Laurent Manchon

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Alternative splicing (AS) plays a central role during cell-fate determination. However, how the core spliceosomal factors (CSFs) are involved in this process is poorly understood. Here, we report the down-regulation of the U2AF1 CSF during stem cell differentiation. To investigate its function in stemness and differentiation, we downregulated U2AF1 in human induced pluripotent stem cells (hiPSCs), using an inducible-shRNA system, to the level found in differentiated ectodermal, mesodermal and endodermal cells. RNA sequencing and computational analysis reveal that U2AF1 down-regulation modulates the expression of development-regulating genes and regulates transcriptional networks involved in cell-fate determination. Furthermore, U2AF1 down-regulation induces a switch in the AS of transcription factors (TFs) required to establish specific cell lineages, and favours the splicing of a differentiated cell-specific isoform of DNMT3B. Our results showed that the differential expression of the core spliceosomal factor U2AF1, between stem cells and the precursors of the three germ layers regulates a cell-type-specific alternative splicing programme and a transcriptional network involved in cell-fate determination via the modulation of gene expression and alternative splicing of transcription regulators.

Many nascent long non-coding RNAs have received considerable attention in recent years because of their major regulatory roles in gene expression and signaling pathways at various levels. Indeed, long non-coding RNAs undergo the same maturation steps as pre-mRNAs of protein coding genes, but they are less efficiently spliced and polyadenylated in comparison to them. Here we focus on a specific human long non-coding RNA and we show the activity of a new candidate drug that potentially affect its splicing and generate an anti-HIV and anti-inflammatory effects driven by upregulation of microRNA biogenesis. To investigate this activity, we combine the use of capture sequencing technology and an ab initio transcript assembly on cells from six healthy individuals. The sequencing depth of capture sequencing permitted us to assemble transcripts exhibiting a complex array of splicing patterns. We revealed that splicing of the long non-coding RNA is activated by the drug whereas this splicing was not present in untreated samples.

The mechanisms that contribute to developmental stability are barely known. Here we show that alternative splicing ofyorkie(yki) is required for developmental stability inDrosophila. Yki encodes the effector of the Hippo pathway that has a central role in controlling organ growth and regeneration. We identify the splicing factor B52 as necessary for inclusion ofykialternative exon 3 that encodes one of the two WW domains of Yki protein. B52 depletion favors expression of Yki1 isoform carrying a single WW domain, and reduces growth in part through modulation ofykialternative splicing. Compared to the canonical Yki2 isoform containing two WW domains, Yki1 isoform has reduced transcriptional and growth-promoting activities, decreased binding to PPxY-containing partners, and lacks the ability to bridge two proteins containing PPxY motifs. Yet, Yki1 and Yki2 interact similarly with transcription factors and can thus competein vivo. Strikingly, flies deprived from Yki1 isoform exhibit increased fluctuating wing asymmetry, a signal of increased developmental noise. Our results identifyykialternative splicing as a new level of control of the Hippo pathway and provide the first experimental evidence that alternative splicing participates in developmental robustness.

Many nascent long non-coding RNAs have received considerable attention in recent years because of their major regulatory roles in gene expression and signaling pathways at various levels. Indeed long non-coding RNAs undergo the same maturation steps as pre-mRNAs of proteincoding genes, but they are less efficiently spliced and polyadenylated in comparison to them. Here we focus on a specific human long non-coding RNA and we show the activity of a new candidate drug that potentially affect its splicing and generate an anti-HIV and anti-inflammatory effects driven by upregulation of microRNA biogenesis. To investigate this activity we combine the use of capture sequencing technology and anab initiotranscript assembly on cells from six healthy individuals. The sequencing depth of capture sequencing permitted us to assemble transcripts exhibiting a complex array of splicing patterns. In essence, we revealed that splicing of the long non-coding RNA is activated by the drug whereas this splicing was not present in untreated samples.

ABX464 is a first-in-class, clinical-stage, small molecule for oral administration that has shown strong anti-inflammatory effects in the DSS-model for inflammatory bowel disease (IBD) and also prevents replication of the HIV virus. ABX464 which binds to cap binding complex (CBC) has demonstrated safety and efficacy in a phase 2a proof-of-concept clinical trial in patients with Ulcerative colitis. Previously, with limited technologies, it was not possible to quantify the effect of ABX464 on viral and cellular RNA biogenesis. Here, using RNA CaptureSeq and deep sequencing, we report that ABX464 enhances the splicing of HIV RNA in infected PBMCs from six healthy individuals and also the expression and splicing of a single long noncoding RNA to generate the anti-inflammatory miR-124 both ex vivo and in HIV patients. While ABX464 has no effect on pre-mRNA splicing of cellular genes, depletion of CBC complex by RNAi leads to accumulation of intron retention transcripts. These results imply that ABX464 did not inhibit the function of CBC in splicing but rather strengthens it under pathological condition like inflammation and HIV infection. The specific dual ability of ABX464 to generate both anti-inflammatory miR-124 and spliced viral RNA may have applicability for the treatment of both inflammatory diseases and HIV infection.

