Gene-gene functional relationships in Alzheimer's disease: CELF1 regulates KLC1 alternative splicing.

The causes of Alzheimer's disease (AD) are poorly understood, although many genes are known to be involved in this pathology. To gain insights into the underlying molecular mechanisms, it is essential to identify the relationships between individual AD genes. Previous work has shown that the splice variant E of KLC1 (KLC1_vE) promotes AD, and that the CELF1 gene, which encodes an RNA-binding protein involved in splicing regulation, is at a risk locus for AD. Here, we identified a functional link between CELF1 and KLC1 in AD pathogenesis. Transcriptomic data from human samples from different ethnic groups revealed that CELF1 mRNA levels are low in AD brains, and the splicing pattern of KLC1 is strongly correlated with CELF1 expression levels. Specifically, KLC1_vE is negatively correlated with CELF1. Depletion and overexpression experiments in cultured cells demonstrated that the CELF1 protein down-regulates KLC1_vE. In a cross-linking and immunoprecipitation sequencing (CLIP-seq) database, CELF1 directly binds to KLC1 RNA, following which it likely modulates terminal exon usage, hence KLC1_vE formation. These findings reveal a new pathogenic pathway where a risk allele of CELF1 is associated with reduced CELF1 expression, which up-regulates KLC1_vE to promote AD.

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.

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.

Fully in vitro iterative construction of a 24 kb-long artificial DNA sequence to store digital information.

In the absence of a DNA template, the ab initio production of long double-stranded DNA molecules of predefined sequences is particularly challenging. The DNA synthesis step remains a bottleneck for many applications such as functional assessment of ancestral genes, analysis of alternative splicing or DNA-based data storage. In this report we propose a fully in vitro protocol to generate very long double-stranded DNA molecules starting from commercially available short DNA blocks in less than 3 days using Golden Gate assembly. This innovative application allowed us to streamline the process to produce a 24 kb-long DNA molecule storing part of the Declaration of the Rights of Man and of the Citizen of 1789 . The DNA molecule produced can be readily cloned into a suitable host/vector system for amplification and selection.

High-Throughput Transcriptomics of Celf1 Conditional Knockout Lens Identifies Downstream Networks Linked to Cataract Pathology.

Defects in the development of the ocular lens can cause congenital cataracts. To understand the various etiologies of congenital cataracts, it is important to characterize the genes linked to this developmental defect and to define their downstream pathways that are relevant to lens biology and pathology. Deficiency or alteration of several RNA-binding proteins, including the conserved RBP Celf1 (CUGBP Elav-like family member 1), has been described to cause lens defects and early onset cataracts in animal models and/or humans. Celf1 is involved in various aspects of post-transcriptional gene expression control, including regulation of mRNA stability/decay, alternative splicing and translation. Celf1 germline knockout mice and lens conditional knockout (Celf1cKO) mice develop fully penetrant cataracts in early postnatal stages. To define the genome-level changes in RNA transcripts that result from Celf1 deficiency, we performed high-throughput RNA-sequencing of Celf1cKO mouse lenses at postnatal day (P) 0. Celf1cKO lenses exhibit 987 differentially expressed genes (DEGs) at cut-offs of >1.0 log2 counts per million (CPM), ≥±0.58 log2 fold-change and <0.05 false discovery rate (FDR). Of these, 327 RNAs were reduced while 660 were elevated in Celf1cKO lenses. The DEGs were subjected to various downstream analyses including iSyTE lens enriched-expression, presence in Cat-map, and gene ontology (GO) and representation of regulatory pathways. Further, a comparative analysis was done with previously generated microarray datasets on Celf1cKO lenses P0 and P6. Together, these analyses validated and prioritized several key genes mis-expressed in Celf1cKO lenses that are relevant to lens biology, including known cataract-linked genes (e.g., Cryab, Cryba2, Cryba4, Crybb1, Crybb2, Cryga, Crygb, Crygc, Crygd, Cryge, Crygf, Dnase2b, Bfsp1, Gja3, Pxdn, Sparc, Tdrd7, etc.) as well as novel candidates (e.g., Ell2 and Prdm16). Together, these data have defined the alterations in lens transcriptome caused by Celf1 deficiency, in turn uncovering downstream genes and pathways (e.g., structural constituents of eye lenses, lens fiber cell differentiation, etc.) associated with lens development and early-onset cataracts.

