The Coevolution of Insect Muscle TpnT and TpnI Gene Isoforms

Raul Herranz, Jesus Mateos, Jose A. Mas, Elena Garcıa-Zaragoza, Margarita Cervera, andRoberto MarcoDepartamento de Bioquımica, Instituto de Investigaciones Biomedicas ‘‘Alberto Sols’’ UAM-CSIC, Facultad de Medicina,Universidad Autonoma de Madrid, C/Arzobispo Morcillo 4, 28029 Madrid, Spain

In bilaterians, the main regulator of muscle contraction is the troponin (Tpn) complex, comprising three closely interactingsubunits (C, T, and I). To understand how evolutionary forces drive molecular change in protein complexes, we havecompared the gene structures and expression patterns of Tpn genes in insects. In this class, while TpnC is encoded bymultiple genes, TpnT and TpnI are encoded by single genes. Their isoform expression pattern is highly conserved withinthe Drosophilidae, and single orthologous genes were identified in the sequenced genomes of Drosophila pseudoobscura,Anopheles gambiae, and Apis mellifera. Apis expression patterns also support the equivalence of their exon organizationthroughout holometabolous insects. All TpnT genes include a previously unidentified indirect flight muscle (IFM)–specificexon (10A) that has evolved an expression pattern similar to that of exon 9 in TpnI. Thus, expression patterns, sequenceevolution trends, and structural data indicate that Tpn genes and their isoforms have coevolved, building species- andmuscle-specific troponin complexes. Furthermore, a clear case can be made for independent evolution of the IFM-specificisoforms containing alanine/proline-rich sequences. Dipteran genomes contain one tropomyosin gene that encodes one ortwo high–molecular weight isoforms (TmH) incorporating APPAEGA-rich sequences, specifically expressed in IFM. Cor-responding exons do not exist in the Apis tropomyosin gene, but equivalent sequences occur in a high–molecular weightApis IFM-specific TpnI isoform (TnH). Overall, our approach to comparatively analyze supramolecular complexes revealscoevolutionary trends not only in gene families but in isoforms generated by alternative splicing.

Introduction

One of the most intriguing aspects of molecular evo-lution is the multiplicity of isoforms produced by genes(Roberts and Smith 2002), the functional significance ofwhich still largely escapes our understanding. Gene diversi-fication has clearly relied on two mechanisms, either entiregenes (or even chromosome fragments) are duplicated gen-erating a gene family, or specific exons are duplicated alongwith the capacity for generating alternatively spliced tran-scripts.Many studies have compared the evolutionary advan-tages of each process (Lynch and Conery 2000; Ohta 2000;Lynch 2002). Exon-duplication events seem to be preferred ifthe result increases the variability of only small domains ina protein (Abi Rached, McDermott, and Pontarotti 1999).

Particularly relevant are the cases where groups of pro-teins interact to form high supramolecular complex struc-tures, especially if the physiological effects of isoformsare well characterized. Our group has been studying thesegene-evolution processes in troponin, a thin-filament com-plex, by focusing on one of the most diversified branches oflife, the insects. The troponin complex comprises three sub-units, namely troponin C (TpnC), troponin I (TpnI), andtroponin T (TpnT), and mediates the thin-filament responseto calcium when striated muscular contraction is initiated.In mammalian systems, extensive studies (Filatov et al.1999; Gordon, Homsher, and Regnier 2000; Gordon,Regnier, and Homsher 2001) have addressed the structure-function relationships between the Tpn complex subunits.As a consequence of Ca21 binding to TpnC, this polypep-tide undergoes a conformational change, altering its in-teraction with TpnI, releasing the inhibitory TpnI-actin

binding, and allowing movements of the troponin-trpomyo-sin complex on the F-actin that increases myosin binding toactin and thus promoting contraction.

The gene numbers for each troponin constituent varyamong the metazoa. While only one copy each of the TpnTand TpnI genes occur in insects, up to three copies are foundin vertebrates. There are only two TpnC genes in mammals(encoding cardiac and striated isoforms), but five to six tro-ponin C genes have been found in holometabolous insects(Herranz, Mateos, and Marco 2005). Nevertheless, eachtroponin subunit exists in multiple isoforms. The singleDrosophila troponin I gene has a total of 13 exons, andup to 10 different TpnI isoforms are produced by alterna-tive splicing processes of some exons (Beall and Fyrberg1991; Barbas et al. 1993). Variability regions are localizedat specific points across the protein encoded by the alterna-tive exons 3 and 9 and by four mutually exclusive exon 6’s.The TpnT gene in both Drosophila melanogaster andDrosophila virilis was described as containing a set of11 exons and up to four alternatively splicing isoforms,generated by the inclusion or exclusion of exons 3, 4,and 5 (Benoist et al. 1998). No splicing variants affectingthe Ct half of the protein were described.

Muscle structure in protostomes is much more diversethan in deuterostomes. For example, holometabolousinsects have two different musculatures, the larval oneformed during embryogenesis, and the adult one formedduring the pupal metamorphosis, when the muscles respon-sible for flight develop in the adult thorax. These musclesare the indirect flight muscles (IFMs), responsible for wingbeating, and the tergal depressor of the trochanter (TDT)muscle, responsible for the jump at take-off of the fly.Stretch activation is an important property of IFM in manyinsects. Its direct consequence is that their oscillatory con-tractions are asynchronous, meaning that the nervousimpulses received by the fibers are more irregular and lessfrequent than the contractions themselves. Specific protein

Key words: troponin, tropomyosin, coevolution, Insecta, Drosophila,Anopheles, Apis, phylogenetics.

E-mail: roberto.marco@uam.es.

