The RNA-binding proteins CELF1 and ELAVL1 cooperatively control the alternative splicing of CD44.

CD44 mRNA contains nine consecutive cassette exons, v2 to v10. Upon alternative splicing, several isoforms are produced with different impacts on tumor biology. Here, we demonstrate the involvement of the RNA-binding proteins CELF1 and ELAVL1 in the control of CD44 splicing. We show by FRET-FLIM that these proteins directly interact in the nucleus. By combining RNAi-mediated depletion and exon array hybridization in HeLa cells, we observe that the exons v7 to v10 of CD44 are highly sensitive to CELF1 and ELAVL1 depletion. We confirm by RT-PCR that CELF1 and ELAVL1 together stimulate the inclusion of these exons in CD44 mRNA. Finally, we show in eight different tumor types that high expression of CELF1 and/or ELAVL1 is correlated with the inclusion of CD44 variable exons. These data point to functional interactions between CELF1 and ELAVL1 in the control of CD44 splicing in human cancers.

Reassembling green fluorescent protein for in vitro evaluation of trans-translation.

In order to discover new antibiotics with improved activity and selectivity, we created a reliable in vitro reporter system to detect trans-translation activity, the main mechanism for recycling ribosomes stalled on problematic messenger RNA (mRNA) in bacteria. This system is based on an engineered tmRNA variant that reassembles the green fluorescent protein (GFP) when trans-translation is active. Our system is adapted for high-throughput screening of chemical compounds by fluorescence.

Synergistic defects in pre-rRNA processing from mutations in the U3-specific protein Rrp9 and U3 snoRNA.

U3 snoRNA and the associated Rrp9/U3-55K protein are essential for 18S rRNA production by the SSU-processome complex. U3 and Rrp9 are required for early pre-rRNA cleavages at sites A0, A1 and A2, but the mechanism remains unclear. Substitution of Arg 289 in Rrp9 to Ala (R289A) specifically reduced cleavage at sites A1 and A2. Surprisingly, R289 is located on the surface of the Rrp9 ß-propeller structure opposite to U3 snoRNA. To understand this, we first characterized the protein-protein interaction network of Rrp9 within the SSU-processome. This identified a direct interaction between the Rrp9 ß-propeller domain and Rrp36, the strength of which was reduced by the R289A substitution, implicating this interaction in the observed processing phenotype. The Rrp9 R289A mutation also showed strong synergistic negative interactions with mutations in U3 that destabilize the U3/pre-rRNA base-pair interactions or reduce the length of their linking segments. We propose that the Rrp9 ß-propeller and U3/pre-rRNA binding cooperate in the structure or stability of the SSU-processome. Additionally, our analysis of U3 variants gave insights into the function of individual segments of the 5'-terminal 72-nt sequence of U3. We interpret these data in the light of recently reported SSU-processome structures.

The RNA-binding protein Celf1 post-transcriptionally regulates p27Kip1 and Dnase2b to control fiber cell nuclear degradation in lens development.

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.

A Point Mutation in a lincRNA Upstream of GDNF Is Associated to a Canine Insensitivity to Pain: A Spontaneous Model for Human Sensory Neuropathies.

Human Hereditary Sensory Autonomic Neuropathies (HSANs) are characterized by insensitivity to pain, sometimes combined with self-mutilation. Strikingly, several sporting dog breeds are particularly affected by such neuropathies. Clinical signs appear in young puppies and consist of acral analgesia, with or without sudden intense licking, biting and severe self-mutilation of the feet, whereas proprioception, motor abilities and spinal reflexes remain intact. Through a Genome Wide Association Study (GWAS) with 24 affected and 30 unaffected sporting dogs using the Canine HD 170K SNP array (Illumina), we identified a 1.8 Mb homozygous locus on canine chromosome 4 (adj. p-val = 2.5x10-6). Targeted high-throughput sequencing of this locus in 4 affected and 4 unaffected dogs identified 478 variants. Only one variant perfectly segregated with the expected recessive inheritance in 300 sporting dogs of known clinical status, while it was never present in 900 unaffected dogs from 130 other breeds. This variant, located 90 kb upstream of the GDNF gene, a highly relevant neurotrophic factor candidate gene, lies in a long intergenic non-coding RNAs (lincRNA), GDNF-AS. Using human comparative genomic analysis, we observed that the canine variant maps onto an enhancer element. Quantitative RT-PCR of dorsal root ganglia RNAs of affected dogs showed a significant decrease of both GDNF mRNA and GDNF-AS expression levels (respectively 60% and 80%), as compared to unaffected dogs. We thus performed gel shift assays (EMSA) that reveal that the canine variant significantly alters the binding of regulatory elements. Altogether, these results allowed the identification in dogs of GDNF as a relevant candidate for human HSAN and insensitivity to pain, but also shed light on the regulation of GDNF transcription. Finally, such results allow proposing these sporting dog breeds as natural models for clinical trials with a double benefit for human and veterinary medicine.

