# Insights into the Evolution and Regulation of Hybrid Internal-Terminal Exons from Tropomyosin Exon 9A in Xenopus laevis

Agnès Méreau, Hubert Lerivray, Justine Viet, Serge Hardy, Luc Paillard, Yann Audic

► To cite this version:

Agnès Méreau, Hubert Lerivray, Justine Viet, Serge Hardy, Luc Paillard, et al.. Insights into the Evolution and Regulation of Hybrid Internal-Terminal Exons from Tropomyosin Exon 9A in Xenopus laevis. Biochimie, 2025, 238, part A, pp.29-42. 10.1016/j.biochi.2025.07.009. hal-05235669

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# Insights into the evolution and regulation of hybrid internal-terminal exons from tropomyosin exon 9A in *Xenopus laevis*

<figure><img src="image_2.png" /></figure>

Agnès Méreau, Hubert Lerivray, Justine Viet, Serge Hardy, Luc Paillard <fnref n="1" />,*, Yann Audic <fnref n="1" />

Univ Rennes, CNRS, IGDR [(Institut de Génétique et Développement de Rennes)] - UMR 6290, F-35000, Rennes, France

## ARTICLE INFO

**Article history:**

Received 7 April 2025

Received in revised form

4 July 2025

Accepted 7 July 2025

Available online 9 July 2025

Handling Editor: Dr B Friguet

**Keywords:**

Alternative splicing

Developmental biology

AG-Independent intron

Cleavage-polyadenylation

Vertebrates

## ABSTRACT

Hybrid internal-terminal exons function as either internal or terminal exons. Their evolutionary origins remain unclear. Here, we investigate the phylogenetic origin and regulation of a hybrid exon, 9A9', in the TPM1 gene encoding tropomyosin alpha-1. We demonstrate that exon 9A9' was originally terminal in non-vertebrate deuterostomes and switched to internal in vertebrates through the exonization of a downstream exon, 9B. While the terminal nature of exon 9A9' was lost in most vertebrates, it was conserved in amphibians and coelacanths where it behaves as a hybrid internal-terminal exon. Using Xenopus laevis as a model, we show that the preservation of terminal exon 9A9' in the tpm1 gene likely arose from evolutionary pressures to mitigate the developmental toxicity linked to exon 9B inclusion during neurulation. We identify two peculiarities of terminal exon 9A9': it lies downstream of an AG-independent intron, and its definition is supported by an intronic cis-regulatory element, the UTE, which enhances recognition of the weak cleavage-polyadenylation site. Our findings characterize the molecular mechanisms underlying the regulation of hybrid internal-terminal exons and reveal how evolutionary pressures can reactivate vestigial traits to resolve developmental challenges. This work broadens our understanding of alternative splicing evolution and its significance in vertebrate development.

© 2025 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY license

(http://creativecommons.org/licenses/by/4.0/).

## 1. Introduction

The maturation of primary transcripts into mature mRNA involves precise splicing reactions to remove introns and ligate exons, and a final step of cleavage and polyadenylation for terminal exons. Internal exons are defined by a splice acceptor (SA) at the 5' end and a splice donor (SD) at the 3' end, while in terminal exons the SD is replaced by a cleavage and polyadenylation site (CPA). An additional site, the branch point (BP), located within the upstream intron generally close to the SA, is crucial for the process. Splicing occurs in two main steps: first, the SD is ligated to the BP, forming a lariat structure. Then, the 3' end of the upstream exon is ligated to the downstream SA, completing the exon junction and releasing the intron [1,2].

Alternative splicing, where specific exons are conditionally

included or skipped, generates multiple mRNA isoforms from a single gene. A comprehensive comparison of 47 eukaryotic genomes and transcriptomes revealed a strong correlation between organism complexity (measured by cell type variety) and alternative splicing prevalence. This contrasts with the weak correlation between complexity and the sheer number of protein-coding genes [3]. For example, the budding yeast *Saccharomyces cerevisiae* contains introns in only 306 of its ~6000 protein-coding genes, with alternative splicing in just 20 % of these [4]. In contrast, more than 92 % of human genes generate multiple mRNA isoforms [5,6]. Interestingly, splicing profiles of the same organs can vary greatly across vertebrates in contrast to the tissue-specificity of gene expression which has been largely conserved [7] [8].

A major driver of the increasing complexity of alternative splicing patterns during evolution is the appearance of new internal exons. They arise from tandem exon duplication [9], or from the "exonization" of previously intronic regions [10,11]. In this process, mutations create new SA and SD sites within introns, and the corresponding intronic regions are retrieved as exons within the mature mRNAs. The mutations that lead to exonization are generally well tolerated because the new splice sites are usually

<footnote>This article is part of a special issue entitled: Structure Integration, Function, and Reactivity of RNA published in Biochimie.</footnote>

<footnote>* Corresponding author.</footnote>

<footnote>E-mail address: luc.paillard@univ-rennes.fr (L. Paillard).</footnote>

<footnote n="1">¹ LP and YA are joint senior authors.</footnote>
less efficient than the canonical ones, and the new exon is only incorporated into a small proportion of the mature mRNA molecules. Over the course of evolution, further mutations that strengthen the new splice sites may be selected if the incorporation of the new exon is advantageous [12]. Transposable elements, like short interspersed nuclear elements (SINE) such as Alu elements in primates, are particularly prone to exonization [13,14]. A key event for Alu exonization is the appearance of a BP upstream of the putative novel exon, which is recognized by the U2AF65 protein. The RNA-binding protein HNRNPC competes with U2AF65 for binding to the potential new branch points, and therefore limits the consequences of Alu exonization [15,16].

In addition to internal exons, exonization of intronic regions downstream of genes can produce alternative terminal exons. This results in proteins with different C-terminal domains and/or mRNAs with different 3' untranslated regions (3'UTR), impacting mRNA translation or stability. As for internal exons, HNRNPC can mitigate the exonization of Alu-derived terminal exons [16]. For any exonization event to create a new terminal exon, mutations must create both SA and CPA sites. Premature cleavage and polyadenylation within introns is frequent [17] but typically repressed by the "telescripting" function of U1 snRNP [18,19]. Terminal exons may thus emerge from intronic regions through SA formation alongside the release of telescripting at a nearby cryptic CPA.