Mol. Biol. Evol. 22(11):2231–2242. 2005doi:10.1093/molbev/msi223Advance Access publication July 27, 2005

� The Author 2005. Published by Oxford University Press on behalf ofthe Society for Molecular Biology and Evolution. All rights reserved.For permissions, please e-mail: journals.permissions@oupjournals.org

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variants are expressed in these muscles, sometimes encodedby different genes, as for example actin (Lovato et al. 2001)or troponin C (Qiu et al. 2003; Herranz et al. 2004), butmore usually as the result of differential splicing of tran-scripts from a single gene. In addition, other muscle-specific components, such as arthrin and troponin H, arethin-filament proteins exclusively expressed in the IFMs.Arthrin is an ubiquitinated actin independently acquiredin several insect orders (Schmitz et al. 2003). TroponinH (Bullard et al. 1988), a high–molecular weight compo-nent of Lethocerus flight muscles, was identified as similarto the heavy tropomyosin isoforms expressed in DrosophilaIFM (Karlik and Fyrberg 1986; Hanke and Storti 1988).Troponin H and heavy tropomyosin (TmH; Mateos et al.,unpublished data) contain alanine/proline-rich extensionsin the Ct of the proteins.

In the present work, we describe how TpnI and TpnTisoforms have coevolved in insects. Our purpose in under-taking a systematic study of the insect troponin genes is tounderstand the coevolutionary mechanisms that led to func-tional divergence of isoform variation generated by the dif-ferential expression of the troponin complex genes. Thespecific repertoire of troponin isoforms expressed in distinctmuscle types or stages of development has been estab-lished. In particular, we have analyzed the TpnI and TpnTgenes of four Drosophilidae species (melanogaster,subobscura, pseudoobscura, and virilis), the evolution ofwhich diverged during the last 60 Myr, as well as inAnopheles gambiae and Apis mellifera that diverged fromthe Drosophilidae around 250 and 300 MYA, respectively(Powell 1997; Dudley 2000; Gaunt and Miles 2002).

With these objectives, our studies have allowed us toidentify previously undescribed features of the troponinsT and I in insects. We have identified a new mutually ex-cluding exon (10A) for the troponin T gene that producesan IFM/TDT differentially expressed isoform, addinga new variability region near the 3# end of the gene.We have refined the information on the TpnI expressionpatterns identifying TpnI exon 9 as more specific thanoriginally described, and as the functional partner of theTpnT exon 10A isoform in the IFM. In contrast to thesituation in Diptera, in Apis the alanine/proline-rich exten-sions do not occur attached to the Tm gene but are en-coded in the TpnI gene, as three APPAEGA-rich 3#exons, H1, H2, and H3.

Materials and MethodsInsect Stocks

The Oregon R strain was used as wild-type source ofD. melanogaster material. Drosophila subobscura was ob-tained from Rosa de Frutos (Valencia, Spain) and D. viriliswas from Manuel Calleja (Madrid, Spain). These speciesdiverged from D. melanogaster around 12 and 50 MYA(Powell 1997; Gaunt and Miles 2002). Apis mellifera sam-ples were obtained in the Summer 2003 from IndustriasAlonso located in El Vellon (Madrid). We have also in-cluded the data obtained from D. pseudoobscura (HumanGenome Sequencing Project at Baylor College of MedicineBlast server) and the mosquito A. gambiae (Holt et al. 2002)genomes.

Nucleic Acid Extraction

Tissues from D. subobscura and D. virilis were ob-tained at different developmental stages as well as adultbody parts (head, thorax, and abdomen). Dissections weredone in cold acetone at �70�C. RNA extractions weremade using TRIZOL Reagent (Invitrogen, Paisley, UK).The same procedure was carried out with A. mellifera(two different larval and two pupal stages, as well as re-cently emerged and mature honeybees, that were dissectedto separate heads, thoraces, and abdomens). Specificmuscles, IFM or TDT, were recovered from animals pre-treated for at least 1 week in dehydrating acetone solutionat �20�C, which facilitates the fiber isolation. GenomicDNA was extracted using a Tris-HCl 10 mM pH 7 homog-enizing solution containing 0.5% sodium dodecyl sulfateand NaCl 60 mM, followed by a standard phenol-based pu-rification (Sambrook, Fritsch, and Maniatis 1989).

Reverse Transcriptase–Polymerase Chain Reaction

We used total RNA (2 lg) and oligo-dT (1 lg) in orderto normalize the amount of cDNA used as template in thepolymerase chain reactions (PCRs). Specific probes weredesigned for each gene amplification, based on knownsequences fromD. melanogaster orA. mellifera. PCRswerecarried out for 30–35 amplification cycles (94�C/30 s, 55–60�C/45 s, and 72�C/45 s) with thermostable polymerase(DyNAzime, Fynnzymes) in aGeneAmp2700System ther-mocycler of PE Applied Biosystems (Foster City, Calif.).For genomic fragment amplifications, hybridization andelongation times were extended up to 1 min and TpnIPCR experiments were performed in the presence of2.5% dimethyl sulfoxide.

Rapid Amplification cDNA Extension

We have determined the 3# untranslated region (UTR)of the different TpnI transcripts in A. mellifera by using theAmbion FirstChoice RLM-RACE Kit, with a TpnI exon8 probe plus the 3# outer and inner kit probes.

Cloning and Sequencing

Standard commercial protocols were followed forDNA extraction (Concert Rapid Gel Extraction, GIBCO),ligations (pGEM-T, Promega, Madison, Wisc.), Escheri-chia coli transformations (DH5a strain), and plasmid puri-fication (Wizard Plus SV minipreps, Promega) sometimesfollowed by a phenol-based concentration step (Sambrook,Fritsch, and Maniatis 1989). Standard probes included inpGEM-T easy plasmid (SP6 and T7) were used for sequenc-ing reactions in an automatic sequencer. All the detected re-verse transcriptase (RT)–PCR products were sequenced inthis way using internal oligonucleotides when necessary.