Robust identification of Ptbp1-dependent splicing events by a junction-centric approach in Xenopus laevis.

Regulation of alternative splicing is an important process for cell differentiation and development. Down-regulation of Ptbp1, a regulatory RNA-binding protein, leads to developmental skin defects in Xenopus laevis. To identify Ptbp1-dependent splicing events potentially related to the phenotype, we conducted RNAseq experiments following Ptbp1 depletion. We systematically compared exon-centric and junction-centric approaches to detect differential splicing events. We showed that the junction-centric approach performs far better than the exon-centric approach in Xenopus laevis. We carried out the same comparisons using simulated data in human, which led us to propose that the better performances of the junction-centric approach in Xenopus laevis essentially relies on an incomplete exonic annotation associated with a correct transcription unit annotation. We assessed the capacity of the exon-centric and junction-centric approaches to retrieve known and to discover new Ptbp1-dependent splicing events. Notably, the junction-centric approach identified Ptbp1-controlled exons in agfg1, itga6, actn4, and tpm4 mRNAs, which were independently confirmed. We conclude that the junction-centric approach allows for a more complete and informative description of splicing events, and we propose that this finding might hold true for other species with incomplete annotations.

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.

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.

The Splicing Factor PTBP1 Represses TP63 γ Isoform Production in Squamous Cell Carcinoma.

The TP63 gene encodes the p63 transcription factor. It is frequently amplified or overexpressed in squamous cell carcinomas. Owing to alternative splicing, p63 has multiple isoforms called α, ß, γ, and δ. The regulatory functions of p63 are isoform specific. The α isoform inhibits the epithelial-to-mesenchymal transition (EMT) and controls apoptosis, while the γ isoform promotes EMT. Using The Cancer Genome Atlas data, we observed that a higher proportion of the TP63γ isoform is a detrimental factor for the survival of patients with head and neck squamous cell carcinoma (HNSCC) and is accompanied by the downregulation of desmosomal genes. By a correlation-based approach, we investigated the regulation of the production of the TP63γ isoform. According to our analysis of GTEx data, the expression of the RNA-binding protein PTBP1 (polypyrimidine tract binding protein 1) is negatively correlated with the abundance of TP63γ in several tissues. Accordingly, we demonstrated that PTBP1 depletion in HNSCC cell lines, keratinocyte or Xenopus embryos leads to an increase in TP63γ isoform abundance. By RNA immunoprecipitation and in vitro interaction assays, we showed that PTBP1 directly binds to TP63 pre-mRNA in close proximity to the TP63γ-specific exon. Intronic regions around the TP63γ-specific exon were sufficient to elicit a PTBP1-dependent regulation of alternative splicing in a splice reporter minigene assay. Together, these results identify TP63γ as an unfavorable prognostic marker in HNSCC, and identify PTBP1 as the first direct splicing regulator of TP63γ production and a potential route toward TP63 isoform control. Significance: Quantifying TP63γ isoforms in patients' tumors could allow for the early detection of patients with HNSCC with an early loss in desmosomal gene expression and poor prognostic. The identification of PTBP1 as a transacting factor controlling TP63γ production may allow to control TP63γ expression.

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.

DHX15-independent roles for TFIP11 in U6 snRNA modification, U4/U6.U5 tri-snRNP assembly and pre-mRNA splicing fidelity.

The U6 snRNA, the core catalytic component of the spliceosome, is extensively modified post-transcriptionally, with 2'-O-methylation being most common. However, how U6 2'-O-methylation is regulated remains largely unknown. Here we report that TFIP11, the human homolog of the yeast spliceosome disassembly factor Ntr1, localizes to nucleoli and Cajal Bodies and is essential for the 2'-O-methylation of U6. Mechanistically, we demonstrate that TFIP11 knockdown reduces the association of U6 snRNA with fibrillarin and associated snoRNAs, therefore altering U6 2'-O-methylation. We show U6 snRNA hypomethylation is associated with changes in assembly of the U4/U6.U5 tri-snRNP leading to defects in spliceosome assembly and alterations in splicing fidelity. Strikingly, this function of TFIP11 is independent of the RNA helicase DHX15, its known partner in yeast. In sum, our study demonstrates an unrecognized function for TFIP11 in U6 snRNP modification and U4/U6.U5 tri-snRNP assembly, identifying TFIP11 as a critical spliceosome assembly regulator.

