

potentially 64 distinct variants of the fast T n T isoform are derived from a single geneby a complex schemeof alternative splicing (7-9). Two different cardiacT n T proteins (10) which may have functional differences (11) are derived from a single gene by alternative RNA splicing (12). We describe here the first sequence information for the slow isoform of T n T a n d propose that, different slow T n T variants feature major differences in an important functional domain of the proteins. EXPERIMENTALPROCEDURES

Cloning and characterization of the two cDNAs described in this study has been performed essentially according to methods described by Maniatis et al. (13). For DNA sequencing analyses the method of Sanger et al. (14) was employed. Specific details for individual experiments are provided inthe figure legends. RESULTSANDDISCUSSION

The two T n T cDNA clones described in this paper, H22h and M1, were isolated from a human adult skeletal muscle cDNA library (15). Clone H22h was obtained as an abundant message clone by using a strategy described elsewhere and Troponin T (TnT)’ is the tropomyosin binding componentrepresents an essentially full length cDNA clone (16). Clone of the troponin complex that regulates the contraction of M1 was selected by using a probe containing the repetitive muscle in response toCa2+ effluxfrom the sarcoplasmic human DNA sequence Hut2 which is described by Hoffmanreticulum (1). Adult cardiac muscle, slow skeletal muscle, and Liebermann et al. (17). Comparison of the locationsof restricfast skeletal muscle contain different isoforms of T n T (2). tion endonuclease cleavage sites in the cDNAs revealed that The domains of T n T involved in critical interactions with both clones are related (Fig. l a ) , and nucleotide sequencing troponin C and troponin I in the troponin complex and with (Fig. l b ) verified these similarities. The major differences are tropomyosin have been characterized in considerable detail that clone H22hcontains two shortinsertions relative to for the fast isoform of T n T by peptide binding studies (3-6). clone M1. The clones contain 5’ untranslated regions of 57 Recently it has been demonstrated that more than 40 and or 58 nucleotides (see legend to Fig. l b ) and are likely to be full length (16). Both clones alsocarry 3”untranslatedregions *This workwas supported in part by grants from the National (86 and 83 nucleotideslongincloneH22h and clone M1, Institutes of Health, the Muscular Dystrophy Association, and the Veterans Administration (to L. K.). The costs of publication of this respectively) that end in a polyadenylic acid tail. T o identify article were defrayed in part by the payment of page charges. This these clones, the aminoacid sequenceswere derived fromboth article must therefore be hereby marked “aduertisement” in accord- cDNAs and compared to sequences of known proteins conance with 18 U.S.C. Section 1734 solelyto indicate this fact. tained in the Protein Identification Resource (NBRF) data The nucleotide sequence(s) reported in this paperhas been submitted base. The search revealed similarities to all representedT n T totheGenBankTM/EMBLDataBankwith accession numbeds) proteins. The similarity to both fast and cardiacT n T amino 5034 76. acid sequences is about 65% in the conserved carboxyl-ter$Held a training grant from the DeutscheForschungsgemeinschaft. minal segment. This result suggests that H22h and M1 repSupported by United States Public Health Service Grant 5 T32 resent T n T cDNAclones, thefirst isolatedfrom human CA 09302 awarded bythe National CancerInstitute. Current address: sources. In addition, the degree of dissimilarity of the polyDNAXResearch Institute ofMolecularandCellularBiology,901 peptide encoded by these clones to cardiac and fast TnT California Ave., Palo Alto, CA 94304. tl Supported by United States Public Health Service Grant 5 T32 proteins (35%),which is like the degree of dissimilarity beCA 09302 awarded by the National Cancer Institute. tween fastandcardiac T n T proteins, suggested thatthe (1 Current address: Childrens Medical Research Foundation, P.B. isolated human clonescorresponded neither to fast nor to Box 61, Camperdown, N. S. W. 2050, Australia. ** To whom correspondence shouldbe addressed (151M) Veterans cardiac T n T but representeda third and distinctclass of T n T Administration Medical Center, Miranda Ave., Palo Alto, CA 94305. isoforms. No direct reference data for slow T n T is available since the protein has not been sequenced, but it seemed likely The abbreviationused is: TnT, troponin T.