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.

Reverse genetics in eukaryotes.

Reverse genetics consists in the modification of the activity of a target gene to analyse the phenotypic consequences. Four main approaches are used towards this goal and will be explained in this review. Two of them are centred on genome alterations. Mutations produced by random chemical or insertional mutagenesis can be screened to recover only mutants in a specific gene of interest. Alternatively, these alterations may be specifically targeted on a gene of interest by HR (homologous recombination). The other two approaches are centred on mRNA. RNA interference is a powerful method to reduce the level of gene products, while MO (morpholino) antisense oligonucleotides alter mRNA metabolism or translation. Some model species, such as Drosophila, are amenable to most of these approaches, whereas other model species are restricted to one of them. For example, in mice and yeasts, gene targeting by HR is prevalent, whereas in Xenopus and zebrafish MO oligonucleotides are mainly used. Genome-wide collections of mutants or inactivated models obtained in several species by these approaches have been made and will help decipher gene functions in the post-genomic era.

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.

Chromosome wide analysis of CUGBP1 binding sites identifies the tetraspanin CD9 mRNA as a target for CUGBP1-mediated down-regulation.

CUGBP1 is an RNA-binding protein controlling alternative splicing, mRNA translation and stability. In this work we used a motif scoring approach to identify putative CUGBP1 binding sites for genes located on the human chromosome 12. This allowed us to identify the gene CD9 as a presumptive target for CUGBP1-mediated regulation. In a number of cancers, the tetraspanin CD9 is down-regulated, an event correlated with a bad prognostic. Using a combination of biochemical approaches and CUGBP1 knockdown, we showed that CUGBP1 directly controls CD9 expression.

Identification of CUG-BP1/EDEN-BP target mRNAs in Xenopus tropicalis.

The early development of many animals relies on the posttranscriptional regulations of maternally stored mRNAs. In particular, the translation of maternal mRNAs is tightly controlled during oocyte maturation and early mitotic cycles in Xenopus. The Embryonic Deadenylation ElemeNt (EDEN) and its associated protein EDEN-BP are known to trigger deadenylation and translational silencing to several mRNAs bearing an EDEN. This Xenopus RNA-binding protein is an ortholog of the human protein CUG-BP1/CELF1. Five mRNAs, encoding cell cycle regulators and a protein involved in the notch pathway, have been identified as being deadenylated by EDEN/EDEN-BP. To identify new EDEN-BP targets, we immunoprecipitated EDEN-BP/mRNA complexes from Xenopus tropicalis egg extracts. We identified 153 mRNAs as new binding targets for EDEN-BP using microarrays. Sequence analyses of the 3' untranslated regions of the newly identified EDEN-BP targets reveal an enrichment in putative EDEN sequences. EDEN-BP binding to a subset of the targets was confirmed both in vitro and in vivo. Among the newly identified targets, Cdk1, a key player of oocyte maturation and cell cycle progression, is specifically targeted by its 3' UTR for an EDEN-BP-dependent deadenylation after fertilization.

ElrA and AUF1 differentially bind cyclin B2 mRNA.

In Xenopus embryos, maternal cyclins drive the first 12 cell divisions after which several cyclins are terminally degraded, including cyclin B2. Cyclin B2 disappearance is due to transcription-mediated mRNA deadenylation at the midblastula transition, when transcription initiates and the cell cycle lengthens. To further define the mechanism, we characterized proteins capable of binding cyclin B2 3'UTR. We show that ElrA and AUF1 compete for binding to regions containing cytoplasmic polyadenylation elements (CPEs), with AUF1 binding increasing at the midblastula transition. Deletion of both CPEs abrogates polyadenylation but has no effect on deadenylation or binding of ElrA or AUF1. Overexpression of ElrA or AUF1 does not alter cyclin B2 mRNA stability. These results show that ElrA and AUF1 bind to cyclin B2 mRNA independent of CPEs and function by binding other elements.

