Agnès Méreau

A Janus Splicing Regulatory Element Modulates HIV-1 tat and rev mRNA Production by Coordination of hnRNP A1 Cooperative Binding

Retroviral protein production depends upon alternative splicing of the viral transcript. The HIV-1 acceptor site A7 is required for tat and rev mRNA production. Production of the Tat transcriptional activator is highly controlled because of its apoptotic properties. Two silencer elements (ESS3 and ISS) and two enhancer elements (ESE2 and ESE3/(GAA)3) were previously identified at site A7. hnRNP A1 binds ISS and ESS3 and is involved in the inhibitory process, ASF/SF2 activates site A7 utilisation. Here, by using chemical and enzymatic probes we established the 2D structure of the HIV-1(BRU) RNA region containing site A7 and identified the RNA segments protected in nuclear extract and by purified hnRNP A1. ISS, ESE3/(GAA)3 and ESS3 are located in three distinct stem-loop structures (SLS1, 2 and 3). As expected, hnRNP A1 binds sites 1, 2 and 3 of ISS and ESS3b, and oligomerises on the polypurine sequence upstream of ESS3b. In addition, we discovered an unidentified hnRNP A1 binding site (AUAGAA), that overlaps ESE3/(GAA)3. On the basis of competition experiments, hnRNP A1 has a stronger affinity for this site than for ESS3b. By insertion of (GAA)3 alone or preceded by the AUA trinucleotide in a foreign context, the AUAGAA sequence was found to modulate strongly the (GAA)3 splicing enhancer activity. Cross-linking experiments on these heterologous RNAs and the SLS2-SLS3 HIV-1 RNA region, in nuclear extract and with recombinant proteins, showed that binding of hnRNP A1 to AUA(GAA)3 strongly competes the association of ASF/SF2 with (GAA)3. In addition, disruption of AUA(GAA)3 demonstrated a key role of this sequence in hnRNP A1 cooperative binding to the ISS and ESS3b inhibitors and hnRNP A1 oligomerisation on the polypurine sequence. Thus, depending on the cellular context ([ASF/SF2]/[hnRNP A1] ratio), AUA(GAA)3 will activate or repress site A7 utilisation and can thus be considered as a Janus splicing regulator.

Bioinformatic prediction, deep sequencing of microRNAs and expression analysis during phenotypic plasticity in the pea aphid, Acyrthosiphon pisum.

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.

PTB Regulates the Processing of a 3-Terminal Exon by Repressing both Splicing and Polyadenylation

ABSTRACT 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 α-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 α-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.

ERK-regulated differential expression of the Mitf 6a/b splicing isoforms in melanoma.

The master regulator of the melanocyte lineage Mitf is intimately involved in development as well as melanoma, controlling cell survival, differentiation, proliferation and metastasis/migration. Consistent with its central role, Mitf expression and Mitf post‐translational modifications are tightly regulated. An additional potential level of regulation is afforded by differential splicing of Mitf exon‐6 leading to the generation of two isoforms that differ by the presence of six amino‐acids in the Mitf (+) isoform and which have differential effects on cell cycle progression. However, whether the ratio of the two isoforms is regulated and whether their expression correlates with melanoma progression is not known. Here, we show that the differential expression of the Mitf 6a/b isoforms is dependent on the MAPKinase signalling, being linked to the activation of MEK1‐ERK2, but not to N‐RAS/B‐RAF mutation status. In addition, quantification of Mitf 6a/b splicing forms in 86 melanoma samples revealed substantially increased levels of the Mitf (−) form in a subset of metastatic melanomas. The results suggest that differential expression of the Mitf 6a/b isoforms may represent an additional mechanism for regulating Mitf function and melanoma biology.

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

The Xenopus αfast-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 α-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.

Implication of the SMN complex in the biogenesis and steady state level of the Signal Recognition Particle

Spinal muscular atrophy is a severe motor neuron disease caused by reduced levels of the ubiquitous Survival of MotoNeurons (SMN) protein. SMN is part of a complex that is essential for spliceosomal UsnRNP biogenesis. Signal recognition particle (SRP) is a ribonucleoprotein particle crucial for co-translational targeting of secretory and membrane proteins to the endoplasmic reticulum. SRP biogenesis is a nucleo-cytoplasmic multistep process in which the protein components, except SRP54, assemble with 7S RNA in the nucleolus. Then, SRP54 is incorporated after export of the pre-particle into the cytoplasm. The assembly factors necessary for SRP biogenesis remain to be identified. Here, we show that 7S RNA binds to purified SMN complexes in vitro and that SMN complexes associate with SRP in cellular extracts. We identified the RNA determinants required. Moreover, we report a specific reduction of 7S RNA levels in the spinal cord of SMN-deficient mice, and in a Schizosaccharomyces pombe strain carrying a temperature-degron allele of SMN. Additionally, microinjected antibodies directed against SMN or Gemin2 interfere with the association of SRP54 with 7S RNA in Xenopus laevis oocytes. Our data show that reduced levels of the SMN protein lead to defect in SRP steady-state level and describe the SMN complex as the first identified cellular factor required for SRP biogenesis.