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Analysis of Human Slow Troponin T cDNAs probe lor RNA blot

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FIG. 1. Restriction endonuclease digest map and nucleotide sequence of the full-length human slow TnT cDNA clone, H22h. a, restriction enzyme map of clone H22h. Open bars indicate 5’- and 3’-untranslated region and solid bars coding region, respectively. The two brackets within the coding region mark the sequences that arenot present in the otherwise very similar cDNA clone M1. Thin lines depict flanking poly dG. C and poly dA.T stretches (left and right borders, respectively). The RsaI and the PuuII fragments used as probes for the RNA and DNA hybridization experiments are indicated. b, nucleotide sequence and derived amino acid sequence of human slow TnT clone H22h. The nucleotide sequence of clone H22h and of the closely related clone M1 were determined on both strands by cloning of restriction fragments from both cDNA clones into M13 cloning vectors and sequencing according to the method of Sanger et al. (14). The first base of the sequence represents the first base in the 5’-untranslated region of clone H22h next to the flanking poly dG.C tail. An additional base (A) is present at the beginning of clone M1. The amino acid sequence deduced from the coding sequence of clone H22h is written beneath the nucleotide sequence. The three differences between the coding regions of cDNA clones H22h and M1 are underlined; base 117 is G (instead of C) in clone M1, leading to a change from aspartic acid to glutamic acid in the corresponding protein. Two stretches of 33 and 48 nucleotides, from about base pair 131 to 163 and from about base pair 670 to 717, respectively, are not present in the sequence of clone M1. The exact sequences and location of the inserted exons relative to M1 are not deducible from the comparison of both cDNA sequences alone. One out of three possible adjacent locations is indicated for the amino-terminal “insert”; one out of four adjacent possible locations is indicated for the carboxyl-terminal “insert.” The triplet ACC in front of the poly dA.dT tail (in parenthses) is present in the sequence of H22h, but not in clone M1. The polyadenylation signal “AATAAA” which iscommon to both sequences is underlined.

that both H22h and M1 a r e t h e first examples of slow TnT

signal only after prolonged exposure on the autoradiogram isoform cDNA clones. (Fig. sa). This result demonstrates that both clones represent In order to unambiguously demonstrate the identity of clone slow isoforms of TnT. H22h and M1 are t h u s the first isolates H22h and clone M1, we performed a blotting analysisof RNA of clones for slow T n T from any source. isolated from adult rabbit soleus, psoas, and heart muscle Comparison of the sequences of the two clones revealed which contain predominantly or exclusively slow, fast, and that two small stretches of 33 a n d 48 nucleotides in length cardiac TnT, respectively (2). The probe used in this experiment, comprising about500 base pairsof the coding sequence are present in clone H22h, but not in cloneM1 (Fig. 1, a a n d of H22h, is depicted in Fig.la. TnT mRNA could be detected b). In addition, the presence of a single nucleotide difference easily in soleus muscle RNA. On the other hand, heart ven- (C uersus G) at base 117 results in the conservative change tricular RNA gave no signaland psoas RNA gave a very faint from aspartic acid to glutamic acid in the corresponding TnT

