Polypyrimidine Tract-binding Protein Is Involved in Vivo in Repression of a Composite Internal/3′ -Terminal Exon of the Xenopus α-Tropomyosin Pre-mRNA

Abstract

The Xenopus ␣ fast -tropomyosin gene contains, at its 3 -end, a composite internal/3 -terminal exon (exon 9A9 ), which is subjected to three different patterns of splicing according to the cell type. Exon 9A9 is included as a terminal exon in the myotome and as an internal exon in adult striated muscles, whereas it is skipped in nonmuscle cells. We have developed an in vivo model based on transient expression of minigenes encompassing the regulated exon 9A9 in Xenopus oocytes and embryos. We first show that the different ␣-tropomyosin minigenes recapitulate the splicing pattern of the endogenous gene and constitute valuable tools to seek regulatory sequences involved in exon 9A9 usage. A mutational analysis led to the identification of an intronic element that is involved in the repression of exon 9A9 in nonmuscle cells. This element harbors four polypyrimidine track-binding protein (PTB) binding sites that are essential for the repression of exon 9A9 . We show using UV cross-linking and immunoprecipitation experiments that Xenopus PTB (XPTB) interacts with these PTB binding sites. Finally, we show that depletion of endogenous XPTB in Xenopus embryos using a morpholinobased translational inhibition strategy resulted in exon 9A9 inclusion in embryonic epidermal cells. These results demonstrate that XPTB is required in vivo to repress the terminal exon 9A9 and suggest that PTB could be a major actor in the repression of regulated 3 -terminal exon.Alternative splicing is a widespread mechanism in metazoans, by which multiple mRNAs can be produced from a single gene. This process is often subjected to tissue or developmental control and allows the production of protein isoforms differing in specific domains.Over the last few years, some general topics have emerged from the intensive studies undertaken to elucidate the mechanism of alternative splicing. It is thus established that regulated exons are generally flanked by suboptimal splice sites whose recognition is modulated by cis-acting regulatory sequences present within exons or introns. In most cases, alternative exons are subject to positive and negative regulation which insures a tight control on their usage (reviewed in Ref.1). Numerous trans-acting factors that modulate alternative splicing have also been characterized. These regulatory factors belong to two broad families of RNA-binding proteins, designated hnRNP proteins and RS-domain proteins. The latter includes the SR protein subfamily (2, 3) that is involved in constitutive or alternative exon recognition through exonic sequence called exonic enhancer (4).Despite this significant breakthrough, the basis of tissue regulation of alternative splicing is still poorly understood in vertebrates. Indeed, contrary to the paradigm described in Drosophila, where many splicing events are controlled by tissue-specific factors (5), to date the implication of such factors appears limited in vertebrates.To overcome this lack of tissue specific factors it was proposed that ubiquitously expressed splicing factors could...

Polypyrimidine Tract Binding Protein Prevents Activity of an Intronic Regulatory Element That Promotes Usage of a Composite 3′-Terminal Exon

“…Plasmid Constructs-The ␣-TM minigenes pBS-SV40.7-9B, pBSActin.7-9B, and pBSKeratin.7-9B have been described previously (17). The mutated minigenes were modified by PCRmediated site-directed mutagenesis and cassette substitutions as described (17).…”

“…The mutated minigenes were modified by PCRmediated site-directed mutagenesis and cassette substitutions as described (17). The different oligonucleotides are given in the supplemental material (supplemental Table S1).…”

“…Each amplimer was cloned into pGEM-T easy vector (Promega) and verified by DNA sequencing. The BamHI/NotI or EcoRV/NotI fragments were then cloned into pT7TS-V5 plasmid in frame with the carboxyl V5 epitope tag (17).…”

“…In non-muscle cells, exon 9A9Ј is skipped, and exon 9D is used as a 3Ј-terminal exon (16). Transient transgenesis of minigenes driven by tissue-specific promoters that recapitulate the specific use of exon 9A9Ј in embryonic myotomal cells, and its repression in embryonic epidermal can be used to study the function and interplay of cis sequence elements within the ␣-TM pre-mRNA (17). Loss or gain of function of trans-acting factors and the consequences on the processing of pre-mRNA derived from minigenes or from the endogenous gene can also be apprehended (17,18).…”

“…Transient transgenesis of minigenes driven by tissue-specific promoters that recapitulate the specific use of exon 9A9Ј in embryonic myotomal cells, and its repression in embryonic epidermal can be used to study the function and interplay of cis sequence elements within the ␣-TM pre-mRNA (17). Loss or gain of function of trans-acting factors and the consequences on the processing of pre-mRNA derived from minigenes or from the endogenous gene can also be apprehended (17,18). Using this model, we showed in previous studies that xPTB, the Xenopus orthologue of the mammalian RNA-binding protein PTB1, represses the usage of the composite exon 9A9Ј as a terminal exon in embryonic non-muscle cells.…”