Identification of post-transcriptionally regulated Xenopus tropicalis maternal mRNAs by microarray.

Cytoplasmic control of the adenylation state of mRNAs is a critical post-transcriptional process involved in the regulation of mRNAs stability and translational efficiency. The early development of Xenopus laevis has been a major model for the study of such regulations. We describe here a microarray analysis to identify mRNAs that are regulated by changes in their adenylation state during oogenesis and early development of the diploid frog Xenopus tropicalis. The microarray data were validated using qRT-PCR and direct analysis of the adenylation state of endogenous maternal mRNAs during the period studied. We identified more than 500 mRNAs regulated at the post-transcriptional level among the 3000 mRNAs potentially detected by the microarray. The mRNAs were classified into nine different adenylation behavior categories. The various adenylation profiles observed during oocyte maturation and early development and the analyses of 3'-untranslated region sequences suggest that previously uncharacterized sequence elements control the adenylation behavior of the newly identified mRNAs. These data should prove useful in identifying mRNAs with important functions during oocyte maturation and early development.

RNA-Seq Analysis of an Antisense Sequence Optimized for Exon Skipping in Duchenne Patients Reveals No Off-Target Effect.

Non-coding uridine-rich small nuclear RNAs (UsnRNAs) have emerged in recent years as effective tools for exon skipping for the treatment of Duchenne muscular dystrophy (DMD), a degenerative muscular genetic disorder. We recently showed the high capacity of a recombinant adeno-associated virus (rAAV)-U7snRNA vector to restore the reading frame of the DMD mRNA in the muscles of DMD dogs. We are now moving toward a phase I/II clinical trial with an rAAV-U7snRNA-E53, carrying an antisense sequence designed to hybridize exon 53 of the human DMD messenger. As observed for genome-editing tools, antisense sequences present a risk of off-target effects, reflecting partial hybridization onto unintended transcripts. To characterize the clinical antisense sequence, we studied its expression and explored the occurrence of its off-target effects in human in vitro models of skeletal muscle and liver. We presented a comprehensive methodology combining RNA sequencing and in silico filtering to analyze off-targets. We showed that U7snRNA-E53 induced the effective exon skipping of the DMD transcript without inducing the notable deregulation of transcripts in human cells, neither at gene expression nor at the mRNA splicing level. Altogether, these results suggest that the use of the rAAV-U7snRNA-E53 vector for exon skipping could be safe in eligible DMD patients.

CUG-BP1/CELF1 requires UGU-rich sequences for high-affinity binding.

CUG-BP1 [CUG-binding protein 1 also called CELF (CUG-BP1 and ETR3 like factors) 1] is a human RNA-binding protein that has been implicated in the control of splicing and mRNA translation. The Xenopus homologue [EDEN-BP (embryo deadenylation element-binding protein)] is required for rapid deadenylation of certain maternal mRNAs just after fertilization. A variety of sequence elements have been described as target sites for these two proteins but their binding specificity is still controversial. Using a SELEX (systematic evolution of ligand by exponential enrichment) procedure and recombinant CUG-BP1 we selected two families of aptamers. Surface plasmon resonance and electrophoretic mobility-shift assays showed that these two families differed in their ability to bind CUG-BP1. Furthermore, the selected high-affinity aptamers form two complexes with CUG-BP1 in electrophoretic mobility assays whereas those that bind with low affinity only form one complex. The validity of the distinction between the two families of aptamers was confirmed by a functional in vivo deadenylation assay. Only those aptamers that bound CUG-BP1 with high affinity conferred deadenylation on a reporter mRNA. These high-affinity RNAs are characterized by a richness in UGU motifs. Using these binding site characteristics we identified the Xenopus maternal mRNA encoding the MAPK (mitogen-activated protein kinase) phosphatase (XCl100alpha) as a substrate for EDEN-BP. In conclusion, high-affinity CUG-BP1 binding sites are sequence elements at least 30 nucleotides in length that are enriched in combinations of U and G nucleotides and contain at least 4 UGU trinucleotide motifs. Such sequence elements are functionally competent to target an RNA for deadenylation in vivo.

