SC35 activates the usage of exon 9A9′ as a 3′-terminal exon through UTE. A, the wild-type minigene driven by the SV40 early promoter was injected into the nucleus of stage VI oocytes into which in vitro transcribed V5-tagged xSC35 RNA (4, 8, and 16 ng) had been previously injected into cytoplasm. V5-tagged xSC35 expression was monitored by Western blotting using a V5 antibody. B, the wild-type (WT) and ΔUTE mutant minigenes were injected into the nucleus of stage VI oocytes into which in vitro transcribed V5-tagged xSC35 RNA had been previously injected into cytoplasm (+) or not (−). A and B, total RNA was subjected to 3′-RACE-PCR using a ³²P-oligonucleotide specific to the SV40 minigene reporter. Quantification was performed on a PhosphorImager to yield the proportion of each product given in the table below. MW, molecular weight markers.

A subclass of SR proteins activate exon 9A9′ splicing through UTE. The wild-type (WT) or ΔUTE mutant constructions were injected into the nucleus of stage VI oocyte alone or in presence of in vitro transcribed RNA encoding V5-tagged SR proteins. A, V5-tagged SR protein expression in cytoplasmic and nuclear fractions was monitored by Western blotting using a V5 antibody. B, total RNA was subjected to 3′-RACE-PCR using a ³²P-oligonucleotide specific to the SV40 minigene reporter. The structure of the amplified fragments is indicated on the right. Quantification was performed on a PhosphorImager to yield the proportion of each mRNA given in the table below. ND, not detected.

xPTB prevents the activity of UTE in non muscle cells. A, mutant minigenes deleted for UTE (ΔUTE) and wild-type (WT) minigenes, driven by the keratin promoter, were injected in Xenopus embryo alone (−) or together with control morpholino (Co Mo) or xPTB-Mo. The steady-state level of xPTB was checked by Western blot using a xPTB antiserum. xPTB is indicated by an arrow, and the nonspecific cross-reacting band indicated by a star was used as a loading control. The mRNA splicing pattern was assayed by 3′-RACE-PCR using a ³²P-oligonucleotide specific to the keratin minigene constructs. B, the wild-type and ΔUTE minigenes driven by the SV40 promoter were injected into oocyte nuclei alone or with increasing amounts of 150PY competitor RNA. The mRNA splicing pattern was assayed by 3′-RACE-PCR using a ³²P-oligonucleotide specific to SV40 minigene constructs. A and B, quantification was performed on a PhosphorImager to yield the proportion of each product given in the table below. ND, not detected.

Differential processing of the 3′-end of the Xenopus α-TM pre-mRNA. A, schematic diagram of the 3′-terminal region of the Xenopus α-TM pre-mRNA and of the different tissue-specific RNA processing patterns. Exons and introns are represented by boxes and horizontal lines, respectively. αe, αa, and αnm represent the poly(A) sites present within exons 9A9′, 9B, and 9D, respectively. The arrows represent the forward and reverse primers used to specifically RT-PCR-amplify the different RNA isoforms. The different RNA products in myotome, embryonic heart, adult striated muscle, and non-muscle tissues are indicated. B, diagrammed representation of the α-tropomyosin minigene and of the different in vivo expression assays using the Xenopus embryo and oocyte. C, schematic representation of the 3′-RACE/PCR assay. RNA was reverse transcribed with an oligo(dT) anchor primer. cDNA was further amplified with a reverse anchor primer and a radiolabeled forward primer that hybridize at the junction of the promoter and exon 7. Primers are indicated by arrows, and an asterisk marks the radiolabeling. The structure and size of all splicing products produced by the different minigenes are given.

UTE activates the splicing and cleavage/polyadenylation of exon 9A9′. RNase protection analysis of RNA extracted from uninjected embryos or embryos injected with UTE-1 Mo, UTE-2 Mo, or control morpholino (Co Mo). The RNase protection probes are presented above. A, UTE activates the cleavage/polyadenylation of exon 9A9′. RNase protection was realized with an RNA probe extending beyond the αe poly(A) site, as presented at the top. The three-nucleotide difference within exon 9′ between the two α-TM genes is indicated by a star. The identity of uncleaved (Uncl.) and cleaved (Cl.) protected fragments is indicated on the right. B, UTE activates the splicing of exon 9A9′. RNase protection was realized with an RNA probe that covered the 3′ splice site of exon 9A9′, as presented at the top. The identity of unspliced and spliced fragments is shown on the right. C, unprocessed RNAs accumulate in UTE Mo-injected embryos. RNase protection was realized with a probe spanning the 3′ splice site and extending beyond the cleavage site of exon 9A9′, as shown on the left. The protected fragment corresponding to uncleaved/unspliced (Uncl./Unspl.) molecules is indicated on the right. A–C, the protected fragments were quantified by PhosphorImager analysis and normalized to uridine content and EF1-α. The α-TM, EF1-α, and MLC probes were used as molecular weight markers (MW).