Analysis of Human Slow cDNAs Troponin T

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FIG. 2. a, demonstration bv Northernblottingthattranscripts corresponding to human T n T cDNA clones are specifically present in RNA from slow-twitch muscles of rabbits. 10 pg of total cellular RNAs from rahbit soleus, psoas, and heart ventricle were separated onan agarose-formaldehyde gel andtransferredonto aBiodyne membraneas described by Thomas(33).RNAonthefilter was hybridized to a nick-translated "'P-labeled probe (34) of an internal RsaI fragment of clone H22h (Fig. la). The region contained in this fragment is highly conserved between different T n T isoforms. The filter was washed in 0.5 X SSC,0.1% sodium dodecyl sulfate a t 55 "C

proteins. There are two possible explanations for these sequence differences. First, the two clones may have been derived from two different but very similar slow T n T genes. Alternatively,they may have beenderived by differential splicing of acommon precursor transcribed from a single gene. A Southernblotanalysis of human genomic DNA demonstrates that the two mRNAs are the product of a single copy gene. Each of five aliquots of a human genomic DNA sample was digestedwith a different restrictionendonuclease. The DNA fragmentswere size-fractionated on anagarose gel a "'P-labeled and transferred atonitrocellulose filter. We used PuuII DNA fragment (Fig. la) common to both cDNAs as a hybridization probe. The autoradiogram from this experiment is shown in Fig. 2b. Single bandswere detected with the probe in all restriction endonuclease-digested DNA samples, and we conclude that there isa single slow T n T gene in the genome. Accordingly, the two cDNAs must be derived from a single genomic transcript which undergoes alternative splicing, although the splicing pattern of slow T n T transcripts could be more complex than the comparisonof the two cDNA clones alone would suggest. The otherdifference betweenthe cDNAs a t base 117 (C versus G ) presumably represents a polymorphism at the humanslow T n T locus. If so, H22h andM1 were derived from differentalleles of this gene. However, the simple explanation of a cloning artifact to explain this single base difference cannot be excluded. The three terminal bases ACC of the 3"untranslated region of H22h are missing in M1 (Fig. lb). Heterogeneity of the length of 3"untranslated sequences seems tobe rare (18)but has been described before for bovine prolactin mRNAs (19) and for mRNA of the mouse ribosomal protein L30 (20). The precise locations of the junctions of the two optional inserts in the commonsequence of slow T n T cannot be assigned withcertainty.Threeadjacentlocations for the amino-terminalinsertand four adjacent locationsfor the carboxyl-terminal insert are possible. Only one of the alternative locations for each insert is designated in Fig. Ib. The more precise localization of the alternative splice sites must await cloning and analysis of the slow troponin T gene. Alternative splicing of a precursor nuclear RNA is well documented for cardiac T n T (12) and fast TnT (8,9,21). The most surprising aspect of alternative splicing in the TnT family is that the variants of all three isoforms are derived from their respective precursors by different schemesof alternative splicing as presented in Fig. 3a. The onlycommon feature is that the insertion sites of the amino-terminal optional exon block of slow TnT andof the setof five alternatively spliced amino-terminal exons of fast TnT are at an equivalent position in thecoding sequence (Fig. 3). With this exception, different regions of the TnT proteins areaffected in each case. and exposed for 8 h at -70 "C. Only one band of hybridization is visible in soleus RNA, but the two different mRNAs would not be distinguished by this assay. A control experiment (not shown) demonstrated that the three RNA preparations used were of comparable quality. b, Southern blotting demonstration that the human TnT gene is single copy. 10 pg of HeLa DNA were digestedwith the restrictionendonucleases EcoRI, HindIII, BarnHI, BgIII, or XbaI. DNA fragments were size-fractionated on an agarose gel and transferred onto a nitrocellulose filter (35).The blotted DNA fragments were hybridized with the :'2P-labeled nick-translated PuuII fragment indicated in Fig. la, comprising the 3"untranslated region and part of thecarboxyl-terminal coding region as well as theflanking poly[d(A-T)]tail of clone H22h.CloneH22h and clone M1 are essentially identical in this region. The filter was washed a t 65 "C in 0.5 X SSC, 0.1% sodium dodecyl sulfate and exposed overnight a t -70 "C. HindIII fragments end labeled with "'P were used as size markers.