An analysis of the sequence requirements of EDEN-BP for specific RNA binding.

EDEN-BP (embryo deadenylation element-binding protein) binds specifically to the EDEN motif in the 3'-untranslated regions of maternal mRNAs and targets these mRNAs for deadenylation and translational repression in Xenopus laevis embryos. EDEN-BP contains three RNA recognition motifs (RRMs) and is related to the elav family of RNA-binding proteins. In the present study we show that the two N-terminal RRMs of EDEN-BP are necessary for the interaction with EDEN as well as a part of the linker region (between RRM2 and RRM3). Using a band shift assay we show that two different complexes are formed according to the size and, therefore, the functional nature of the EDEN motif. Finally, we show that EDEN-BP can form a dimer in a two-hybrid assay. Accordingly, we suggest that the functional configuration of EDEN-BP is a dimer.

Identification of CELF1 RNA targets by CLIP-seq in human HeLa cells.

The specific interactions between RNA-binding proteins and their target RNAs are an essential level to control gene expression. By combining ultra-violet cross-linking and immunoprecipitation (CLIP) and massive SoliD sequencing we identified the RNAs bound by the RNA-binding protein CELF1, in human HeLa cells. The CELF1 binding sites deduced from the sequence data allow characterizing specific features of CELF1-RNA association. We present therefore the first map of CELF1 binding sites in human cells.

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.

zfp36 expression delineates both myeloid cells and cells localized to the fusing neural folds in Xenopus tropicalis.

Regulatory RNA binding proteins allow for specific control of gene expression in a very dynamic manner. In mammals ZFP36, formerly known as Tristetraprolin, controls the inflammatory response by binding to an AU-rich element located in the 3' untranslated region of its target mRNAs. The developping embryo relies on a population of primitive macrophages to ensure proper immunity. Although the role of zfp36 in adult immunity has been extensively studied, its expression in the developing immune system has been poorly documented. Here, we have used whole mount in situ hybridization with a 3' UTR specific probe to address the expression of zfp36 in developing Xenopus tropicalis embryos. We have shown that zfp36 is expressed in two distinct cellular populations. First, it is a new marker of primititive myeloid cells, being coexpressed with the myeloid marker mpo. Therefore this early expression may suggest a role for zfp36 in macrophage differentiation and activation. In addition, a second cell population was found to transiently express zfp36, but not mpo, along the fusing neural folds and may correspond to cells undergoing autophagy during neural tube closure.

Expression analysis of the polypyrimidine tract binding protein (PTBP1) and its paralogs PTBP2 and PTBP3 during Xenopus tropicalis embryogenesis.

The PTB (polypyrimidine tract binding protein) family of RNA-binding proteins plays a critical role in development through the regulation of post-transcriptional events. We have determined expression patterns of the three members of this gene family ptbp1, ptbp2 and ptbp3 during Xenopus tropicalis embryogenesis using whole-mount in situ hybridization. Our results show that each paralog presents a unique pattern of expression. ptbp1 is the prevalent maternal mRNA and is differentially expressed in the three germ layers. Later in development, it is widely expressed in the embryo including the epidermis, the dermatome, the intermediate mesoderm, the lateral plate mesoderm and the neural crest. ptbp2 expression is restricted to the nervous system including the brain, the neural retina and the spinal cord and the intermediate mesoderm. In addition to being expressed in erythroid precursors, ptbp3 is present in specific subdomains of the brain and the spinal cord, as well as in the posterior part of the notochord, suggesting it may play a role in the patterning of the nervous system. In the eye, each of the three genes is expressed in a specific structure which emphasizes their non-redundant function during development. Strickingly, our experiments also revealed that none of the three paralogs was expressed in the myotome, suggesting that the absence of PTB activity is a key determinant to display myotomal splicing patterns.