A, targeting the endogenous UTE generates α2Δ9A mRNA, a potential non-stop decay substrate. Endogenous α7, α2, and 05 RNAs were analyzed by RT-PCR using a ³²P-labeled sense primer specific for exon 8 and reverse primers specific for exon 9′, 9B, and 9D, respectively. The PCR products were quantified by PhosphorImager analysis and normalized to EF1-α. The bar diagram shows the log₂ mean -fold change ± S.D. of the different α-tropomyosin isoforms in morpholino-injected embryos relative to uninjected embryos (n = 3 independent experiments). B, reduction of the α2Δ9A mRNA is translation-dependent. A morpholino complementary to the region encompassing the start codon of the muscle α-tropomyosin mRNAs (TM Mo) was injected alone or in combination with UTE-1 Mo. The steady-state level of muscle α-tropomyosin was studied by Western blot with the monoclonal anti-tropomyosin antibody (clone TM311). Non-muscle tropomyosin was used as a loading control. Endogenous α2 and α2Δ9A RNAs were analyzed by RT-PCR using primers specific for exon 7 and 9B. The PCR products were quantified by PhosphorImager analysis and normalized to EF1-α. Data were presented as mean -fold change ± S.D. of α2Δ9A RNA in morpholino-injected embryos relative to uninjected embryos (n = 3 independent experiments).

Morpholino-targeted inhibition of the endogenous UTE modifies the 3′-end processing of the α-TM pre-mRNA. A, partial sequence of the α-TM pre-mRNA upstream of exon 9A9′. Exon sequences are indicated in capital letters, and intron sequences are in lowercase letters. The distal −274 branch point is shown in boldface characters, and the high affinity PTB binding sites are framed. UTE is delimited by brackets, and the targeting positions of UTE-1 and UTE-2 Mo are underlined. B, uninjected embryos or embryos injected with control Mo (Co Mo) or UTE-1 Mo or UTE-2 Mo were analyzed by a RNase protection assay using the probe illustrated at the top. Sequences derived from vector are represented by black rectangles. EF1-α and MLC1/3 probes were included in the assay as control for RNA recovery and embryo staging, respectively. The identity or composition of each protected product is indicated on the right. α7 RNA produced two protected fragments because of three nucleotide differences within exon 9A9′ between the two α-tropomyosin genes.

An intronic sequence upstream of exon 9A9′ contributes to its splicing as a 3′-terminal exon in muscle cells. A, schematic map of the intronic region upstream of exon 9A9′. The distant −274 branch site and its associated polypyrimidine tract are represented by a black circle and a black rectangle, respectively. The intronic sequence from −204 to −165 upstream of exon 9A9′ is shown. The generated mutations are shown below the wild-type sequence. B, the wild-type (WT) and the indicated mutant minigenes under the control of the cardiac actin promoter were injected into Xenopus embryos. The mRNA splicing pattern was assayed by 3′-RACE-PCR using a ³²P-oligonucleotide specific to the cardiac actin minigene constructs in the presence (+) or absence (−) of reverse transcriptase. The structure of the amplified fragments is indicated on the right. Quantification was performed on a PhosphorImager to yield the proportion of each mRNA. The percentage of each mRNA is given in the table below a representative experiment as mean ± S.D. (n = 20 for the wild type construct and n = 3 for the mutants). C, the distant −274 branch site and UTE are functionally coupled. The Δ−204−165 mutation (ΔUTE) and the mutation of the distant nucleotide −274 branch site (mutBS) were associated with the 3′ cons mutant in which seven consecutive pyrimidine near the dinucleotide AG were introduced or to the SPA mutant in which a strong synthetic poly(A) site was substituted to the αe poly(A) site. The wild type and the indicated mutant minigenes under the control of the cardiac actin promoter were injected into Xenopus embryos. Samples were processed as for B. The percentage of each mRNA is given in the table below a representative experiment as mean ± S.D. (n = 20 for the wild-type construct, and n = 3 for the mutants).