Analysis of splicing patterns by pyrosequencing

Several different mRNAs can be produced from a given pre-mRNA by regulated alternative splicing, or as the result of deregulations that may lead to pathological states. Analysing splicing patterns is therefore of importance to describe and understand developmental programs, cellular responses to internal or external cues, or human diseases. We describe here a method, Pyrosequencing Analysis of Splicing Patterns (PASP), that combines RT–PCR and pyrosequencing of PCR products. We demonstrated that: (i) Ratios of two pure RNAs mixed in various proportions were accurately measured by PASP; (ii) PASP can be adapted to virtually any splicing event, including mutually exclusive exons, complex patterns of exon skipping or inclusion, and alternative 3′ terminal exons; (iii) In extracts from different organs, the proportions of RNA isoforms measured by PASP reflected those measured by other methods. The PASP method is therefore reliable for analysing splicing patterns. All steps are done in 96-wells microplates, without gel electrophoresis, opening the way to high-throughput comparisons of RNA from several sources.

Xenopus as a model to study alternative splicing in vivo.

An increasing number of genes are being identified for which the corresponding mRNAs contain different combinations of the encoded exons. This highly regulated exon choice, or alternative splicing, is often tissue‐specific and potentially could differentially affect cellular functions. Alternative splicing is therefore not only a means to increase the coding capacity of the genome, but also to regulate gene expression during differentiation or development. To both evaluate the importance for cellular functions and define the regulatory pathways of alternative splicing, it is necessary to progress from the in vitro or ex vivo experimental models actually used towards in vivo whole‐animal studies. We present here the amphibian, Xenopus, as an experimental model highly amenable for such studies. The various experimental approaches that can be used with Xenopus oocytes and embryos to characterize regulatory sequence elements and factors are presented and the advantages and drawbacks of these approaches are discussed. Finally, the real possibilities for large‐scale identification of mRNAs containing alternatively spliced exons, the tissue‐specific patterns of exon usage and the way in which these patterns are modified by perturbing the relative amount of splicing factors are discussed.

Ptbp1 and Exosc9 knockdowns trigger skin stability defects through different pathways.

In humans, genetic diseases affecting skin integrity (genodermatoses) are generally caused by mutations in a small number of genes that encode structural components of the dermal-epidermal junctions. In this article, we first show that inactivation of both exosc9, which encodes a component of the RNA exosome, and ptbp1, which encodes an RNA-binding protein abundant in Xenopus embryonic skin, impairs embryonic Xenopus skin development, with the appearance of dorsal blisters along the anterior part of the fin. However, histological and electron microscopy analyses revealed that the two phenotypes are distinct. Exosc9 morphants are characterized by an increase in the apical surface of the goblet cells, loss of adhesion between the sensorial and peridermal layers, and a decrease in the number of ciliated cells within the blisters. Ptbp1 morphants are characterized by an altered goblet cell morphology. Gene expression profiling by deep RNA sequencing showed that the expression of epidermal and genodermatosis-related genes is also differentially affected in the two morphants, indicating that alterations in post-transcriptional regulations can lead to skin developmental defects through different routes. Therefore, the developing larval epidermis of Xenopus will prove to be a useful model for dissecting the post-transcriptional regulatory network involved in skin development and stability with significant implications for human diseases.

Polypyrimidine tract binding protein prevents activity of an intronic regulatory element that promotes usage of a composite 3'-terminal exon.

Alternative splicing of 3′-terminal exons plays a critical role in gene expression by producing mRNA with distinct 3′-untranslated regions that regulate their fate and their expression. The Xenopus α-tropomyosin pre-mRNA possesses a composite internal/3′-terminal exon (exon 9A9′) that is differentially processed depending on the embryonic tissue. Exon 9A9′ is repressed in non-muscle tissue by the polypyrimidine tract binding protein, whereas it is selected as a 3′-terminal or internal exon in myotomal cells and adult striated muscles, respectively. We report here the identification of an intronic regulatory element, designated the upstream terminal exon enhancer (UTE), that is required for the specific usage of exon 9A9′ as a 3′-terminal exon in the myotome. We demonstrate that polypyrimidine tract binding protein prevents the activity of UTE in non-muscle cells, whereas a subclass of serine/arginine rich (SR) proteins promotes the selection of exon 9A9′ in a UTE-dependent way. Morpholino-targeted blocking of UTE in the embryo strongly reduced the inclusion of exon 9A9′ as a 3′-terminal exon in the endogenous mRNA, demonstrating the function of UTE under physiological circumstances. This strategy allowed us to reveal a splicing pathway that generates a mRNA with no in frame stop codon and whose steady-state level is translation-dependent. This result suggests that a non-stop decay mechanism participates in the strict control of the 3′-end processing of the α-tropomyosin pre-mRNA.