In addition to strictly first, internal and terminal exons, hybrid exons are widespread in humans [20]. While the evolution of hybrid first-internal exons has been recently investigated [21], the evolutionary origins of hybrid internal-terminal exons remain unclear. Here, we explored the hybrid internal-terminal exon 9A9' of *tmp1* in *Xenopus laevis* to trace its evolutionary history. *tmp1* encodes tropomyosin alpha-1, a major component of the cytoskeleton. In non-muscle cells, *tmp1* exon 8 is spliced to distal exon 9D (Fig. 1A). Muscle cells use a more proximal exon, 9A, to produce two muscle-specific isoforms: 8-9A-9B, in adult skeletal muscles and embryonic hearts, and 8-9A9', in embryonic somites (future skeletal muscles). These two isoforms diverge in the use of either a SD at the 3' end of exon 9A, enabling the junction of exon 9A to exon 9B, or of a CPA 71 nucleotides downstream of exon 9A, defining exonic region 9' (Fig. 1A). Hence, exon 9A9' is terminal and exon 9A is an internal exon spliced to exon 9B [22]. The two isoforms 9A-9B and 9A9' differ only in their 3'UTR and the encoded proteins are the same except for the final amino acid [23].

Here, through transcriptomic database analysis and splicing reporter assays in *Xenopus laevis*, we discovered that exon 9A is purely internal in most vertebrates and hybrid only in amphibians and coelacanths. However, it is purely terminal in non-vertebrate deuterostomes. This suggests that this exon was initially terminal, then turned to internal in vertebrates by the emergence of downstream exon 9B. The initial property of being used as terminal was then lost or kept only as a vestigial trait in vertebrates, except in species like *Xenopus laevis* where it was reactivated. Cross-species analysis indicated that reactivation of exon 9A9' correlates with the presence of a downstream CPA, and of a specific intronic sequence, UTE. Functional assays using morpholino antisense oligonucleotides to alter endogenous *tmp1* splicing pattern and mRNA injections revealed that the accumulation of exon 9B-containing *tmp1* mRNA disrupts neurulation in *X. laevis*, likely driving the selective reactivation of exon 9A9' as a terminal exon. Mechanistic analysis combining injections of morpholino antisense oligonucleotides with RT-PCR and RNase protection assays further revealed that UTE replaces the traditional SA in defining exon 9A9', marking a unique regulatory adaptation for hybrid exons. Together, these findings shed light on the evolution and regulatory mechanisms of a hybrid internal-terminal exon, illustrating the selective pressures that led to its reactivation in

specific vertebrate lineages.

## 2. Materials and methods

### 2.1. Comparative genomic analysis

The availability of transcriptomic dataset covering multiple tissues and developmental stages was evaluated for each species by querying GEO datasets (https://www.ncbi.nlm.nih.gov/gds) with the query ("expression profiling by array"[DataSet Type]) OR "expression profiling by high throughput sequencing"[DataSet Type]) AND *genus species* [Organism]).

Sequence data for *TPM1* ortholog genes were collected for 43 species representing ray-finned fishes, sarcopterygians and amniotes. When multiple loci for *TPM1* were identified they were reported as distinct loci. All the sequences, annotations and accession numbers are given in Table S1. When available, synteny was evaluated using the *TLN2* and *LACTB* genes surrounding the *TPM1* locus. For non-vertebrate species, *tropomyosin* genes were identified based on genome wide tblastn searches using previously annotated tropomyosin protein in the respective species. Exonic annotation was manually curated for each species based on available transcriptomic data and is reported in Table S1. The properties of the branch points were evaluated with SVM-BPfinder [24]. For plotting, the bp are such that bp_score >0 and svm_score>0 and located not further away than 9 nt before the AGEZ. The properties of the 5' splice sites and cleavage-polyadenylation signals were evaluated with MaxEntScan [25] and polyA_SVM [26]. For plotting the score is represented as 2 to the power of (-score) to visualize bigger circles for better poly(A) sites. UTE scoring was performed using PWMEnrich in R [27]. For plotting the circle size is relative to the log odd_score.

### 2.2. Plasmids construction

The pBS-Actin-*tmp1a*-7-9B splicing reporter gene has been described previously, along with reporter genes with mutant 5' or 3' splice sites [22]. The transcription of the *X. laevis* *tmp1* genomic region from exon 7 to exon 9B was driven in embryonic myotomal cells by the cardiac actin promoter. In compound reporter genes, the region between the SnaB1 (20 nucleotides downstream of exon 8) and Kpn1 (310 nucleotides downstream of exon 9A) sites was replaced by equivalent sequences from *N. parkeri*, *L. chalumnae*, *G. gallus*, *G. seraphini*, *R. bivittatum*, *N. forsteri* or *P. annectens*, between around 600 nucleotides upstream and around 300 nucleotides downstream of exon 9A. These DNA sequences were obtained as geneblocks (IDT, Table S2). Luciferase reporter LucGb plasmid was generated by cloning BahmH1-Spe1 fragment from pmirGlo vector (Promega) into pT7TS vector linearized using HindIII. The resulting construction was used to generateLuciferase reporter Luc9' and Luc9B plasmid using Gibson Assembly master mix (NEBE2611 S/L). LucGb, Luc9B and Luc9' mRNA were produced by *in vitro* transcription using the mMessage mMachine T7 kit (Ambion) from SalI-linearized pT7TS-Luciferase reporters [28].

### 2.3. *Xenopus laevis* embryo culture and injections

*X. laevis* embryos were obtained by *in vitro* fertilization of eggs from laboratory-reared females (APAFIS project #7109-2016100416077554). Embryo injections were carried out as described previously [22]. For splicing reporter gene injection, 250 pg of supercoiled DNA were injected into one blastomere at the two-cell stage. The sequences of the morpholinos are given in Table S2. We injected from 0.5 pmol to 3 pmol morpholinos (depending on injection conditions) or 1 fmol of Luciferase mRNA
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<figure><img src="image_7.png" /><figcaption>**Fig. 1.** 9A9' is a hybrid terminal-internal exon in amphibians and coelacanth. A, Structure of *tpm1* terminal exons in *Xenopus laevis*. Exons are represented as boxes and introns as lines. The splicing events are indicated above the respective exons and introns as dashed lines linking the exons. Exon 8 is spliced to exon 9D in non-muscle cells and to exon 9A in muscle cells. In muscle cells, exon 9A functions either as a terminal exon with exonic region 9' (forming 9A9') or as an internal exon joined to exon 9B via its splice donor site. This dual functionality makes it a regulated hybrid internal-terminal exon. SA, splice acceptor. SD, splice donor. CPA, cleavage and polyadenylation site. B, Strategy to identify *TPM1* exonic region 9' and exon 9B across vertebrate species. C, Landscape of *TPM1* exonic region 8, 9A, 9' and exon 9B across vertebrates based on transcriptomic databases. The phylogenetic tree on the left is from the NCBI taxonomy database. D, Splicing reporter assay. The upper panel shows the splicing reporter plasmid containing the *X. laevis* genomic</figcaption></figure>
into both blastomeres of two-cell embryos. The embryos were cultured until Nieuwkoop and Faber stages 17 or 30.