Analysis of Human Slow Troponin T cDNAs a

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T n T variants present in the fast leg muscles of the adult chicken (7). Similarly differential expression during differentiation cannot be the only reason for the existence of the two slow T n T isoform variants discussed in this report since both were recovered from an adult human skeletal muscle cDNA library. The carboxyl-terminal70-80% of T n T has been conserved during evolution between all threeisoforms (about 65%, comparison not shown) and within the sameisoform in different species. For example, comparison of the rat fast TnT gene (8) and quail fast T n T cDNAs (21) reveals a very similar coding sequence and perfect conservation of the splicing pattern in b 1 LTXPKIPEGEKMFDDIQKKRQNKDLHELQALIDSHFEARKKEEEELIALKERIEKRRAERAEPQRIRAE this segmentof the gene. Peptide binding studies suggest that 2 LTRPKIPEGEKMFDDIQKKRQNKDLHELQALIDSHFE~KEEEELIALKERIEK~~PQRI~ interactions of fast T n T with tropomyosin, troponin C, and 3 LVPPKIPDGERLDFDDIHRKRMEKDLNELQALIEAHFESRKKEEEELISLKDRIEQRRAERAEPQRIRSE 4 LIPPKIPEGERMFDDIHRKRKDLLELQTLIDVHFEQRKKEEEELVALKERIE~SERAEQQ~RTE troponin I occur in this conserved carboxyl-terminal domain. 5 LIPPKIPEGERMFDDIHRKRMEKDLLELQTLIDVHFEQRKKEEEELVALKERIERRRSERAEPQRFRTE Tropomyosin binds to a rabbit fast T n T peptide comprising amino acids 71-151 (3, 4), and both troponinC and troponin 1 2 I bind to peptides containing amino acids152-259 ( 5 , 6 ) in a 3 Ca2+-sensitive manner(23). Altogether these datasuggest that 4 5 this conserved carboxyl-terminal region contains important functional domains in all the T n T isoforms. The initial de1 RKPLNIDHLSDDKLR----------------DKAKEtl*D scriptions of alternative splicing of T n T isoforms (8, 9, 12, 2 R K P L N I D H L S D D K L R - - - - - - - - - - - - - - - - D K A K E L W D T ~ 3 RKPLNIDHLSEDKLR----------------DKAKELWQTIRDLEAEKFDLQEKFKRQKYEINYLRNRVS 21) did not reveal significant variation in this carboxyl-ter4 KKPLDIDYHGEEQLRARSAWLPPSQPSCPAREKAQELSDWIHQLESEKFDLHAKLKPQKYEINVLYNRIS minal region and were consistent with this hypothesis. Car5 KKPWIDYHGEEQLR----------------EKAQELSDWIHQLESEKFDLHAKLKPQKYEINVLYNRIS diac TnT, forexample,reveals no variation in this region 1 WSKKAGATAKGKVGGRWK (12). In addition, although fast T n T uses one out of two 2 QAQUiSKKAGATAKGKVGGRWK available exons encoding amino acids 229-242 in thecarboxyl3 DHQKVKGS-GKTWGGRWK 4 HAQKFRKGAG-”KGRVGGRWK terminal region, these exons are quite similar in amino acid 5 HAQKFRKGAG---KGRVGGRWX sequence (9, 21). In contrast, the slow T n T gene described FIG. 3. a. schematic comDarison of alternative exon uatterns of the TnT gene family.The cdding regionsof rat fast TnT IS), human here generates radically different variant proteins by its opslow TnT (this paper), and chicken cardiac TnT (12) are compared. tional use of exons in the very heart of the evolutionarily Evolutionarily conserved carboxyl-terminal and divergedamino-ter- conserved carboxyl-terminal region as well as by use of opminal parts of the T n T isoforms are represented by block and open tional exons in the amino-terminal region (Fig. 3a). bars, respectively. The relative locationsof alternatively splicedexons Asequence comparison for the evolutionarilyconserved in the basic sequences are indicated. Facultative exons are depicted segments of the three TnTisoforms is shown in Fig. 3b. The by stippled boxes. The majority of the possible exon combinations of the five optional amino-terminalexons of fast TnT seems actually to carboxyl-terminal “insertion site” of the alternative exon in 198/199 in thereference have been identified in fast skeletal muscle (7). The