Bioinformatics Tools

Sequences were obtained and preliminarily comparedby National Center for Biotechnology Information Blast(Altschul et al. 1997). Sequence information was processedusing GeneJockey II software. Phylogenetic trees wereobtained from sequence comparisons and alignments based

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on ClustalW with BIOEDIT (Hall 1999) software using‘‘Fitch-Margoliash and least-squares distance methods’’ al-gorithm or remotely with Phylodendrom version 0.8d, beta1999, and ClustalW version 1.75 (neighbor-joining methodwith 1,000 bootstraps [Thompson, Higgins, and Gibson1994]). Tree output was rendered with TreeView version1.6.6 (Page 1996). Branches with bootstrap values below70%have been collapsed. The 2D structure predictionswereobtained from the ExPASy web server using the PSIPREDmethod (Jones 1999; McGuffin, Bryson, and Jones 2000).

Accession Numbers

Sequences obtained or annotated by us have beensubmitted to the corresponding section of the GenBankwith the following accession numbers: D. melanogasterand D. virilis TpnT gene, AY439172 and AY439178–9;TpnT and TpnI Drosophilidae transcripts, AY439173–7,AY439180–4, and BK001654–5; and A. gambiae and A.mellifera TpnT and TpnI genes, BK005279–82. This infor-mation has also been prepared in a Supplementary MaterialTable A (Supplementary material online) that also includesthe accession numbers of the rest of the sequences usedin this work. All gene annotations contain a detailed de-scription of all the identified transcripts including theirsequences and expression patterns.

ResultsConservation of Orthologous TpnI and TpnT GeneStructures in Insects

We have used the previously reportedD. melanogasterTpnT and TpnI gene sequences (Barbas et al. 1993; Benoistet al. 1998) to find putative orthologues in theAnopheles andApis genomes. The structural organization of these geneshas been preserved to a high degree in insects (fig. 1). Ingeneral, we observe an increase in gene size, mainly dueto intron length expansion from the smallest genome—178Mb in D. melanogaster (Adams et al. 2000)—to those ofAnopheles and Apis that are almost double in size (Holtet al. 2002). Chromosomal location is not a broadly pre-served feature in insects, neither for TpnC genes (Herranz,Mateos, andMarco 2005) nor for those of TpnI or TpnT. TheTpnT and TpnI genes are both located on the D. melano-gaster X chromosome, but the Anopheles TpnT gene islocated cytologically on chromosome arm 2R, while TpnIremains to be localized (Mongin et al. 2004).

We have cloned and sequenced the transcripts of thesegenes in the drosophilid species and Apis, paying specialattention to the alternatively spliced exons. In the case oftroponin T, the differentially spliced Nt exons are presentin all the analyzed species, although in Apis the TpnT geneshows an additional alternatively spliced exon, named 5#.The Apis equivalent to the dipteran exon 6 is divided intofive constitutive exons (a, b, c, d, and e). Sequencing oftranscripts identified a new variable exon 10, that had beenoverlooked in previous studies of D. melanogaster andD. virilis sequenced TpnT transcripts (Benoist et al.1998). Comparison of the TpnT genomic sequences in in-sects showed that two different exons, named 10A and 10B,are present in all holometabolous species. Both are 79-ntlong, encoding 26 aa. Another difference in the TpnT gene

structure is found in exon 11, which encodes a long poly-glutamic tail with variable size in all studied protostomespecies (Benoist et al. 1998).

The constitutive exons and exon 9 of TpnI gene arepresent in all analyzed insects (fig. 1), but exon 3 is not,to the extent that the Drosophila orthologous exons 2 and4 are fused in a unique exon in Anopheles, but not in Apiswhere a smaller exon 3 is found the main variability of Tro-ponin I arises from mutually exclusive exon 6’s. In the caseofAnopheles, we detected three exon 6 variants by sequencehomology, while four were detected in Apis. Interestingly,additional exons, that we have designated H1, H2, and H3,were found downstream of exon 10 in the Apis TpnI gene,encoding the APPAEGA-repetitive sequences (see below).

Sequence Variations of the Alternatively Spliced Exonsin TpnT and TpnI Genes

Amino acid sequences from TpnT and TpnI constitu-tive exons are conserved almost perfectly in insects. Asshown in figure 2A, even the sequences of the alternativelyspliced exons (3, 4, and 5) from TpnT are well conservedamong the Drosophilidae. Exons 3 and 4 are also conservedin A. gambiae and A. mellifera while a higher degree ofvariation is found in exon 5, reflecting the evolutionary dis-tance between these insects. In this region, the Apis TpnTgene has an additional alternatively spliced exon. Exons 3and 4 are clearly conserved, but exons 5, 5#, and the con-stitutive exon 6a have very different sequences, being moresimilar to sequences in the orthopteran Periplaneta (Wolf1999) and the odonate Libellula TpnT exons (Fitzhugh andMarden 1997; Marden et al. 1999, 2001) despite the muchlarger evolutionary distance of these species from holome-tabolous insects.

The sequences of the mutually exclusive TpnT exons10A and 10B (fig. 2B) are also well preserved among Dro-sophilidae. Larger differences are found in Anopheles andApis. Nonequivalent variations in exon 10A are equally dis-tributed throughout the exon, but less so in exon 10B. In-terestingly, a higher number of threonine residues appear ininsect exon 10A than in exon 10B, but only the last thre-onine of exon 10A is conserved in all the insects analyzed.Drosophila TpnT isoforms are readily phosphorylated invivo (Domingo et al. 1998), precisely in the exon 10A–encoded sequence (Nongthomba and Sparrow, personalcommunication).