## 2.4. Isolation of RNA, analysis by RT-PCR, RT-qPCR and RNase protection assay

RNA extracted from embryos was analysed by an RT-PCR assay as described previously [29]. PCR amplification was performed with a³²P-labeled forward primer in *tpm1* exon 8 (endogenous transcript), or a³²P-labeled forward primer at the actin promoter/ exon 7 junction (splice reporter gene), and reverse primers in exons 8, 9' or 9B. Amplimeres were resolved on 8% polyacrylamide gels and quantified by PhosphorImager analysis (Amersham Biosciences). Real-time PCR was performed using the Power SYBR green PCR master mix in an ABI Prism 7900 (Applied Biosystems). The RNA probes used for the detection of endogenous *tpm1*, *eef1a1* and *myl1* transcripts, and cleaved and uncleaved *tpm1* transcripts were described previously [29]. RNase protection assays were performed using 10 µg of total RNA as described previously [28]. The sequences of the oligonucleotides used for RT-PCR and RT-qPCR are given in Table S2.

## 2.5. Antibodies and western blot analysis

Western blot analysis was carried out as described previously [29]. Blots were detected with the Odyssey infrared imaging system (Li-COR Biosciences) and the following primary antibodies: monoclonal anti-tropomyosin clone TM311 (catalog number T2780, Sigma) and monoclonal anti-beta tubulin clone AA2 (catalog number T4026, Sigma).

# 3. Results

## 3.1. Exploring the exonic region 9' and exon 9B in vertebrates

In *Xenopus laevis*, three alternative RNA isoforms of *tpm1* that differ by their final exons have been identified: 8-9D in non-muscle cells, 8-9A9' in somites and 8-9A-9B in embryonic hearts and adult muscles. Isoforms 8-9A9' and 8-9A-9B diverge in the use of either a splice donor (SD) at the 3' end of exon 9A, or of a cleavage and polyadenylation site (CPA) 71 nucleotides downstream of exon 9A (Fig. 1A). They have distinct 3' untranslated regions (3'UTR) but the encoded proteins are the same except for the final amino acid [23].

We investigated whether the exonic region 9' and exon 9B are present in other vertebrate species (Fig. 1B). Using Blastn and Genomicus for synteny analysis [30], we identified *TPM1* orthologs across 44 vertebrate genomes. This corresponds to 51 genomic loci because of whole genome duplications implying that two *tpm1* loci are found in some species. We pinpointed exon 9A through peptide homology (tblastn, the sequences of exon 9A in several species are in Fig. S1A). Exon 9A was consistently identified in all analysed vertebrates except *Petromyzon marinus* (lamprey). However, exonic region 9' and exon 9B, both non-coding regions, are poorly conserved and therefore challenging to detect in genomic databases. To address this, we queried transcriptomic databases

using sequences from exon 9A of each species. We defined exonic region 9' and exon 9B based on their presence in transcripts: exonic region 9' as the ~70 nucleotide long sequence immediately downstream of exon 9A in the genome, and exon 9B as a more distal region separated from exon 9A by an intronic region not retrieved in transcripts (Fig. 1B). This analysis was restricted to species with at least 10 available GEO datasets to minimize the risk of false-negatives due to limited transcriptome coverage (Table S3).

The results of this analysis are shown in Fig. 1C. All vertebrate species with a sufficient transcriptomic coverage have exon 9B, composed solely of a stop codon and a 3' UTR (Fig. 1C-S1A). This indicates that exon 9A can be used as internal. In addition to *Xenopus laevis*, we identified exonic region 9' in two other amphibians, *Xenopus tropicalis* and *Ambystoma mexicanum*. Exonic region 9' only consists in a stop codon and a 3' UTR. In contrast, most bony fishes (*Gasterosteus aculeatus*, *Oryzias latipes*, *Gadus morhua*) and amniotes (birds: *Gallus gallus*, *Meleagris gallopavo*; mammals: *Monodelphis domestica*, *Canis lupus familiaris*) lack this region indicating that exon 9A is systematically joined to exon 9B. In some species including human, we found a few ESTs containing a genomic region immediately downstream of exon 9A. Yet these sequences terminate irregularly across the genome and lack CPA, likely representing random premature cleavage rather than true exonic region 9'. These situations are annotated as "distal CPA" in Fig. 1B-C.

Since exon 9A9' is detected only in embryos, at least in *Xenopus laevis*, its apparent absence in most other species could, in theory, reflect a lack of embryonic tissue representation in transcriptomic databases. However, transcriptomic data from embryonic tissues are abundant for species with the highest coverage, such as human and mouse (see Table S3). Despite this, we were unable to detect a bona fide exon 9A9' in these species, that is a transcript with a consistent cleavage and polyadenylation site approximately 70 nucleotides downstream of exon 9A, supported by a canonical CPA motif. We therefore believe it is unlikely that exon 9A9' was missed in human or mouse due to sampling limitations; rather, we conclude that this exon is absent in these species. Consistent with this, among species with moderate to high transcriptome coverage, exonic region 9' appears to be restricted to *X. laevis*, *X. tropicalis*, and *Ambystoma mexicanum*. We extended this analysis to species with limited transcriptomic data using an experimental approach to assess inclusion in transcripts of the region immediately downstream of exon 9A in developing *X. laevis* somites. Tested species include amphibians (Anura: *Nanorana parkeri*; Caecilians: *Rhinatrema bivittatum*, *Geotrypetes seraphini*), coelacanth (*Latimeria chalumnae*) and lungfish (*Protopterus annectens*, *Neoceratodus forsteri*).

We constructed plasmids with the *X. laevis* genomic region between exons 7 and 9B under the control of a somite-specific promoter (actin). We replaced the ~1000 nt region overlapping *Xenopus* exon 9A9' with equivalent regions from test species or *Gallus gallus* (negative control). We also engineered variants in *Xenopus* to inactivate the splice acceptor at the 5' end of exon 9A (iSA) or enhance the splice donor at the 3' end (eSD). Plasmids were injected into one blastomere of 2-cell *Xenopus* embryos.