two carboxyl- slow T n T corresponds to amino acids terminal exons (aand 0) of rat fast TnT are used alternatively and rabbit fast T n T protein (24) and also represents aknown are, therefore, marked differently by hatched boxes. The correspond- splice site (corresponding to exons 14 and 15) in fast and ing amino acid region in cardiac TnT, for which only one exon exists, cardiac T n T (8, 12). Four proline residues and one cysteine and in slow TnT, for which only one exon has been found so far, are residue are found among the 16 amino acids inserted in the marked by hatched boxes as well. These regions in slow and cardiac TnT are 64% similar and are slightly more similar to the 0-exon of carboxyl terminus of the protein(Fig. I b ) . Proline is normally rat (8)(50 and 43%)than to the corresponding a-exon (29 and 36%). very rare ( d % ) in the conserved carboxyl-terminal domain The splicing pattern shown for human slowTnT is based on the two of any T n T (8,21,22,24).The only cysteine residues detected cDNA clones described in this report, but the pattern could be even in TnT, so far, are found in quail (21) and chicken fast T n T more complex than the figure suggests. b, protein sequence alignment (25), located in alternative exons, and close to the amino for evolutionarily conserved parts of slow, fast, and cardiac troponin T proteins (symbolized by block bars in a). The evolutionarily con- terminus of bovine cardiac T n T (10). Thus, all the known serued parts of the three troponin T isoforms are compared I , fast cysteine residues in theconserved part of T n T proteins appear TnT (rat) with a-exon (underlined),amino acids 44-259 according to to beencoded by alternative exons raising the possibility that Breitbart et al. (8);2, fast TnT (rat)with 0-exon(underlined),amino introduction of the cysteine-bearing peptide imparts imporacids 44-259 according t o Breitbart et al. (8); 3, cardiac TnT tantfunctional differences tothevariantproteins in the (chicken), amino acids 88-302 according to Cooper and Ordahl (12); troponin complex. A computer analysis using the Chou and 4, slow TnT (human, H22h), aminoacids 50-278; 5, slow TnT Fasman algorithm (26, 27) predicts that the “insert” of 16 (human, Ml),amino acids 50-262. amino acids in human slow T n T would severely disrupt its These considerations raise two important questions. Why conformation by introducing a turn into an a-helical domain are there three isoforms of TnT andso many variants thereof? of the protein (data not shown). The capacity of troponin I And how were two strategies, gene duplication and alternative (6) and, in particular, of troponin C ( 5 ) t o bind to thisregion of slow T n T is likely to be altered. splicing, combined during evolution to generate this unique Alternative splicing is widely found in animal viruses (28), collection of protein variants? One attractive explanation for the existence of many var- and in mammalian species it is especiallycommon among iant TnT proteins emphasizes the possibility that different muscle-specific transcripts (29-32). This efficient strategy of variants of T n T might be necessary during muscle differen- evolutionprovides an alternative to gene duplicationas a while preserving a gene for The levels of individualvariants of means of creating variant proteins tiation (8, 9,12,22). chicken cardiacTnT and rat fast TnT mRNAs change during an essential function. The carboxyl-terminal alternative exon embryogenesis (8,9, 12,22).But a developmental role cannot of slow T n T provides an example of such a splicing scheme explain the surprisingly high number of more than 40 fast that appears tohave allowed maintenance of conserved funcfasl