With regard to the TpnI genes, constitutive exons arealmost strictly conserved and the sequence of alternativeexons 9 and 10 (fig. 2C) reflect a situation similar to thatof TpnT exons 10A and 10B. Even though we know thatthe alternatively spliced sequences are short, phylogenetictrees based on the comparison among the sequences encodedby these exons can be constructed. Although this informa-tion is not sufficient to establish the evolutionary origin ofthese exons, it can be used to detect how they have beenchanging in different insect groupswhen the data of all exonsare analyzed together. In both Tpn genes, one of the dupli-cated exons (TpnT exon 10A and TpnI exon 9) appears tobe less conserved than the other (TpnT exon 10B and TpnIexon 10), showing similar evolutionary patterns in each ofthe three insect groups analyzed. It is therefore likely that

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the duplication of these TpnT and TpnI alternative exonsmay have occurred at the origin of the holometabolous-typeof development. In accordance with this idea, only a singleTpnT exon 10 with intermediate sequence features has beenfound in the hemimetabolous Periplaneta americana andLibellula pulchella. Furthermore, only a single exon 9/10has been found in the arachnid Haemaphysalis longicornisTpnI gene (You et al. 2001). Interestingly, TpnI exon 9 inthe Drosophilidae does not contain a complete stop codon,which is formed by the splicing of its last nucleotide withthe two first bases of exon 10 (Barbas et al. 1993). Thisfeature is not observed in Anopheles or Apis, where stopcodons and 3# UTRs are found both in exons 9 and 10.

The TpnI mutually exclusive exon 6 sequences havealso been analyzed. In a consensus phylogenetic tree based

on exon 6 nucleotide sequences (fig. 3A), the low bootstrapvalues at the base of the insect tree suggest an ancient originfor these duplication events, but the tree supports a cleardifferentiation of exon 6a’s from exon 6b’s. The 6b exonsare variable enough to be phylogenetically discriminative.The 6a exons are so different among themselves that theApis paralogous exons 6a1 and 6a2 are as related to eachother as to their putative drosophilid orthologous exons(see the polytomy in the 6a exon branch). The presenceof a single exon 6a in Anopheles and the loss of the exon6a2 canonical donor-splicing signal in the Drosophilidaeare also noteworthy. In a sequence alignment (fig. 3B), itcan be seen that each Anopheles and Apis exon 6 is asclosely related to each other as it is to those from the Dro-sophilidae. Finally, the Drosophilidae TpnI exon 3 (fig. 4A)

FIG. 1.—Troponin T (top) and troponin I (bottom) gene structures in holometabolous insects. Genes are labeled with the species binomial two-letterabbreviation. Noncoding (white), constitutive (gray), alternative (black) or mutually exclusive (diagonal stripes), and the PAANGKA- or APPAEGA-containing (horizontal stripes) alternative exons are indicated. Light shaded exon 6’s in TpnI have not yet been detected in RT-PCR experiments. Exonnumbers in Anopheles and Apis genes have been assigned by homology to those of their Drosophila counterparts. Genomic DNA is represented bya continuous line that is therefore missing in gene sequences derived from cDNA data. Gene organization is well preserved overall, and gene sizecorrelates with the organism genome size. The main difference affects alanine/proline-rich sequences including the TpnI exon 3 encoding the PAANGKAsequences in the Drosophilidae, but not in Anopheles or Apis. Several Apis TpnI gene exons encode an APPAEGA-containing sequence in their 3# region.

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are essentially alanine/proline-rich sequences. All show thePAANGKA motif which appears repeated several times atdifferent positions in the D. melanogaster, D. subobscura,and D. virilis extensions. This domain is absent in Anoph-eles and in Apis TpnIs, where a very short constitutive exon3 is found.

Three new exons have been located downstream fromexon 10 in the Apis TpnI gene. These exons can be splicedtogether to produce an Apis-specific TpnI isoform contain-ing a Ct extension encoding a repetitive APPAEGA se-quence, through a new GT-splicing donor site in exon10, located 25 bp before the stop codon. This extensionis clearly similar to the alanine/proline-rich extension of

the large–molecular weight IFM-specific tropomyosin inthe Diptera, both in sequence and protein length (fig. 4B).Interestingly, the Apis tropomyosin Tm1 gene lacks thistype of sequence (Mateos et al., unpublished results). Asimilar repetitive alanine/proline extension is found atthe 3# end of a TpnI gene transcript from the hemipteranLethocerus (Qiu et al. 2003 and sequence AJ621044).

Expression Pattern of TpnI and TpnT Genes AreConserved in Drosophilidae

The expression profiles of TpnI and TpnT isoforms inD. subobscura and D. virilis were detected using RT-PCR

FIG. 2.—Alternatively spliced exons in TpnT and TpnI genes. (A) Alignment of the 5# region of the troponin T transcripts in insects. Amino acidconservation (d), silent variation ( ) (cDNA), equivalent variation ( ), and nonequivalent variation ( ) in relation to the Drosophila melanogaster se-quence are shown. These sequences, labeled with the two-letter species abbreviations and the corresponding exon number (E10A Dmmeans exon 10A inD. melanogaster for instance), are conserved in the four drosophilid species (only silent variation appears) and also in the Anopheles andApis exons exceptexon 5. The Libellula (AF133521) and Periplaneta (AF133520) TpnT sequences (obtained from GenBank) retain the same properties although thealternative exon sequences show higher levels of variation. (B) Alignment and phylogenetic tree of the alternative TpnT exon 10 sequences in insects.Branches with bootstrap values below 70% have been collapsed in this cDNA-based tree. Most changes affect the number of phosphorylable residues,indicated at the right of the alignment, with the exception of the conserved final threonine marked with an asterisk. (C) Alignment and phylogenetic tree ofthe alternative TpnI exon 9 and 10 sequences in insects. The Haemaphysalis longicornis TpnI sequence (AB051079), in which exon 9 has not beendescribed, was used as out-group.