region from exon 7 to exon 9B, cloned downstream of an actin promoter (active in somites). In the iSA construct, the splice acceptor immediately upstream of exon 9A is inactivated. In the eSD construct the splice donor immediately downstream of exon 9A is enhanced. In compound constructs, the original *X. laevis* region covering exon 9A9' and portions of the flanking introns (red box and lines, ~1000 nucleotides) is replaced by orthologous regions from other species (Np *Nanorana parkeri*, *Lc Latimeria chalumnae*, *Gg Gallus gallus*, *Gs Geotrypetes seraphini*, *Rb Rhinatrema bivittatum*, *Nf Neoceratodus forsteri*, *Pa Protopterus annectens*). RT-PCR primers targeting the actin sequence (forward primer a), exonic region 9' (reverse primer b), exon 9B (reverse primer c) and exon 8 (reverse primer d) are indicated. The lower panels show the RNA maturation patterns produced by the reporter constructs following embryo injection, analysed by RT-PCR. Bands identities were confirmed by sequencing. E, Ability of the genomic regions encompassing exon 9A9' and parts of the flanking introns from the indicated species to promote the inclusion of terminal exon 9A9', as demonstrated by the splicing reporter assay shown in D.
Following development up to stage 28, we ran RT-PCR with a single forward primer (a) and either of three reverse primers (b to d, Fig. 1D upper panel). The forward PCR primer is specific to an actin sequence present in the plasmid and cannot amplify endogenous *tpm1* mRNA (Fig. 1D, middle and lower panels, lane 1; Fig. S2A, lane 1). The primer targeting exon 8 confirmed effective transcription (Fig. S2A). Consistent with previous findings [31] three mRNA isoforms were produced from the wild-type (WT) *Xenopus* construct: 7-8-9A-9B, 7-8-9B (Fig. 1D, middle panel, lane 2) and 7-8-9A9' (Fig. 1D, lower panel, lane 2). Isoform 7-8-9B is only detected when it is produced from the injected plasmid as its endogenous equivalent is highly unstable [32]. As expected, inactivating the splice acceptor at exon 9A (iSA) completely excluded exon 9A (Fig. 1D, lane 3), while enhancing the splice donor (eSD) led to exon 9A being included only as an internal exon (lane 4). *G. seraphini* and *N. forsteri* compound plasmids produced isoforms similar to the wild-type *Xenopus* gene, but the amount of isoform 7-8-9A9' was lower (Fig. 1D, lanes 8 and 10). With *N. parkeri* or *R. bivittatum*, additional products marked by asterisks were also detected (lanes 5 and 9). Sequencing these PCR products revealed that they result from cryptic splice donors within intron 8 (Fig. S2B-C). The *L. chalumnae* construct mainly produced the 7-8-9A9' isoform (Fig. 1D, lane 6). No or very faint 7-8-9A9' isoform was detected from the *G. gallus* or *P. annectens* constructs (lanes 7 and 11). These findings reveal a functional hierarchy in the capacity of exonic regions overlapping exon 9A to drive the inclusion of terminal exon 9A9', where *X. laevis*, *N. parkeri*, *R. bivittatum*, and *L. chalumnae* regions are more efficient than *G. seraphini* and *N. forsteri*, and *P. annectens* and *G. gallus* regions are entirely ineffective (Fig. 1E).

## 3.2. Cis elements controlling TPM1 maturation in vertebrates

These data show that while exon 9B is universally present in vertebrates, exonic region 9' is actively incorporated into mature mRNAs only in coelacanths (*L. chalumnae*) and amphibians (Fig. 1C and E). In lungfish, exonic region 9' is either absent (*P. annectens*) or incorporated inefficiently (*N. forsteri*). It is notably absent in bony fishes and amniotes. This raises questions about the genomic sequences controlling the inclusion of exonic region 9' and the capacity of exon 9A9' to function as a terminal exon. Firstly, we checked that the genomic organisation of the terminal exons of *TMP1* is the same across 43 vertebrate species with genomic data available (Fig. 2A). The genomic coordinates and accession numbers of the retrieved sequences are given in Table S1. Indeed, all these vertebrate species have exons 8 and 9A. Most of them also have exon 9D. In addition, another coding exon, 9C, localized in the genome between exons 9B and 9D, is absent in amphibians but present in most other vertebrates. All species have at least one terminal exon among 9C and 9D except *P. annectens* (Fig. 2A). Sequence alignment and naive clustering (Fig. S1) of the encoded peptide sequences confirm the correct assignment of these terminal exons. Secondly, we scored the strength of several splicing-related sites: the branch points in the introns upstream of exons 9A and 9B using BP-finder [33], the cleavage and polyadenylation site at the 3' ends of exons 9A9' and 9B with poly(A)SVM [34], and the splice donor site between exon 9A and exonic region 9' with MaxEntScanscore5ss [25]. Additionally, we analysed the UTE in intron 8, an intronic enhancer previously shown in *X. laevis* to direct the cleavage/polyadenylation machinery to the CPA of exon 9' [29]. We scored the UTE motif defined from the two *X. laevis* UTE sequences (corresponding to the two *tpm1* homeologous genes) in the 300 nucleotides upstream of exon 9A. Our analysis found no correlation between the ability of exon 9A9' to function as a terminal exon and the strength of the branch point or CPA of exon 9B

(Fig. S3A), the branch point strength upstream of exon 9A (Fig. S3B), or the splice donor strength at exon 9A's 3' end (Fig. S3C). However, species where exon 9A9' efficiently functions as a terminal exon (blue boxes in Fig. 2A) consistently have both a UTE and a strong CPA in exon 9A9'. This pattern is observed in *X. laevis*, *X. tropicalis*, *N. parkeri*, *A. mexicanum*, *R. bivittatum*, and *L. chalumnae*. Genomic data strongly suggest that *Rana temporaria* and *Pleurodeles walzt* also includes a terminal exon 9A9', but additional transcriptomic data are required to test this prediction. The UTE sequences from these species with a strong CPA are aligned and shown in Fig. 2B. Importantly, there is no predicted CPA in *G. seraphini* and *N. forsteri*, where the inclusion of terminal exon 9A9' is less efficient. Species that do not use terminal exon 9A9' similarly have only one or none of these two regulatory elements (Fig. 2A).

## 3.3. Evolutionary origin of TPM1 terminal exons

Our findings indicate that exon 9A acts as a hybrid exon, functioning as both an internal exon (with exon 9B) and a terminal exon (with exonic region 9') in species like *X. laevis* and *L. chalumnae*, which possess both a UTE in intron 8 and a CPA downstream of exon 9A. In species lacking one or both of these elements, exon 9A functions exclusively or essentially as an internal exon. This raises questions about the ancestral nature of exon 9A: was it initially a terminal exon or an internal one?

