We report the discovery of mRNA 5-leader trans-splicing (SL trans-splicing) in the chordates. In the ascidian protochordate Ciona intestinalis, the mRNAs of at least seven genes undergo trans-splicing of a 16-nucleotide 5-leader apparently derived from a 46-nucleotide RNA that shares features with previously characterized splice donor SL RNAs. SL trans-splicing was known previously to occur in several protist and metazoan phyla, however, this is the first report of SL trans-splicing within the deuterostome division of the metazoa. SL trans-splicing is not known to occur in the vertebrates. However, because ascidians are primitive chordates related to vertebrate ancestors, our findings raise the possibility of ancestral SL trans-splicing in the vertebrate lineage.[ mRNA 5Ј-leader trans-splicing is a mode of gene expression reported in several organisms in which the original 5Ј ends of pre-mRNAs are discarded and are replaced by the 5Ј segment of a spliced leader (SL) RNA (Bonen 1993;Blumenthal 1995;Davis 1996). The function of SL transsplicing is not clear in every case and may vary. Multiple roles have been proposed, including mediation of mRNA stability or translatability (Maroney et al. 1995), resolution of polycistronic pre-mRNAs (Agabian 1990;Blumenthal 1995), and production of functional mRNAs from RNA polymerase I transcripts (Lee and Van der Ploeg 1997). In some organisms, only a subset of mRNAs undergo SL trans-splicing but in others, most or all do (Agabian 1990;Bonen 1993;Davis 1996). SL trans-splicing occurs alongside of the conventional cis-splicing process that removes introns from pre-mRNAs (Bonen 1993;Blumenthal 1995;Mair et al. 2000). There are mechanistic parallels between SL trans-splicing and conventional cis-splicing, including the use of the same set of nucleotide sequence features to mark splice donor and acceptor sites, and a strong resemblance of SL RNAs to spliceosomal U snRNAs (Agabian 1990;Bonen 1993;Nilsen 1993). These similarities imply a close evolutionary relationship between cis-splicing and SL trans-splicing, but the nature of this relationship and the overall evolutionary history of SL trans-splicing are not clear, in part because the phylogenetic distribution of SL transsplicing has not been clearly delineated.The known phylogenetic distribution of SL transsplicing is uneven and includes several protist and metazoan groups (Bonen 1993;Blumenthal 1995;Davis 1996). It was first discovered in a protist group, the trypanosomes (Campbell et al. 1984;Kooter et al. 1984;Milhausen et al. 1984), then subsequently in two protosotome metazoan phyla, Nematoda (Krause and Hirsh 1987) and Platyhelminthes (flatworms) (Rajkovic et al. 1990) and in Euglena, a protist distantly related to trypanosomes (Tessier et al. 1991). SL trans-splicing has not been reported in advanced protostome phyla, that is, the arthropods, annelids, or molluscs, nor among the deuterostomes, the great division of the metazoa that includes chordates/vertebrates. However, because each discovery of SL trans-splicing was a...

The activity of myogenic regulatory factor (MRF) genes is essential for vertebrate muscle development, whereas invertebrate muscle development is largely independent of MRF function. This difference indicates that myogenesis is controlled by distinct regulatory mechanisms in these two groups of animals. Here we used overexpression and gene knockdown to investigate the role in embryonic myogenesis of the single MRF gene of the invertebrate chordate Ciona intestinalis (Ci-MRF). Injection of Ci-MRF mRNA into eggs resulted in increased embryonic muscle-specific gene activity and revealed the myogenic activity of Ci-MRF by inducing the expression of four muscle marker genes, Acetylcholinesterase, Actin, Troponin I, and Myosin Light Chain in non-muscle lineages. Conversely, inhibiting Ci-MRF activity with antisense morpholinos down-regulated the expression of these genes. Consistent with the effects of morpholinos on muscle gene activity, larvae resulting from morpholino injection were paralyzed and their "muscle" cells lacked myofibrils. We conclude that Ci-MRF is required for larval tail muscle development and thus that an MRF-dependent myogenic regulatory network probably existed in the ancestor of tunicates and vertebrates. This possibility raises the question of whether the earliest myogenic regulatory networks were MRF-dependent or MRF-independent.