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techniques (unpublished data). All the transcripts previ-ously identified in D. melanogaster (Barbas et al. 1993;Benoist et al. 1998) were detected in the second instar lar-vae and late pupae stages of these drosophilids. Combina-tions of the troponin T alternative exons 3, 4, and 5appeared in larvae or in adult abdominal muscle transcriptsin patterns characteristic for each muscle. The major tran-scripts of the adult thoracic muscles do not contain any ofthese alternative exons. Troponin I exon 9 inclusion isfound exclusively in transcripts of the adult musculature,

being accompanied by exon 3 in the major thoracic tran-script. The four exon 6 types were detected at varying levelsin all the tissues studied, those containing exon 6b1 beingthe more highly expressed in IFM (Barbas et al. 1993).

The discovery of the newTpnT exon 10A led to a studyof its expression pattern.A single probe for constitutive exon6 and four specific probes for exons 10A or 10B (fig. 5A)were used for RT-PCR. In embryos, only the 10B exon-containing transcript was detected, while in whole adultRNAs, transcripts containing either 10A or 10B appear.

Detection of exon 10A was particularly strong in thethorax, suggesting a specific role of the sequence encodedby this exon in the flight-related musculature (data notshown). Exon 10A- and 10B-specific probes detected, byRT-PCR, the classic 10B exon in the head, abdomen,and dissected TDT fibers (fig. 5B). Exon 10A expressionwas found almost exclusively in the RT-PCR product fromRNA extracted from IFM and TDT fibers although someresidual expression was also detected in abdomens. Con-sequently, IFM TpnT contains sequences encoded onlyby exon 10A, while TDT muscles contain a mixture oftranscripts with exon 10A or 10B. The same thoracicenrichment of exon 10A transcripts was obtained inD. subobscura and D. virilis.

TpnI alternatively spliced exons were detected usingseveral probe combinations. Exon 9 is present in TDTand IFM transcripts (fig. 5C), but while in IFMs it is theonly one expressed, in TDT it is almost completely replacedby the exon 10–containing transcripts. Using probes for thecomplete coding region (exon 2 to exon 9/10) in the adultbody parts showed that the alternatively spliced exon 3 isexpressed in TDT/IFM, but is only present in transcriptsthat also incorporate exon 9 (fig. 5D).

Expression Profile of the TpnT and TpnI Genes inA. mellifera

The same procedure has been carried out with thehoneybee to discover if the same patterns of isoform abun-dance occur. The expression profile of the TpnT transcriptsduring A. mellifera development (fig. 6A) shows that larvalmuscles contain a mixture of transcripts with alternativeexons 3, 4, and 5# or exons 3 and 4. The IFMs containan isoform encoded by a transcript lacking all the 5# regionalternatively spliced exons. Other adult muscles contain amixture of transcripts containing the exons 3, 4, 5, and 5#or 3, 4, and 5. In relation to the 3# half of the genes, exon10B–containing transcripts were found in all muscles exceptin IFM, where it is only marginally detected (dorsoventralindirect flight muscle) or not at all (dorsolateral indirectflight muscle). Exon 10A–containing transcripts are adultthorax-specific, similar to the drosophilid results (seeabove).

The TpnI expression pattern detected using RT-PCR isshown in figure 6B. Probes for the region between exons 4and 9 or exons 4 and 10 revealed that transcripts containingexon 9 or exon 10 are detected in the different developmen-tal stages and in all muscles of the adult Apis musculature.Despite inherent limitations to these PCR experiments,exon 9–containing transcripts seem to appear later thanthose containing exon 10. Using probes for each specific

FIG. 3.—Phylogenetic tree and alignment of the differentially splicedTpnI exon 6’s. (A) Branches with bootstrap values below 70% have beencollapsed in this cDNA-based tree. Exons 6b1 and 6b2 are clearly sepa-rated, but an exon 6a phylogeny cannot be extracted from the tree. (B)Alignment of the translated exon 6 sequences (34 aa translated fromthe last nucleotide of exon 5 plus the exon 6 nucleotides), flanked bythe splicing sequences when available. The sequences are designatedby the two-letter species abbreviation and the corresponding exon number(Dm E6b1 for instance). The 6a exons are the more variable ones, havingeven lost the canonical splicing donor sequence (bold and italics letters inthe figure) in the drosophilid 6a2 exons.

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exon 6 and exon 8 in an inner PCR of the previous amplifiedproducts, the four exon 6’s in both exon 9– and exon 10–containing transcripts were detected. Although quantitativePCR was not carried out, the results indicate that maximumexpression levels of exons 6a and 10 occur during larvalstages, while those of 6b and 9 exons occur in adults.