To explore this, we examined the structure of tropomyosin genes in non-vertebrate deuterostome lineages, including *Strongylocentrotus purpuratus* (sea urchin, echinoderm, Spur_5.0), *Branchiostoma lanceolatum* (amphioxus, cephalochordate, BraLan3) and *Ciona intestinalis* (a tunicate, HT genome version). These lineages diverged before the vertebrate-specific whole-genome duplication [35], resulting in a more limited tropomyosin gene repertoire. Through tandem duplication, the tropomyosin gene family expended to three genes in *C. intestinalis*, four in *B. lanceolatum* (including one with 4 alternative terminal exons) and two in *S. purpuratus* (including one with 3 alternative terminal exons, Fig. 3A). All terminal exons in these species contain a coding region (Fig. 3B) and a 3' UTR, with no indication of a junction to a putative alternative 3'UTR within a hypothetical exon 9B. In addition, by MaxEntScanscore5ss scoring [25], we failed to identify potential SD sites within the terminal exons of non-vertebrates as strong as those found in vertebrates (Fig. 3C). Altogether, our data show that exon 9A functions solely as a terminal exon in echinoderms, cephalochordates and tunicates.

## 3.4. Evolutionary pressure for retaining terminal exon 9A9'

Based on the above findings, a plausible scenario is that exon 9A was initially terminal (see Discussion). It then switched to almost only internal in vertebrates except in lineages like amphibians where its capacity to act as terminal was retained. These findings raise questions about the evolutionary pressures that led to keeping or reactivating terminal exon 9A9' in these lineages. Given that the protein products of the 9A9' and 9A-9B isoforms differ by only a single amino acid, the pressure likely relates to differences in their 3'UTRs (9' versus 9B). Specifically, the reactivation of terminal exon 9A9' may address a developmental need to avoid the accumulation of *tpm1* mRNA containing exon 9B 3'UTR at specific stages. To explore this, we examined the effect of promoting the accumulation of the 9B 3'UTR during early embryonic stages in *Xenopus laevis*.

We blocked the CPA at the 3' end of exon 9A9' by injecting embryos with complementary antisense morpholino oligonucleotides (pAMo). As expected, this drastically reduced the mRNA
<figure><img src="image_8.png" /><figcaption>**Fig. 2.** Regulatory elements controlling *TMP1* terminal exons. **A**, Left panel: presence of *TPM1* exons 8, 9A, 9C, 9D (from genomic database searches) and 9' (from the results shown in Fig. 1C and E) in the indicated species. Right panel: scoring of the UTE and of the cleavage-polyadenylation site (CPA) at the 3' end of exonic region 9'. The size of the circles reflects the predicted strength of each regulatory site. In the UTE panel, the grey rectangles represent the AG exclusion zone. In the CPA panel, the grey rectangles represent exon 9A. **B**, Alignment of UTE-like motifs identified in *TPM1* intron 8, located within the 400 nucleotides upstream of the splice acceptor site, across the indicated species.</figcaption></figure>

isoform containing terminal exon 9A9', favoring instead the 9A-9B isoform (Fig. 4A, left). The pAMo morpholino only slightly reduced the accumulation of Tpm1 protein, in contrast to the complete depletion achieved with a translation-inhibiting morpholino targeting the *tmp1* initiation codon (Fig. 4A, right). Hence, the switch from the 9' to the 9B 3'UTR elicited by pAMo morpholino has minimal effect on translation. However, pAMo-injected embryos exhibited dramatic neurulation defects (Fig. 4B). Normally, neural folds elevate from the neural plate and progressively fuse in a posterior to anterior direction to form the neural tube between stages 15 and 19 (Fig. 4B, left). In contrast, pAMo-injected embryos displayed poor convergence of neural folds, resulting in neural tube malformations or absence (Fig. 4B, right). Almost four fifths of pAMo-injected embryos exhibited these defects, compared to a small fraction in control embryos (Fig. 4C lanes 1–3).

We conducted a rescue experiment to confirm the specificity of this effect. We reasoned that a second antisense morpholino

targeting exon 9B (9BSAMo, directed at its splice acceptor site) would reduce the level of exon 9B-containing isoform. As previously, the pAMo increased the abundance of the 7-8-9A-9B isoform and reduced that of the 7-8-9A9' (Fig. 4D, lanes 1–2). The total level of the 7-8-9A RNA, which encompasses all *tpm1* muscular isoforms, appears unaffected by the addition of the pAMO (Fig. 4D, lower panel). This indicates that while the pAMO shifts the relative proportions among the muscular isoforms, it does not alter the overall balance between muscular and non-muscular isoforms. Increasing concentrations of 9BSAMo progressively reversed this effect (lanes 3–4). Again, the total level of the 7-8-9A RNA was not affected. Importantly, 9BSAMo reduced in parallel the neurulation defects elicited by the pAMo (Fig. 4C, lane 4). This strongly suggests that the premature accumulation of exon 9B-containing mRNA leads to neurulation defects. The protein isoforms produced by the 8-9A9' and 8-9A9B transcripts differ only by a single, C-terminal amino acid. AlphaFold 3 modeling did not reveal any
A

Ciona intestinalis

<figure><img src="image_9.png" /></figure>

Branchiostoma lanceolatum

<figure><img src="image_10.png" /></figure>

Strongylocentrotus purpuratus

NW_022145615.1

<figure><img src="image_11.png" /></figure>

B

<figure><img src="image_12.png" /></figure>

C

<figure><img src="image_13.png" /><figcaption>**Fig. 3.** Terminal exons of *TMP1* in non-vertebrate deuterostome lineages. **A.** *Tropomyosin* gene loci in non vertebrate deuterostome lineage: *Ciona intestinalis* (3 genes), *Branchiostoma lanceolatum* (4 genes) and *Strongylocentrotus purpuratus* (2 genes). Arrows indicate the orientation and locations of the genes. The number of alternative terminal coding exons for each gene is indicated below the arrows. **B.** Carboxy-terminal peptide sequences of tropomyosin proteins from non-vertebrate deuterostome species, corresponding to the last exon of the various tropomyosin genes. **C.** Strenght of the SD (splice donor) within the last exons (exon 9A in vertebrates) of the indicated tropomyosin genes, MaxEntScanscore5ss analysis. The highest scores are shown: they correspond to the bona fide SD sites in vertebrates. The low scores in non-vertebrates suggest that no functional SD site exists on terminal exon in these species.</figcaption></figure>

structural differences between the two proteins (data not shown). This suggests that the toxicity associated with forced accumulation of the 8-9A-9B isoform in embryos is likely attributable to the 9B 3'UTR, rather than differences in protein structure or function. To confirm this, we injected an mRNA made of the luciferase

coding sequence followed by either exon 9B, exon 9', or the Globin (Gb) 3'UTR as a control. About 80 % of embryos injected with the Luc9B mRNA had neurulation defects similar to those observed for the embryos injected with the pAMo. This was considerably more than embryos injected with Luc9' or LucGb (Fig. 4E). These findings
<figure><img src="image_14.png" /><figcaption>A</figcaption></figure>