RNA-Seq enables the generation of extensive transcriptome information providing the capability to characterize transcripts (including alternative isoforms and polymorphism), to quantify expression and to identify differential regulation in a single experiment. To reveal the capacity of new anti-HIV ABX464 candidate in modulating the expression of genes, datasets were generated and validated using RNA-seq approach. This comprehensive dataset will be useful to deepen the comprehensive understanding of the progression of human immunodeficiency virus (HIV) associated with mucosal damage in the gastrointestinal (GI) tract and subsequent inflammation, providing an opportunity to generate new therapies, diagnoses, and preventive strategies.

Trisomy 21 (T21), or Down syndrome (DS), is the most frequent and recognizable cause of intellectual disabilities. The level of disability, as evaluated by the intelligence quotient (IQ) test, varies considerably between patients independent of other factors. To determine the genetic or molecular basis of this difference, a high throughput transcriptomic analysis was performed on twenty T21 patients with high and low IQ, and 10 healthy controls using Digital Gene Expression. More than 90 millions of tags were sequenced in the three libraries. A total of 80 genes of potential interest were selected for the qPCR experiment validation, and three housekeeping genes were used for normalizing purposes. HLA DQA1 and HLA DRB1 were significantly downregulated among the patients with a low IQ, the values found in the healthy controls being intermediate between those noted in the IQ+ and IQ- T21 patients. Interestingly, the intergenic region between these genes contains a binding sequence for the CCCTC-binding factor, or CTCF, and cohesin (a multisubunit complex), both of which are essential for expression of HLA DQA1 and HLA DRB1 and numerous other genes. Our results might lead to the discovery of genes, or genetic markers, that are directly involved in several phenotypes of DS and, eventually, to the identification of potential targets for therapeutic interventions.

Background: The shell of the pearl-producing bivalvePinctada margaritiferais composed of an organic cell-free matrix that plays a key role in the dynamic process of biologically-controlled biomineralization. In order to increase genomic resources and identify shell matrix proteins implicated in biomineralization inP. margaritifera, high-throughput Expressed Sequence Tag (EST) pyrosequencing was undertaken on the calcifying mantle, combined with a proteomic analysis of the shell.Results: We report the functional analysis of 276 738 sequences, leading to the constitution of an unprecedented catalog of 82P. margaritiferabiomineralization-related mantle protein sequences. Components of the current "chitin-silk fibroin gel-acidic macromolecule" model of biomineralization processes were found, in particular a homolog of a biomineralization protein (Pif-177) recently discovered inP. fucata. Among these sequences, we could show the localization of two other biomineralization protein transcripts,pmarg-aspeinandpmarg-pearlin, in two distinct areas of the outer mantle epithelium, suggesting their implication in calcite and aragonite formation. Finally, by combining the EST approach with a proteomic mass spectrometry analysis of proteins isolated from theP. margaritiferashell organic matrix, we demonstrated the presence of 30 sequences containing almost all of the shell proteins that have been previously described from shell matrix protein analyses of thePinctadagenus. The integration of these two methods allowed the global composition of biomineralizing tissue and calcified structures to be examined in tandem for the first time.Conclusions: This EST study made on the calcifying tissue ofP. margaritiferais the first description of pyrosequencing on a pearl-producing bivalve species. Our results provide direct evidence that our EST data set covers most of the diversity of the matrix protein ofP. margaritiferashell, but also that the mantle transcripts encode proteins present inP. margaritiferashell, hence demonstrating their implication in shell formation. Combining transcriptomic and proteomic approaches is therefore a powerful way to identify proteins involved in biomineralization. Data generated in this study supply the most comprehensive list of biomineralization-related sequences presently available among protostomian species, and represent a major breakthrough in the field of molluskan biomineralization.

Recombinant human erythropoietins (rHuEPOs) are glycoproteins drugs, produced by the pharmaceutical industry to restore production of red blood cells by stimulating human bone marrow for which this pathology has been diagnosed. It is suspected that these molecules are diverted as doping agents in horseracing to enhance oxygen transport and aerobic power in racehorses. Although indirect double‐blotting or direct liquid chromatography‐mass spectrometry (LC‐MS) methods have been developed to confirm the presence of rHuEPO in a sample, the short detection time (48 h) is still a problem for doping control. In this context, gene profiling investigation through Serial Analysis of Gene Expression (SAGE) has been conducted on seven thoroughbreds treated with Eprex^®. This functional genomic method has been performed from total blood cells collected from each animal to assess the mRNA expression consecutive to rHuEPO injections. Sample pooling was chosen as a powerful, cost‐effective, and rapid means of identifying the most common and specific changes in terms of gene expression profile and to eliminate individual variation. Consequently, three SAGE libraries were constructed, before, during, and after Eprex^® treatment. More than 71 440 mRNA signatures were observed and subjected to statistical analysis; 49 differentially expressed genes were identified and analyzed by qPCR. From the selected gene list, were defined as potential biomarkers in terms of their low inter‐individual variation and capacity as strong markers of rHuEPO administration up to 60 days after the beginning of the doping period. In this paper, a new strategy is proposed to the horseracing industry to prevent rHuEPO abuse. Copyright © 2010 John Wiley & Sons, Ltd.