Fertilized eggs of the ascidian, Ciona intestinalis, were prevented from undergoing cytokinesis but not nuclear division by treatment with cytochalasin B. After appropriate times, such cleavage-arrested multinucleate zygotes developed acetylcholinesterase of larval tail muscle and an alkaline phosphatase ordinarily localized in the larval endoderm tissues. Separate histochemical reactions on one of a pair of samples taken from the eggs of single animals provided examples (6/34) in which the numbers of cytochalasin-treated embryos displaying the respective reaction product overlapped sufficiently (15-29%) to indicate that some of the zygotes had developed both enzymes in the same uncleaved single cell. With an actual dual-staining technique that can be applied to single cleavage-arrested zygotes, 62% of those developing a strong alkaline phosphatase reaction also had a strong acetylcholinesterase reaction. In other experiments, quantitative measurements of enzyme activity in homogenates of 114 single cleavage-arrested zygotes confirm directly that 18% of the zygotes produce both enzymes. There was no obligatory mutual exclusion of the potential for simultaneous expression of two tissue-specific characteristics that would ordinarily be segregated into different lineages during early cleavages. The cytoplasmic determinants believed responsible for these histotypic expressions can apparently function independently in the same cell.

The expression pattern of CiMDF, the MyoD-family gene of Ciona intestinalis, was analyzed in unmanipulated and microsurgically derived partial embryos. CiMDF encodes two transcripts during development (coding for distinct proteins), the smaller of which, CiMDFa, was detected in maternal RNA. Zygotic activity of CiMDF initiated in cleaving embryos of 32-64 cells. Both CiMDFa and CiMDFb transcripts were detected at this time; however, CiMDFa accumulated more rapidly before declining in abundance such that, by the early tail-formation stage, CiMDFb was more prevalent. Microsurgical isolations of various lineage blastomeres from the eight-cell stage were used to analyze CiMDF expression in the two embryonic lineages that give rise to larval tail muscle-autonomously specified primary cells and conditionally specified secondary cells. CiMDFa and CiMDFb transcripts were detected in both lineages, suggesting that neither functioned in a lineage-specific manner. The data also demonstrated that CiMDF expression was autonomous in the primary lineage (i.e., cells derived from the B4.1 blastomeres) and correlated with histospecific differentiation of muscle. In the secondary lineage (i.e., cells derived from the A4.1 and b4.2 blastomeres), CiMDF expression was conditional and, as in the primary lineage, correlated with muscle differentiation. These experiments reveal similar patterns of CiMDF activity in the primary and secondary muscle lineages and imply a requirement for the expression of this gene in both lineages during larval tail muscle development.

We have characterized the embryonic muscle cell cholinesterase of the solitary ascidian, Ciona intestinalis (L.). The effects of selective enzyme inhibitors and the inhibition of enzyme activity at high concentrations of substrate suggest that the muscle cell enzyme is an acetylcholinesterase (E.C. 3.1.1.7). After gastrulation and before hatching, acetylcholinesterase activity increased 35- to 40-fold; after hatching (18 hours postfertilization) this activity continued to increase, leveling off at about 36 hours of development. Histochemical observations showed that before hatching acetylcholinesterase was located principally in the muscle cells of the tail and, after hatching, it began to develop in cells of the adult musculature and brain. Inhibition of protein syntnesis by puromycin and of RNA synthesis by actinomycin D, suggest that both protein and RNA synthesis were required for the increase in acetylcholinesterase activity observed in unhatched embryos. Although the continued increase in enzyme activity duirng embryonic development was sensitive to puromycin at all times tested, the actinomycin D sensitivity of this increase was restricted to a discrete time that was completed by about 11 hours of development.