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Analysis of Human Slow Troponin T cDNAs

Ginard, B. (1984) Cell 38,409-421 tion while allowinga variantto arise. Thisevolutionary 10. Rlsnik, V. V., Verin, A. D., and Gusev, N. B. (1985) Biochem. J . 225,549552 mechanism does not explain theorigin of alternative splicing Tobacman, L. S.,and Lee R. (1987) J. Biol. Chem. 262,4059-4064 in the highly variable amino-terminal segmentsof the three 11. 12. Cooper, T. A,, and OrdahL C. P. (1985) J. Biol. Chem. 2 6 0 , 11140-11148 T n T isoforms. In any case,evolution appears to haveselected 13. Maniatis, T.,Fritsch, E. F., and Sambrook, J. (1982) Molecular Cloni A fmboratory Manual, Cold Spring HarborLaboratory, Cold Spring Haxor, for or accepted an extraordinarydegree of hypervariability in NV 14. Sak&r, F., Nicklen, S., and Coulson, A. R. (1977) Proc. Natl. Acad. Sei. U. this region. S. A. 7 4 , 5463-5467 When in evolution was alternative splicing adopted and 15. Gunning, P., Ponte, P., Okayama, H., Engel, J., Blau, H., and Kedes, L. (1983) Mol. Cell. Biol. 3 , 787-795 used to generate TnT variant proteins? Was alternative splic- 16. Garrison, J. C., Hardemann, E., Wade, R., Kedes, L., and Gunning, P. ing already used by the putative single ancestral T n T gene (1985) Gene (Amst.) 38,177-188 17. Hoffman-Liebermann,B., Liebermann, D., Troutt, A., Kedes, L. H., and that was duplicated and subsequently modified? Or did the Coben, S.N. (1986) Mol. Cell. Biol. 6,3632-3642 individual genes for slow, fast, and cardiac TnT adopt differ- 18. Birnstiel, M. L., Busslinger, M., and Strub, K. (1985) Cell 4 1 , 349-359 N. L., Smith, M., Gillam, S., Woychick, R. P., and Rottman, F. ential splicing independently after their derivation from an 19. Sasavage, M. (1982) Proc. Natl. Acad. Sei. U. S. A. 79,223-227 ancestral gene that did not create variant proteins? Unfortu-20. Wiedemann, L. M., and Per R. P. (1984) Mol. Cell. Biol. 4 , 2518-2528 K. E. M., Bucher, F A . , and Emerson, C. P. (1985) J. B~ol.Chem. nately the available data cannot distinguish these possibili- 21. Hastings, 260.13699-13703 ties. Both the evolutionary and functional aspects of the TnT splicing question are similarly puzzling. Why the TnT gene family as a groupdeveloped such a unique collection of mechanisms for production of variant proteins remainsa key issue in understanding the functionof T n T in muscle. REFERENCES 1. Squire, J. (1981) The Structural Basis of Muscular Contraction, pp. 193200, Plenum Publishing Corp., New York 2. Dhoot, G.K., Frearson, N., and Perry, V. S. (1979) Exp. Cell Res. 1 2 2 , 339-350 3. Pearlstone, J. R., and Smillie, L. B. (1977) Can. J . Biochem. 55,1032-1038 4. Mak, A. S.,and Smillie, L. B. (1981) J. Mol. Biol. 1 4 9 , 541-550 5. Pearlstone, J. R., and Smillie, L. B. (1978) Can. J. Biochem. 56,521-527 6. Pearlstone, J. R., and Smillie, L. B. (1980) Can. J. Biochem. 58,649-654 7. Imai, H., Hirai, S., Hirono, H., and Hirabayashi, T. (1986) J. Biochem. (Tokyo) 99,923-930 8. Breitbart, R. E., Nguyen, H. T., Medford, R. M., Destree, A. T., Maldavi, V., and Nadal-Ginard, B. (1985) Cell 41,67-82 9. Medford, R. M., Nguyen, H. T., Destree, A. T., Summers, E., and Nadal-