Chicken Perinatal Troponin Ts Are Generated by a Combination of Novel and Phylogenetically Conserved Alternative Splicing Pathways

Abstract

During perinatal development, avian and mammalian skeletal muscles express a novel set of troponin T (TnT) isoforms with higher M(r)s and more acidic pIs than their adult counterparts. In mammals, these TnTs result from the incorporation of a developmentally regulated 5'-fetal exon into fast TnT mRNAs. To determine whether chicken perinatal TnTs are generated by a similar mechanism, we sequenced 40 chicken TnT 5'-cDNAs from perinatal and adult pectoralis. Evidence for five 5'-exons not present in the mammalian gene was found, including one, y, whose splicing is developmentally regulated. Although none of these new exons are homologous with the mammalian fetal exon, their alternative splicing, superimposed on three conserved splicing patterns of the phylogenetically shared 5'-exons, generates the chicken perinatal TnT isoforms. These observations indicate that chicken, and most likely other avian species, evolved an independent 5'-alternative splicing mechanism to generate perinatal TnTs that have the same biophysical, and presumably functional, properties as their mammalian homologs.

Structure and Evolution of the Alternatively Spliced Fast Troponin T Isoform Gene

“…The 31 pectoral cDNAs that bear the His-rich peptide were identical for inclusion of p1-5. This is significant since Schachat and colleagues (38) reported chicken pectoral isoforms that have variant numbers of repeats of the His-rich peptide including a form that has two more His peptide repeats than we find in the qTnTf gene (38). Finally, consistent with protein studies (39), leg forms are found in our pectoral cDNA sample, but these were recovered at a relatively low frequency (3/33).…”

“…1 and 2). The existence of exons encoding a histidine-rich (His-rich) peptide specific to avian pectoral muscle has been previously demonstrated by sequencing of chicken TnTf protein and cDNAs (35)(36)(37)(38). Our primer extension analysis of quail pectoral RNA strongly supported the existence of these pectoral isoforms in quail; however, such clones were not recovered from pectoral cDNA libraries (6).…”

“…Thus, these latter two forms were called qLeg2 and qLeg2a (Table I). Although qLeg2 is not represented in our limited quail leg cDNA set, it is equivalent to a reported chick leg form (38). The nine qPec forms vary by the inclusion or exclusion of peptides encoded by exons 4, w, 7, and 8.…”

“…Finally, consistent with protein studies (39), leg forms are found in our pectoral cDNA sample, but these were recovered at a relatively low frequency (3/33). The postembryonic pectoral forms show a transition to forms lacking exon 4, which is considered an embryonic exon (38).…”

“…Developmental Expression of qTnTf Isoforms-We compared our set of clones with a set of 40 chicken fetal, perinatal, and adult TnTf cDNAs that was reported by Schachat et al (38) and an independent set of 40 chicken cDNAs (20 adult pectoral and 20 adult gastrocnemius) that was reported by Ogut and Jin (40). The set of chicken cDNAs from by Schachat and co-workers (38) was made from mRNA isolated from fetal (16 and 19 day embryos), perinatal (day 5), and adult chicken pectoral muscles and fell into 16 independent, alternatively spliced sequences that the authors placed into two broad classes based on length (Table I).…”