**Fig. 4.** The abnormal accumulation of exon 9B is harmful to *Xenopus laevis* embryos. **A.** We injected two-cell embryos in both blastomeres with 20 ng (2.5 pmol) of antisense morpholino oligonucleotide (pAMo) to block recognition of the cleavage and polyadenylation signal (CPA) delineating exonic region 9'. Controls included non-injected embryos (NI), embryos injected with a 2.5 pmol of a control morpholino (coMo), or a translation-blocking morpholino against *tpm1* (tpmMo). We allowed them to develop until stage 28. We assessed the splicing pattern of endogenous *tpm1* mRNA by RT-PCR using primers in exon 7, exon 9A, exonic region 9' or exon 9B (middle panel). Muscular Tpm1 protein levels were evaluated by western blot (right panel). Tubulin was used as a loading control (Tub). Quantification of 3 independent experiments is shown below the blot as mean ± standard deviation. **B.** Two-cell embryos injected in both blastomeres with pAMo or left non-injected were allowed to develop until stage 17. Representative images of neurulae are shown. Observed defects in pAMo-injected embryos include improper elevation of neural folds and failure in the convergence and closure of the neural folds. **C.** Quantification of neurulation defects following injection of the indicated morpholino oligonucleotides. Each embryo was injected with 25 ng morpholinos: 25 ng control morpholino (coMo), 15 ng pAMo with 10 ng control morpholino (pAMo), 15 ng pAMo with 10 ng 9BSAMo (paMo+9BSAMo), 10 ng 9BSAMo with 15 ng control morpholino (9BSAMo). *p* values, Fisher's exact test. Shown are the results of a single experiment which was reproduced at least twice. **D.** The splicing pattern of endogenous *tpm1* mRNA was analysed by RT-PCR as in **A** following injection of the indicated amounts (ng) of coMo, pAMo and9BSAMo. **E.** Quantification of neurulation defects following injection in both blastomeres of 2 cell embryos of 1 fmole of luciferase mRNA with the following 3'UTR: globin (Gb), 9' or 9B.
strongly suggest a functional link between accumulation of 9B 3'UTR and defective neurulation observed in *Xenopus* embryos. From these results, we propose that terminal exon 9A9' was reactivated or maintained in amphibians and coelacanths to prevent the accumulation of exon 9B-containing *tpm1* mRNA, which is harmful during neurulation.

### 3.5. Peculiarities of terminal exon 9A9'

We explored whether the complex regulation of exon 9A9' as a hybrid terminal-internal exon in *Xenopus laevis* relied on any specific molecular features. The splice acceptor site is the first AG dinucleotide downstream of the branch point creating an “AG exclusion zone” (AGEZ) between the branch point and the acceptor site [36]. The AGEZ upstream of exon 9A is unusually long (Fig. S3B; compare with the AGEZ upstream of exon 9B, Fig. S3A). This suggests that the branch point of intron 8 may be located farther from the splice acceptor site than usual (typically 20–50 nucleotides upstream). To verify this, we used a nested RT-PCR based method [37]. The reverse primer (R2) is located between the exon-intron junction and the putative branchpoint. The PCR primers are designed to amplify the lariat of intron 8 across the branch point (primers R1 and F2) or the exon-intron junction (F1 and R1, Fig. 5A). The size (178 nucleotides) and the sequence of the amplimeres indicate that the branch point is the adenine nucleotide located 274 nucleotides upstream of the acceptor site (Fig. 5A).

According to the exon definition model, exons are marked by a cross-exon complex connecting their ends. Disrupting exon definition causes exon skipping [38–40]. To investigate the definition of exon 9A9', we designed a morpholino antisense oligonucleotide targeting the branch point (bpMo), predicting that it would disrupt exon 9A9' definition and result in exon skipping. Consistently, bpMo reduced isoforms 8–9A9' and 8-9A-9B while increasing 8–9B (Fig. 5B, lane 4). In line with previous findings (Fig. 4A), the pAMo increased 8-9A-9B at the expense of 8–9A9' (Fig. 5B, lane 3). A morpholino targeting the splice acceptor site of exon 9A (9ASAMo) had a distinct effect from bpMo (lane 5). It reduced both 8–9A9' and 8-9A-9B, but did not increase 8–9B. This suggests that, while bpMo impairs exon definition resulting in the accumulation of the isoform resulting from exon skipping (8–9B), exon 9A9' or 9A are still defined despite 9ASAMo. Of course an accessible splice site is necessary at a later stage to splice exon 8 to exon 9A or 9A9'.

To confirm these data, we used RT-qPCR to quantify exon-intron junctions in intron 7 (junctions of exon 7 to intron 7, e7i7, and of intron 7 to exon 8, i7e8) and intron 8 (e8i8 and i8e9A, Fig. 5C, upper panel). Fig. S4 shows the amounts of downstream junctions (i7e8 and i8e9A) plotted against the respective amounts of upstream junctions (e7i7 and e8i8). The junctions relative to intron 7 and the junctions relative to intron 8 in embryos injected with the coMo or the pAMo had a low abundance due to efficient splicing. By contrast, the abundance of both upstream and downstream junctions of intron 8 was high in bpMo-injected embryos, as intron 8 excision is highly reduced due to the poor definition of exons 9A and 9A9' when the branch point is blocked. Finally, for intron 8 in 9ASAMo-injected embryos, the downstream junction (i8e9A) was more abundant than the upstream junction (e8i8). This is confirmed by plotting the ratios of downstream to upstream junctions (Fig. 5C). The most likely explanation is that 9ASAMo blocks the second transesterification step, trapping non-excised lariats. That the first transesterification step occurs normally implies that 9ASAMo does not disrupt exon 9A definition. This supports again the idea that exon 9A9' is defined independently of the splice acceptor site. In dissected hearts where the isoform 8-9A-9B is predominant, the 9ASAMo similarly reduced the abundance of both 8–9A9' and 8-9A-9B isoforms without

increasing the abundance of isoform 8–9B (Fig. S4). Therefore the internal exon 9A is defined independently of the splice acceptor site, in the same way as the terminal exon 9A9'. Together, these data show that intron 8 has a distant branch point, and that the definition of exons 9A or 9A9' does not rely on the recognition of the AG acceptor splice site by U2AF1 (U2AF35). Intron 8 can therefore be described as AG-independent [38–40].

### 3.6. The role of UTE in defining terminal exon 9A9'

Given the independence of definition of exons 9A and 9A9' from the splice acceptor site, we investigated if the UTE plays a role in exon 9A9' definition. Injecting embryos with a morpholino targeting the UTE (UTEMo) significantly reduced 8–9A9' but not 8-9A-9B (Fig. 6A, lanes 1–2). This finding was confirmed in dissected somites (Fig. 6A, lanes 5–6) and dissected hearts (Fig. 6A, lanes 9–10; the absence of *myl1* in the heart fraction confirms the correct dissection). This shows that the UTE is essential for defining exon 9A9' as terminal, but not 9A as internal.

