No abstract

No abstract

No abstract

An equilibrium between spliced and unspliced primary transcripts is essential for retrovirus multiplication. This equilibrium is maintained by the presence of inefficient splice sites. The A3 3-splice site of human immunodeficiency virus type I (HIV-1) is required for Tat mRNA production. The infrequent utilization of this splice site has been attributed to the presence of a suboptimal polypyrimidine tract and an exonic splicing silencer (ESS2) in tat exon 2 ϳ60 nucleotides downstream of 3-splice site A3. Here, using site-directed mutagenesis followed by analysis of splicing in vitro and in HeLa cells, we show that the 5 extremity of tat exon 2 contains a second exonic splicing silencer (ESS2p), which acts to repress splice site A3. The inhibitory property of this exonic silencer was active when inserted downstream of another HIV-1 3-splice site (A2). Protein hnRNP H binds to this inhibitory element, and two Uto-C substitutions within the ESS2p element cause a decreased hnRNP H affinity with a concomitant increase in splicing efficiency at 3-splice site A3. This suggests that hnRNP H is directly involved in splicing inhibition. We propose that hnRNP H binds to the HIV-1 ESS2p element and competes with U2AF 35 for binding to the exon sequence flanking 3-splice site A3. This binding results in the inhibition of splicing at 3-splice site A3.Because the unique transcript produced from the integrated proviral cDNA of retroviruses serves as the genome for newly synthesized virions and also for the production of mRNAs by alternative splicing, retrovirus multiplication depends upon an equilibrium between spliced and unspliced primary transcripts. To ensure this equilibrium, retroviral RNAs generally have splice sites that are used with low efficiencies. In human immunodeficiency virus type I (HIV-1), 3Ј-splice sites (3Јss) 1 and several central 3Јss (A3, A4a, A4b, A4c, and A5) compete with each other (Fig. 1A). Site A3 is required for production of tat mRNAs, sites A4a, b, and c for production of rev and env mRNAs and site A5 for production of nef and env mRNAs (Fig. 1A) (1). Metazoan 3Јss consist of three critical elements: the branchpoint sequence (2, 3), a polypyrimidine tract (PPT) sequence (4, 5) and an AG dinucleotide at the 3Ј-end of the intron (for reviews, see Refs. 6 -9). HIV-1 branchpoint sequences are highly divergent in comparison to the metazoan consensus sequence (10 -12), and HIV-1 PPTs are suboptimal (short and interspersed by purines) (13-15). The affinity of factor U2AF for the PPT depends upon the presence of a long stretch of U residues (9, 16). Factor U2AF consists of two proteins, U2AF 65 and U2AF35 , with molecular weight of 65 and 35, respectively (17). Introns with suboptimal PPTs, like those in HIV-1 RNA, require binding of U2AF 35 at the intron-exon junction for stable interaction of U2AF 65 with the PPT (9, 18, 19). Furthermore, suboptimal 3Јss are often the subject of positive or negative regulation by cis-regulatory elements, which are frequently located in the 3Ј exon. Exonic splicing en...

BackgroundPost-transcriptional regulation in eukaryotes can be operated through microRNA (miRNAs) mediated gene silencing. MiRNAs are small (18-25 nucleotides) non-coding RNAs that play crucial role in regulation of gene expression in eukaryotes. In insects, miRNAs have been shown to be involved in multiple mechanisms such as embryonic development, tissue differentiation, metamorphosis or circadian rhythm. Insect miRNAs have been identified in different species belonging to five orders: Coleoptera, Diptera, Hymenoptera, Lepidoptera and Orthoptera.ResultsWe developed high throughput Solexa sequencing and bioinformatic analyses of the genome of the pea aphid Acyrthosiphon pisum in order to identify the first miRNAs from a hemipteran insect. By combining these methods we identified 149 miRNAs including 55 conserved and 94 new miRNAs. Moreover, we investigated the regulation of these miRNAs in different alternative morphs of the pea aphid by analysing the expression of miRNAs across the switch of reproduction mode. Pea aphid microRNA sequences have been posted to miRBase: http://microrna.sanger.ac.uk/sequences/ConclusionsOur study has identified candidates as putative regulators involved in reproductive polyphenism in aphids and opens new avenues for further functional analyses.

Opacification of the ocular lens, termed cataract, is a common cause of blindness. To become transparent, lens fiber cells undergo degradation of their organelles, including their nuclei, presenting a fundamental question: does signaling/transcription sufficiently explain differentiation of cells progressing toward compromised transcriptional potential? We report that a conserved RNA-binding protein Celf1 post-transcriptionally controls key genes to regulate lens fiber cell differentiation. Celf1-targeted knockout mice and celf1-knockdown zebrafish and Xenopus morphants have severe eye defects/cataract. Celf1 spatiotemporally down-regulates the cyclin-dependent kinase (Cdk) inhibitor p27Kip1 by interacting with its 5’ UTR and mediating translation inhibition. Celf1 deficiency causes ectopic up-regulation of p21Cip1. Further, Celf1 directly binds to the mRNA of the nuclease Dnase2b to maintain its high levels. Together these events are necessary for Cdk1-mediated lamin A/C phosphorylation to initiate nuclear envelope breakdown and DNA degradation in fiber cells. Moreover, Celf1 controls alternative splicing of the membrane-organization factor beta-spectrin and regulates F-actin-crosslinking factor Actn2 mRNA levels, thereby controlling fiber cell morphology. Thus, we illustrate new Celf1-regulated molecular mechanisms in lens development, suggesting that post-transcriptional regulatory RNA-binding proteins have evolved conserved functions to control vertebrate oculogenesis.

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...

The polypyrimidine tract binding protein (PTB) has been described as a global repressor of regulated exons. To investigate PTB functions in a physiological context, we used a combination of morpholino-mediated knockdown and transgenic overexpression strategies in Xenopus laevis embryos. We show that embryonic endoderm and skin deficient in PTB displayed a switch of the alpha-tropomyosin pre-mRNA 3' end processing to the somite-specific pattern that results from the utilization of an upstream 3'-terminal exon designed exon 9A9'. Conversely, somitic targeted overexpression of PTB resulted in the repression of the somite-specific exon 9A9' and a switch towards the nonmuscle pattern. These results validate PTB as a key physiological regulator of the 3' end processing of the alpha-tropomyosin pre-mRNA. Moreover, using a minigene strategy in the Xenopus oocyte, we show that in addition to repressing the splicing of exon 9A9', PTB regulates the cleavage/polyadenylation of this 3'-terminal exon.