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 alpha-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 alpha-tropomyosin pre-mRNA.

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

BACKGROUND: Post-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. RESULTS: We 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/. CONCLUSIONS: Our 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.

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.

MicroRNAs in Drosophila: the magic wand to enter the Chamber of Secrets?

MicroRNAs are small non-coding RNAs that are now recognised as key regulators of gene expression in eukaryotes. Over the past few years, hundreds of miRNAs have been identified from various organisms including vertebrates, nematodes, insects and plants. A high level of conservation of some miRNAs from animals to plants underlines their crucial role in eukaryotes. Although biogenesis and mode of action of miRNAs are now quite well understood, their numerous and specific regulatory functions remain to be unravelled. In this review, we summarise the current knowledge on miRNAs in insects, which was mainly acquired through the study of the fruit fly, Drosophila melanogaster.

Mammalian CELF/Bruno-like RNA-binding proteins: molecular characteristics and biological functions.

In mammals, the CELF/Bruno-like family of RNA-binding proteins contains six members. The founder members of the family are the CUG-BP1 (CELF1) and ETR-3 (CELF2) proteins. Four other members have been identified mainly by sequence similarity. The founder members were cloned or identified in a number of laboratories which has lead to a profusion of names and two separate naming systems. In addition, different members of the CELF/Bruno-like protein family have been shown to be implicated in two major post-transcriptional regulatory processes, namely the alternative splicing and the control of translation and stability of target mRNAs. Several studies have indicated a certain functional redundancy between the CELF proteins in fulfilling these functions. The multiplicity of gene names and the eventual functional redundancy is a source of potential confusion in published work. We present here a synthetic picture of the present situation and, where possible, models are proposed that can account for the data obtained in the various laboratories with different biological models. Furthermore, we have highlighted some important questions that still need to be resolved.

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.

A posttranscriptional mechanism that controls Ptbp1 abundance in the Xenopus epidermis.

The output of alternative splicing depends on the cooperative or antagonistic activities of several RNA-binding proteins (RBPs), like Ptbp1 and Esrp1 in Xenopus. Fine-tuning of the RBP abundance is therefore of prime importance to achieve tissue- or cell-specific splicing patterns. Here, we addressed the mechanisms leading to the high expression of the ptbp1 gene, which encodes Ptbp1, in Xenopus epidermis. Two splice isoforms of ptbp1 mRNA differ by the presence of an alternative exon 11, and only the isoform including exon 11 can be translated to a full-length protein. In vivo minigene assays revealed that the nonproductive isoform was predominantly produced. Knockdown experiments demonstrated that Esrp1, which is specific to the epidermis, strongly stimulated the expression of ptbp1 by favoring the productive isoform. Consequently, knocking down esrp1 phenocopied ptbp1 inactivation. Conversely, Ptbp1 repressed the expression of its own gene by favoring the nonproductive isoform. Hence, a complex posttranscriptional mechanism controls Ptbp1 abundance in Xenopus epidermis: skipping of exon 11 is the default splicing pattern, but Esrp1 stimulates ptbp1 expression by favoring the inclusion of exon 11 up to a level that is limited by Ptbp1 itself. These results decipher a posttranscriptional mechanism that achieves various abundances of the ubiquitous RBP Ptbp1 in different tissues.

Investigating alternative RNA splicing in Xenopus.

Alternative splicing, the process by which distinct mature mRNAs can be produced from a single primary transcript, is a key mechanism to increase the organism complexity. The generation of alternative splicing pattern is a means to expand the proteome diversity and also to control gene expression through the regulation of mRNA abundance. Alternative splicing is therefore particularly prevalent during development and accordingly numerous splicing events are regulated in a tissue or temporal manner. To study the roles of alternative splicing during developmental processes and decipher the molecular mechanisms that underlie temporal and spatial regulation, it is important to develop in vivo whole animal studies. In this chapter, we present the advantages of using the amphibian Xenopus as a fully in vivo model to study alternative splicing and we describe the experimental procedures that can be used with Xenopus laevis embryos and oocytes to define the cis-regulatory elements and identify the associated trans-acting factors.

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.