U1 snRNP binding is known to suppress downstream cleavage and polyadenylation in some mRNAs [41–45]. We tested if a similar mechanism applied to *tpm1* mRNA by using a morpholino against the internal splice donor site of exon 9A9' (SDMo). As expected, the SDMO reduced the amount of 8-9A-9B isoform in whole embryos, dissected somites and hearts, demonstrating that it efficiently inhibits the definition of internal exon 9A (Fig. 6A lane 3, 7 and 11; see quantifications, right panel). Co-injecting SDMo mitigated the UTE-mediated increase in 8-9A-9B isoform (Fig. 6A, compare lanes 2–4, 6–8, 10–12). Yet, it did not restore high 8–9A9' levels. Rather, it caused exon skipping with increased 8–9B. These observations rule out a simple mechanism of competition between the formations of 8–9A9' and 8-9A-9B isoforms. Rather, they demonstrate that the definition of 9A9' as a terminal exon is an active process that requires the UTE even in the absence of competition with the splice donor site downstream of exon 9A.

To analyse the accumulation of splicing reaction intermediates, we conducted an RNase protection assay (Fig. 6B) with a probe spanning exon 9A9' and adjacent introns (Fig. 6B, upper panel). We focused our analysis on the *tpm1L* homeologous gene (colored lines in Fig. 6B), as hybridization of the probe with the *tpm1S* RNA results in an additional cleavage within exon 9' due to a single nucleotide variation between the 2 homeologous genes. Non-injected and control MO-injected embryos (lanes 3–4) exhibited protected fragments corresponding to mature 8–9A9' and 8-9A-9B isoforms (152 and 79 nucleotides, respectively). pAMO (lane 7) increased the 8-9A-9B isoform, consistent with previous findings (Fig. 4A), but also of an intermediate matured at its 5' end but not its 3' end (216 nucleotides). Conversely, in 9ASAMo-injected embryos, a 3' processed but 5' unprocessed intermediate was detected (185 nucleotides, lane 6). Morpholino targeting the branch point or the UTE led to a predominance of fully unprocessed RNA (249 nucleotides, lanes 5 and 8). Even if fully unprocessed RNA is accumulated, both exon-intron boundaries are accumulated at the same level. This is consistent with the results observed in Fig. 5C where the i8e9A/e8i8 ratio is unchanged in bpMo-injected embryos. However, while mature isoform 8-9A-9B production (79 nucleotides) was strongly reduced by the morpholino against the branch point, it was not affected by the morpholino against the UTE. This confirms that the UTE is crucial for defining terminal exon 9A9' but not internal exon 9A. In addition, these results reveal that inhibiting the UTE abrogates both splicing of exon 8 to exon 9A and cleavage at the 3' end of exon 9'. Hence, the UTE couples splicing with cleavage and polyadenylation.
<figure><img src="image_15.png" /></figure>

<figure><img src="image_16.png" /></figure>

<figure><img src="image_17.png" /></figure>
**Fig. 5.** The definition of exons 9A and 9A9' is independent of the splice acceptor site. **A.** Mapping of the *in vivo* branch point in intron 8. Left, positions of the primers used to locate the branch point in intron 8. Primer R2 is used for reverse transcription. Primers R1 and F1 amplify the cDNA obtained after reverse-transcription of *tpm1* pre-mRNA (150 nucleotides). Primers R1 and F2 are divergent on *tpm1* pre-mRNA and do not yield an amplimere, but are convergent on the intron 8 lariat, producing amplimeres that encompass the branch point. Right, RT-PCR products obtained from RNA extracted from stage 28 whole embryos (E) and dissected somites (S), using the indicated primers. Bottom, sequence of *tpm1* intron 8. Sequencing of the amplimere produced by the R1 and F2 primers identified the branch point at single-nucleotide resolution (stars indicate branch points ligated to the donor site in sequenced R1-F2 amplimeres). **B.** Two-cell embryos were left non-injected (NI) or injected in both blastomeres with the indicated morpholinos: coMo, control; pAMo, morpholino against the CPA at the 3' end of exon 9A9'; bpMo, against the branch point in intron 8; 9ASAMo, against the splice acceptor site at the 5' end of exon 9A. At stage 28 total RNA was extracted and analysed by RT-PCR using a forward primer in exon 8 and reverse primers in exonic region 9' (top) or exon 9B (bottom). We assessed RNA recovery and embryo staging by amplification of *eef1a1* and *myl1* cDNA, respectively. Right panel, quantification of at least 3 independent independent experiments similar to that shown in the left panel. Fold changes are shown relative to NI embryos. **C.** Real-time RT-PCR with primers overlapping exon/intron boundaries in exon 7 and intron 7 (e7i7), intron 7 and exon 8 (i7e8), exon 8 and intron 8 (e8i8), and intron 8 and exon 9A (i8e9A). The ratios of the downstream junctions (i7e8 and i8e9A) to their respective upstream junctions (e7i7 and e8i8) are shown. Each dot represents a single embryo injected with the morpholino indicated by the color code on the right. **p.** Student's t-test.

## 4. Discussion

In this study, we used the highly conserved exon 9A of the mRNA encoding alpha-tropomyosin as a model to explore the evolutionary origin of hybrid internal-terminal exons. Our findings reveal that exon 9A is purely terminal in non-vertebrate deuterostomes, including *Echinoidea* (echinoderms), *Cephalochordata* and *Tunicata*. Within *Vertebrata* (vertebrates) however, exon 9A can function as either a purely internal exon or as a hybrid internal-terminal exon. Given that *Cephalachordata* and *Tunicata* share a closer evolutionary relationship with *Vertebrata* (all three belonging to the phylum *Chordata*) than with *Echinoidea*, the most parsimonious hypothesis is that exon 9A was originally terminal in deuterostomes. The adaptation of exon 9A as an internal exon likely occurred early in vertebrate evolution, driven by the emergence of an internal splice donor site (separating the 9A from the 9' regions) and of a non coding exon 9B. Over time, most vertebrate lineages appear to have lost the ancestral function of exon 9A as a terminal exon. However, sporadic retention or reactivation of exon 9A9' usage as a terminal exon in some species (see Fig. 1C-E) suggest that this trait persists as a vestigial feature. Notably, salamanders (*A. mexicanum*), frogs (*X. laevis*, *R. temporaria*, *N. parkeri*) and coelacanths (*L. chalumnae*) exhibit strong utilization of exon 9A9' as a terminal exon, suggesting that this vestigial trait was reactivated in their ancestors. Whether this reactivation arose independently in amphibians and coelacanths or stems from a shared ancestry is an intriguing question. As the reactivation depends on the conserved sequence known as the UTE (see Fig. 2), we think it more likely that it occurred in a common ancestor (early sarcopterygian, following the divergence from bony fishes) with a subsequent loss in lungfish, caecilians and amniotes. This evolutionary scenario - where a once-terminal exon switched to a hybrid exon through the acquisition of an internal splice donor site and the emergence of a downstream exon - may also apply to other hybrid exons.