Finally, RT-PCR using probes designed to amplify theregion between exon 4 and exon H1 of the Apis TpnI geneshowed, as expected, that a heavy Apis TpnI (troponin H) isexpressed exclusively in the IFM (fig. 6B). This band wascloned and tested for its exon 6 content, using an exon 8probe in combination with a specific probe for each oneof the four exon 6. Sequencing of 14 clones confirmed that7 clones contained exon 6b1 and the other 7 clones con-tained exon 6b2. In addition, these TpnH transcripts includea shorter spliced version of exon 10 that could not be de-tected in earlier RT-PCR assays using the exon 10 probe. Inorder to locate the 3# ends of the TpnI transcripts, we haveperformed rapid amplification cDNA extension experi-ments with three representative Apis RNA fractions(fig. 6C). We found up to eight different transcript endsfor the TpnI gene, but only three major ones occur in mRNAfrom the thoracic muscles and IFM samples. Two of themare 3# to exon 9 and exon 10, produce the standard TpnItranscripts, and are also used in the larval muscles. Theother end incorporates the initial part of exon 10 but extendsinto exons H1, H2, and H3, producing a TpnH isoformwith a long alanine/proline-rich extension. We have alsodetected a transcript in which exon H1 does not use its

acceptor-splicing site and this would produce a TnHisoform with a shorter alanine/proline-rich extension.

Discussion

It is generally accepted thatmuscle tissue arose early onin the evolution of bilateria. Muscle structure and its mech-anism of function have been retained relatively unchangedsince that time, although individual muscle tissues are fine-tuned to reflect their physiological functions. Because themyofibril comprises a series of supramolecular complexes,studying the evolution of an individual protein must be car-ried out in the context of the complex inwhich it is a part.Wehave initiated this analysis by focusing on one of the better-characterized substructures, the thin filament, starting withits main regulatory switch, the troponin complex. In thiswork, a comparative approach has been used to expandthe description of how troponin genes have evolved ininsects to reach their final tissue-specific isoform repertoires.

Similarly Evolved Troponin Complex IsoformsShare a Similar Expression Pattern

Comparing gene sequences in the four drosophilid spe-cies has made us aware of a new set of isoforms in both tro-ponin I and T genes and we have established their specificexpression in the IFM. Alternative splicing of TpnT exon10A increases variability at the 3# end of these transcripts.Interestingly, the existence of two variable regions at both

FIG. 4.—Alignment of the IFM-specific troponin I and tropomyosin alanine/proline-rich extensions. (A) Although the TpnI exon 3 sequences are notperfectly conserved even in the Drosophilidae, the heptad PAANGKA (shaded box) is conserved. It is repeated in some species surrounded by an alanine/proline-rich region. This sequence is absent in Anopheles and Apis because their TpnI genes lack an exon 3 with these properties. (B) The TpnH extensionis encoded by a single Tm1 gene exon in Anopheles, while in the Apis TpnI gene it involves up to three gene 3# exons (separated in the three rows of thealignment), all of them encoding an alanine/proline-rich sequence with the APPAEGAmotif (shaded box). An equivalent sequence has been found in theLethocerus TpnI gene (AJ621044) and is included as the final sequence in the alignment.

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the Nt and Ct of the proteins causes the distribution ofvariability in insect TpnT and TpnI alternatively splicedregions to resemble more closely the situation of the verte-brate genes.

Table 1 highlights the coordinate expression of differ-ent transcripts of the three Tpn subunits in various droso-philid muscle types. In particular, it exposes the expressionpattern coincidences between the TpnI exon 6’s and theTpnC genes, and between TpnI exon 9/10 and TpnT exon10A/B, also observed in Apis. All these expression partnersseem to have evolved in a similar way as a result of theirinteractions in the thin-filament structure. Insect TpnCgenes also show a complex evolutionary pattern, involvingboth divergence and convergence events (Herranz, Mateos,and Marco 2005). For instance, Type I TpnC genes, ex-pressing larval hypodermic isoforms, have evolved inde-

pendently in holometabolous insects, leading to tworecently acquired isoforms in the drosophilid species,only one isoform in Anopheles, and two distantly relatedisoforms in Apis. TpnI 6a exons, the transcripts of whichare mainly expressed in larval hypodermic muscles, mayhave evolved similarly. There is just one in Anophelesand two 6a exons in the Drosophilidae and Apis thatcould have been obtained independently in both insect or-ders. A different case of coupled evolution affects the TpnIexon 9 and TpnT exon 10A pair so that after appearing,probably at the beginning of the holometabolous insectbranching, they became coexpressed in the very specializedIFM. In fact, this pair of exons shows higher sequence var-iability than its counterpart, the TnI exon 10 and TnT exon10B pair. This higher variability in IFM-expressed isoformscan be related with the strong selection forces under thesemuscles.

Alternatively Spliced Exon Coevolution Can BeLinked to the Tpn Complex Structure

Following these ideas, our aim was to explore how fara functional basis of this coexpression/coevolutionary trendcould be established. Comparing the secondary structurepredictions of the insect and human orthologous troponinproteins led to conclusions that, although the sequenceconservation between deuterostomes and protostomestroponins is not very high, the structural predictions are con-served. For example, as can be seen in figure 7, the second-ary structure predictions of Drosophila IFM TpnI isoformand the human cardiac TpnI are remarkably conserved. Thisconservation includes the interaction sites of TpnI withTpnC, Tm, TpnT, and actin as previously described in mam-mals (Filatov et al. 1999; Gordon, Homsher, and Regnier2000; Gordon, Regnier, and Homsher 2001) and recentlywas confirmed in a crystal structure of half of a cardiac tro-ponin complex (Takeda et al. 2003). In particular, the insectTpnI alternative exon 6b2 sequence is equivalent to that ofthe cardiac TpnI exon interacting with the TpnCNt sequenceor actin, depending on the calcium concentration. All theseinformations, together with structural data from vertebratesand from the insect Lethocerus (Wendt, Guenebaut, andLeonard 1997; Wendt and Leonard 1999), lead to a modelfor the troponin complex interactions in Drosophila thattakes into account possible effects of isoform variability.Complex integrity may be stabilized by the interaction ofthe TpnI and TpnT variable Ct regions (exon 9/10 and exon10A/10B, respectively) and TpnINt regionwith the globularCt domain of TpnC. TpnT interacts with Tm mainly throughits central domains, but its Ct polyglutamic tail and itsNt variability region (also polyglutamic rich) probably con-tribute to stabilize this interaction.