The regulation of several enzymes involved in one-carbon metabolism was studied in a mutant of Escherichia coli K-12 defective in S-adenosylmethionine synthetase. The mutant that was reported to have a low endogenous concentration of S-adenosylmethionine had elevated levels of N-5, 10-methylene tetrahydrofolate reductase and serine transhydroxymethylase, but the level of N-5, 10methylene tetrahydrofolate dehydrogenase was not altered. These results suggest that S-adenosylmethionine plays a role in the regulation of one-carbon production and utilization. An enzyme system that cleaved glycine to one-carbon units was demonstrated. The enzymes responsible for the cleavage of glycine were induced by exogenous glycine but were independent of S-adenosylmethionine or purine levels in the cell.The regulation by methionine of several enzymes involved in one-carbon metabolism has been demonstrated by several authors (1,5,10,11,16,26). Generally, their approach was to use methionine-cyanocobalamin (B 12) auxotrophs, whose requirement for methionine was furnished by cyanocobalamin. By increasing the concentration of methionine in the medium, they were able to demonstrate a corresponding decrease in levels of certain enzymes involved in one-carbon metabolism, especially methionine biosynthesis. However, these methods do not distinguish between the effect of methionine and S-adenosylmethionine (SAM) levels, which presumably were increased with increasing methionine concentrations. Unfortunately, the effect of SAM on these enzymes is not directly ascertainable, since it is relatively impermeable to the cells. Recently, however, Greene and co-workers (7,9) and Ahmed (1) isolated mutants of Escherichia coli K-12 defective in SAM synthetase with lowered levels of SAM and increased levels of methionine. With these mutants, one may examine the possibility that SAM levels regulate one-carbon formation and distribution. They may also provide the means to resolve the apparent contradictions between the results of Mansouri et al. (16) and Taylor et al. (26) as to whether the supply of methionine controls the levels of serine transhydroxymethylase (EC 2.1.2.1) and obtain information on the interrelationship between the metabolism of one-carbon units and methionine. Our studies with these mutants showed a pronounced effect of SAM levels on N-5, 10-methylene tetrahydrofolate reductase (EC 1.1.1.68), a much smaller effect on serine transhydroxymethylase, and no effect on N-5, 10-methylene tetrahydrofolate dehydrogenase (EC 1.5.1.5). These results indicate that SAM rather than methionine plays a role in controlling serine transhydroxymethylase levels. They also confirm the results of Kung et al. that N-5, 10-methylene tetrahydrofolate reductase levels are regulated by SAM rather than by methionine (12).As suggested by earlier workers, an alternative means of generating one-carbon units is by cleavage of glycine (23). Serine auxotrophs which cannot synthesize serine via the normal phosphorylated pathway may have their requirement for serine su...

Ascidians are protochordates related to vertebrate ancestors. The ascidian larval tail, with its notochord, dorsal nerve cord, and flanking rows of sarcomeric muscle cells, exhibits the basic chordate body plan. Molecular characterization of ascidian larval tail muscle may provide insight into molecular aspects of vertebrate skeletal muscle evolution. We report studies of the Ci-TnI gene of the ascidian Ciona intestinalis, which encodes the muscle contractile regulatory protein troponin I (TnI). Previous studies of a distantly related ascidian, Halocynthia roretzi, showed that different TnI genes were expressed in larval and adult muscles, the larval TnI isoforms having an unusual C-terminal truncation not seen in any vertebrate TnI. Here we show that, in contrast with Halocynthia, Ciona does not have a specialized larval TnI; the same TnI gene that is expressed in the heart and body-wall muscle of the sessile adult is also expressed in embryonic/larval tail muscle cells. Moreover the TnI isoform produced in embryonic/larval muscle is identical to that produced in adult body-wall muscle, i.e., a 182-residue protein with the characteristic chain length and overall structure of vertebrate skeletal muscle TnI isoforms. Phylogenetic analyses indicate that the unique features of Halocynthia larval TnI likely represent derived features, and hence that the vertebrate-skeletal-muscle -like TnI of Ciona is a closer reflection of the ancestral ascidian larval TnI. Our results indicate that characteristics of vertebrate skeletal muscle TnI emerged early in the evolution of chordate locomotory muscle, before the ascidian/vertebrate divergence. These features could be related to a basal chordate locomotory innovation-e.g., swimming by oscillation of an internal notochord skeleton-or they may be of even greater antiquity within the deuterostomes.

Ci-MRF is the sole myogenic regulatory factor (MRF) of the ascidian Ciona intestinalis, an invertebrate chordate. In order to investigate its properties we developed a simple in vivo assay based on misexpressing Ci-MRF in the notochord of Ciona embryos. We used this assay to examine the roles of three structural motifs that are conserved among MRFs: an alanine-threonine (Ala-Thr) dipeptide of the basic domain that is known in vertebrates as the myogenic code, a cysteine/histidine-rich (C/H) domain found just N-terminal to the basic domain, and a carboxy-terminal amphipathic α-helix referred to as Helix III. We show that the Ala-Thr dipeptide is necessary for normal Ci-MRF function, and that while eliminating the C/H domain or Helix III individually has no demonstrable effect on Ci-MRF, simultaneous loss of both motifs significantly reduces its activity. Our studies also indicate that direct interaction between CiMRF and an essential E-box of Ciona Troponin I is required for the expression of this muscle-specific gene and that multiple classes of MRF-regulated genes exist in Ciona. These findings are consistent with substantial conservation of MRF-directed myogenesis in chordates and demonstrate for the first time that the Ala/Thr dipeptide of the basic domain of an invertebrate MRF behaves as a myogenic code.