In addition to being used as internal, exon 9A9' functions as terminal due to two cis-acting elements within the *tpm1* pre-mRNA: a weak cleavage-polyadenylation site located a few dozen nucleotides downstream of exon 9A, and the UTE. RNase protection assays reveal that the UTE plays a key role in facilitating the recognition of this weak cleavage-polyadenylation site, which is essential for defining exon 9A9' and initiating splicing and cleavage. Interactions between splicing and cleavage-polyadenylation factors are well-documented. For instance, U2AF65 and/or the U2 snRNP physically interact with several cleavage-polyadenylation factors, including the carboxy terminal region of the poly(A) polymerase [46], the cleavage-polyadenylation stimulating complex CPSF [47], or the cleavage factor I CF1(m) [48]. Similarly, CPSF enhances splicing in the case of interleukin 7 receptor exon 6 [49]. These interactions underline the tight coupling of splicing and cleavage-polyadenylation. Given that the UTE lies in close proximity to the branch point, which binds the U2 snRNP, it is plausible that yet unknown UTE-binding

factors interact with the U2 snRNP and/or cleavage-polyadenylation factors to strengthen communication between the 5' and 3' boundaries of exon 9A9'. Interestingly, exon 9A is downstream of an AG-independent intron with a distant branch point. The large region between the branch point and the 3' splice site offers significant potential for intronic cis-regulatory elements of pre-mRNA maturation. The UTE is in that region, and it may also be the case for cis-regulatory elements of other hybrid exons. Future research should systematically evaluate whether hybrid internal-terminal exons preferentially evolve downstream of AG-independent introns with a distant branch point.

While the nature of exon 9A (or 9A9') essentially switched from terminal to internal in vertebrates, its ability to function as a terminal exon persisted in certain lineages, making it a hybrid exon. This raises the question of what evolutionary pressures favoured keeping or reactivating the capacity of exon 9A to be terminal. By using a morpholino antisense oligonucleotide to switch the endogenous *tpm1* splicing pattern from 8- to 9A9' to 8-9A9-8B as well as RNA injection, we demonstrated that mRNAs containing exon 9B, which are produced when exon 9A acts as an internal exon, are toxic in *Xenopus* tadpoles. We hypothesize that the observed neural fold defects result from exon 9B acting as a molecular sponge for regulatory RNA-binding molecules, such as miRNAs and/or RNA-binding proteins. Exon 9B is longer than exon 9' (256 vs 71 nucleotides in *Xenopus laevis*), potentially offering a greater number of binding sites for regulatory factors. Since the morpholino-induced accumulation of exon 9B is restricted to *tpm1*-expressing somitic cells, the presence of exon 9B in somites is likely the primary cause of the neural fold defects. Consequently, the regulatory molecules sequestered by exon 9B must also be present in somites. We queried RBPDB [50] to identify candidate RNA-binding proteins that might interact with exon 9B. By restricting this analysis to proteins present in muscle tissues based on GTEx data [51], we identified three high-interest candidates: MBNL1, KHSRP and RBMX. In parallel, several muscle-specific miRNAs (myomiRs) were identified in vertebrates, like *miR-1*, *miR-95*, *miR-128a*, *miR-133a*, *miR-206*, *miR-214* and *miR-499* [52]. Further experiments will be needed to test whether any of these regulatory molecules physically interact with exon 9B, and whether their titration underlies the observed neurulation defects in *Xenopus* embryos with elevated exon 9B levels.

### CRediT authorship contribution statement

**Agnès Méreau:** Writing – review & editing, Methodology, Investigation, Formal analysis, Conceptualization. **Hubert Lerivray:** Investigation. **Justine Viet:** Methodology, Investigation, Formal analysis. **Serge Hardy:** Funding acquisition, Data curation. **Luc Paillard:** Writing – original draft, Visualization, Validation, Supervision, Data curation, Conceptualization. **Yann Audic:** Writing – review & editing, Resources, Methodology, Investigation, Formal analysis, Conceptualization.
<figure><img src="image_18.png" /><figcaption>A</figcaption></figure>

<figure><img src="image_19.png" /><figcaption>B</figcaption></figure>

<figure><img src="image_20.png" /></figure>

**Fig. 6.** The UTE controls the definition of terminal exon 9A9', but not internal exon 9A.Two-cell embryos were injected in both blastomeres with the indicated antisense morpholinos (SDMo, morpholino against the splice donor site between exon 9A and exonic region 9'; other morpholinos, same as in Fig. 5), or left non-injected (NI), and allowed to develop. A, *tpm1* splicing end products. Total RNAs were extracted from whole embryos, dissected somites or hearts, as indicated, and *tpm1* splicing analysed by RT-PCR using a forward primer in exon 8 and reverse primers in exonic region 9' (top) or exon 9B (bottom). We assessed RNA recovery, embryo stages and embryo dissection by amplifying *eef1a1* and *myl1* cDNA, respectively. Left, one representative experiment, right quantification of at least 3 independent independent experiments (mean ± s.d.). B, *tpm1* splicing intermediates and end products. Total RNAs were extracted from whole embryos and analysed by RNase protection assays. Lane 1, undigested probe mix; the positions of undigested probes for *tpm1* and *myl1* are indicated on the left. Right, schematic representation of the 9A9' pre-mRNA region, showing the *tpm1* probe and the sizes of protected fragments. Colored lines indicate the positions of protected probe fragments for *tpm1.L* RNA (fully complementary to the probe). Grey lines show the positions of probe fragments protected by *tpm1.S* RNA, which were not included in the quantification. Lower panel, quantification of the intermediates and end-products of TPM1 maturation from at least 3 independent injection experiments are shown in the lower panel (mean ± s.d.).
## Funding

This work was supported in part by a grant from AFM-Telethon to Serge Hardy.

## Declaration of competing interest

The authors declare no conflict of interest.

## Acknowledgments

The authors gratefully acknowledge the help of Stéphane Deschamps, Margaux Véron and Claire Bertrand with the experiments.

## Appendix A. Supplementary data

Supplementary data to this article can be found online at https://doi.org/10.1016/j.biochi.2025.07.009.

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