The expression patterns (table 1), sequence evolution-ary trends (figs. 2 and 3), and the secondary structure pre-dictions (fig. 7) taken together indicate that the alternativelyspliced exons inTpnI andTpnTgenes have evolved in a con-certed way, a result consistent with the interactions of theirproducts in the troponin complex. However, this has oc-curred independently in the different insect orders, at leastfor TpnC genes (Herranz, Mateos, and Marco 2005) andfor TmH/TpnH (Mateos et al., in press). The relationship

FIG. 5.—Differences in the expression profile of TpnT and TpnI tran-scripts in the Drosophila melanogaster musculature. Agarose 1.2% gelseparations of RT-PCRs. Each transcript detected by PCR as a band ismarked with an arrow labeled with the name of the amplified gene andits exon composition. (A) Expression pattern of mutually exclusive exon10’s detected in adult or embryonic RNA extractions with four exon 10A-or 10B-specific primers (10A1, 10A2, 10B1, and 10B2). (B) Patterns of ex-pression in adult body parts (head, IFM, TDT, and abdomen). Using probesfor TpnT exon 6 and 10A (10A2 probe) or 10B (10B1 probe), the exon 10expression pattern is shown. Exon 10B is expressed in all muscles in theadult except in the IFM. Exon 10A is the only one expressed in the IFM,but it is also expressed in the TDT muscles. (C) Probes for the 3# region ofthe TpnI gene were used in RT-PCRs (exons 8–9 and 8–10). Exon 9 usesthe first two residues of exon 10 to generate a stop codon, so the exon 10probe detects it as a lower band, the transcripts lacking exon 9, and asa higher band, the transcripts containing exon 9. So exon 9 is expressedmainly in IFM and only weakly in TDT muscles. (D) Using exon 2 andexon 9 probes for TpnI, it can be seen that exon 3 only appears heavilyexpressed in thoracic muscles including always exon 9. RT-negativecontrol lanes are not shown.

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between the variable regions of the troponin complexproteins and their putative interactions with coexpressedisoforms in different muscles or developmental stages arerepresented in figure 8 as an evolutionary/functional net-work for the different insect groups. The interactions amongthe different isoforms of the troponin-tropomyosin complexcomponents produced by alternative splicing (TpnI andTpnT) or differential gene expression are indicated in thefigure. The overall conservation of the relationships ofTpnI with TpnC and TpnT can be observed among theinsects studied. The main exception lies in the switchingof the TmH for TnH in Apis, as discussed below. Further-more, in the drosophilids, a TpnI exon 3 incorporating aPAANGKA heptad repeat is also only expressed in Dro-sophila IFM because Anopheles and Apis lack this exon.The reasons for this variability remain to be clarified.

Troponin H Alanine/Proline-Rich Extensions Absent inApis Tm1 Gene Are Located in the TpnI Gene

APPAEGA heptad repetitions are encoded in the IFM-specific exons (16 and 17) of the Drosophila tropomyosinTm1 gene that produces two heavy tropomyosins (TmH 33and 34) with long Ct extensions. In the Anopheles Tm1gene, only one exon containing this kind of sequencehas been located. Interestingly, in Apis the tropomyosingenes lack these long Ct extensions (Mateos et al., unpub-lished data) but a similar region is present in the TpnI genewhere it is encoded by exons H1, H2, and H3 spliced to-gether to produce a similarly sized, APPAEGA-rich exten-sion (figs. 1 and 4). We have also found a transcriptcontaining only the first 30% of the alanine/proline-rich ex-tension in the IFM. The presence of these TpnH extended

FIG. 6.—Expression profile of TpnI and TpnT transcripts in Apis mellifera. Each transcript detected by PCR as a band is marked with an arrow labeledwith the name of the amplified gene and its exon composition. Adjacent empty lanes to each PCR lane are negative controls (mRNA without reversetranscription). (A) Troponin T expression profile during development (larval L1 and L2 and pupal P1 and P2 stages) and in the thorax muscles (dor-soventral indirect flight muscles [DV-IFM], dorsolateral indirect flight muscles [DL-IFM], legs, and other thoracic muscles) of Apis. Apis TpnT showsa similar expression pattern to that described in Drosophila. (B) The TpnI expression pattern in the same sample is shown. The alternative exons, 9 and 10,both containing a proper stop codon and terminator signals, are expressed in different levels in all muscles and stages. Using probes from exon 4 and exonH1 of the Apis TpnI gene, we have detected an IFM-specific expression pattern for this TpnH isoform in hymenopterans. The four exon 6’s identified areexpressed in the TpnI transcripts (detected by inner PCR using internal exon 6 type-specific probes in a second PCR using the P2 fraction band as substrateDNA), except in the IFM-specific transcript where only 6b exons are detected. (C) The 3# rapid amplification cDNA extension (RACE) experiments wereperformed with thorax, IFM, and larval samples to detect the transcription stops in the Apis TpnI gene. Transcripts representing eight different stop siteshave been proportionally represented on the left, with their size indicated and different arrows styles (depending on the transcripts last exon, dotted line forexon 9, continuous line for exon 10, and broken line for exon H’s) signaling the corresponding band in the RACE electrophoresis gels. Three of them (onetranscript for each group) are the major ones used in the thoracic muscles (their lengths are shown inside circles), but the transcript containing the TpnHexons is absent in larval muscles. RACE-negative control lanes are shown (� lanes).

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isoforms in the Apis TpnI gene does not affect the standardrepertoire of TpnIs, which are similarly processed and ex-pressed as in dipterans.

Asynchrony as a Trigger for Independent Coevolutionof the Troponins

The main variability in the isoforms’ expression andsequences occurs in the asynchronous IFM. Two ideasare generally accepted in relation to these muscles. First,no single biochemical feature is known that completely cor-relates with the stretch-activation phenomenon that is alsoobserved in skeletal and cardiac muscles of vertebrates(Steiger 1977). The phenomenon is much stronger and

persistent in insect IFM (Linari et al. 2004). Second, theIFM asynchrony has evolved independently several timesduring insect evolution (Cullen 1974; Pringle 1981; Dudley2000; Josephson, Malamud, and Stokes 2000). Moleculardata are no exception. The interspecies-independent butintraspecies-adapted evolution of both the TpnC genes(Herranz, Mateos, and Marco 2005) and the TpnT and TpnIgene–splicing variants could have played important rolesin how the IFMs have achieved their special properties.The possible functional relevance of these alanine/proline-rich extension sequences to the evolution of the stretch-activation phenomenon has been discussed elsewhere(Mateos et al., unpublished data).

FIG. 7.—Comparison between the secondary structure predictions of Drosophila IFM and human cardiac TpnI isoforms. Predictions were obtainedand represented using the PSIPRED software (McGuffin, Bryson, and Jones 2000). The explanation of symbols appears in the inset. Conserved features ofthe sequences appear boxed showing the vertebrate-known interactions among thin-filament components. The Drosophila TpnI alternative exons 6b2 and9 are also indicated. For clarity, the long random-coiled predicted Drosophila exon 3 sequence, absent in other organisms, has not been included in thefigure. The regions of interactions with other components of the vertebrate thin filament appear inside broken line contours. The conservation of thesecondary structure is also remarkable in the rest of the Tpn isoforms predictions (data not shown).

Table 1Summary of the Revised Drosophilid TpnT, TpnI, and TpnC Gene Expression Patterns

Main Expression(Drosophilidae)

TpnT Alternative Exons in theCorresponding Isoformsa

TpnI Alternative Exons inthe Corresponding

IsoformsaTpnCGenes3 4 5 10 3 6 9

Larvae 1 � 1 10B � 6a1 � IbAdult (hypodermic) 1 1 1 10B � 6b2/6a2 � IaUnknown location

(minimally expressed) 1 � � 10B � 6a2 � IIAdult (TDT) � � � 10B � 6b2 6 IIIaAdult (IFM) � � � 10A 1 6b1 1 IIIb

a The alternatively spliced exons found in the main transcripts detected at different stages or muscles are defined as present

(1) or absent (�). The6 indicates that transcripts with and without the exon are detected. The expression profiles are derived from

previously published works (Barbas et al. 1993; Benoist et al. 1998; Lovato et al. 2001; Herranz et al. 2004) in Drosophila mel-

anogaster, from studies of two more drosophilids, and information of TpnT exon 10 variability described in this report. The Apis

mellifera isoform expression patterns are not included in the table but are almost identically expressed except for an additional

hypodermic adult muscle TpnT isoform (containing both 5 and 5# exons), the absence of a TpnI exon 3, and the presence in the IFMof TpnH isoforms encoded by the TpnI gene.

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The Evolution of Supramolecular Complexes and theFunctional Role of Isoform Patterns

Previously published work has addressed the issue ofisoform variability and function in relation to its evolution-ary conservation (e.g., Robson et al. 2000; Shagin et al.2004), but, to our knowledge, a comprehensive analysisof the type performed in this article has not been attemptedyet. Together, the higher variability of muscle structures inprotostomes and the increasing availability of genome infor-mation across taxa open the way to understanding the inde-pendent coevolution of geneswhose proteins are involved insupramolecular complexes. Our approach reveals coevolu-tionary trends in components of the complexes sharingtissue expression patterns. The advent of whole-genomesequences from further insect species will help in extendingand refining ourmodel for the evolution of the troponin com-plex. Some splicing isoforms or even complete genes thatare weakly expressed or expressed in a very restricted pat-tern of tissues can be overlooked during a conventionalstudy. What are the functional consequences of the expres-sion of minor isoforms in a supramolecular complex? Itcould just provide a background repertoire of isoforms toimprove and be used in remodeling evolutionary processesbut its conservation across different species suggests a more

direct role. In the case of insect thin filaments, the tropomy-osin-troponin complex has been coevolving to respond tothe functional necessities of the asynchronous flight muscu-lature in different orders. Some alanine/proline-rich motifshave appeared and have become associated with differentpolypeptides in the complex. Different alternatively splicedexons encoding parts of different subunits of the complex,some of them also independently evolved, correlate with thetype and function of the muscle. The sequencing effort indifferent organisms offers new opportunities to test, amongother things, gene annotation accuracy, but more impor-tantly the correlation of genotype changes and the functionalphenotypic features that have evolved in particular groups oforganisms.

Supplementary Material

Table A (GenBank accession numbers) and the un-rooted trees in a PHYLIP format (showing bootstrappingvalues for TreeView application) used in this work areavailable at Molecular Biology and Evolution online(http://www.mbe.oxfordjournals.org/).

Acknowledgments

Weare especially grateful to R. Cripps,M.Manzanares,and J. Sparrow for correcting the English and for criticalreading of the manuscript as well as two anonymous refer-ees who have greatly helped in improving the quality of themanuscript. This research was supported by grants from theSpanish Government PB96-0069, ESP1999-0379-C02 andPNE-008/2001-C, BMC2001-1454.

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Accepted July 7, 2005

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