New function for the RNA helicase p68/DDX5 as a modifier of MBNL1 activity on expanded CUG repeats

Myotonic Dystrophy type I (DM1) is caused by an abnormal expansion of CTG triplets in the 3′ UTR of the dystrophia myotonica protein kinase (DMPK) gene, leading to the aggregation of the mutant transcript in nuclear RNA foci. The expanded mutant transcript promotes the sequestration of the MBNL1 splicing factor, resulting in the misregulation of a subset of alternative splicing events. In this study, we identify the DEAD-box RNA helicase p68 (DDX5) in complexes assembled onto in vitro-transcribed CUG repeats. We showed that p68 colocalized with RNA foci in cells expressing the 3′UTR of the DMPK gene containing expanded CTG repeats. We found that p68 increased MBNL1 binding onto pathological repeats and the stem–loop structure regulatory element within the cardiac Troponin T (TNNT2) pre-mRNA, splicing of which is misregulated in DM1. Mutations in the helicase core of p68 prevented both the stimulatory effect of the protein on MBNL1 binding and the colocalization of p68 with CUG repeats, suggesting that remodeling of RNA secondary structure by p68 facilitates MBNL1 binding. We also found that the competence of p68 for regulating TNNT2 exon 5 inclusion depended on the integrity of MBNL1 binding sites. We propose that p68 acts as a modifier of MBNL1 activity on splicing targets and pathogenic RNA

On the local geometry of the pet reconstruction problem

International audienceIntrinsic uncertainties related to the ill-posedness of the PET image reconstruction inverse problem are investigated in this work. These uncertainties could lead to instabilities in the reconstructed images in particular when Deep Learning approaches are employed. We propose a framework, based on a Bayesian hypothesis testing, enabling to define various distinguishability measures between PET images and propose a local analysis of such a measure in a neighborhood of a reference image. We test numerically this approach in a synthetic experiment of a Biograph TruePoint TrueV acquisition using a 3D brain PET phantom derived from a [18F]-FDG exam with 100 anatomo-functional regions extracted. Our analysis allows us to highlight the key factors impacting the detectability of variations in a region of interest and to exhibit concrete examples of directions along which variations may not be detectable, and instabilities could appear in PET image reconstruction. In addition, it allows us to conduct a quantitative analysis of the role played by the injected radiotracer dose, and to define thresholds below which clinically significant variations cannot be detected

The Polypyrimidine Tract Binding Protein (PTB) Represses Splicing of Exon 6B from the β-Tropomyosin Pre-mRNA by Directly Interfering with the Binding of the U2AF65 Subunit

Splicing of exon 6B from the β-tropomyosin pre-mRNA is repressed in nonmuscle cells and myoblasts by a complex array of intronic elements surrounding the exon. In this study, we analyzed the proteins that mediate splicing repression of exon 6B through binding to the upstream element. We identified the polypyrimidine tract binding protein (PTB) as a component of complexes isolated from myoblasts that assemble onto the branch point region and the pyrimidine tract. In vitro splicing assays and PTB knockdown experiments by RNA interference demonstrated that PTB acts as a repressor of splicing of exon 6B. Using psoralen experiments, we showed that PTB acts at an early stage of spliceosome assembly by preventing the binding of U2 snRNA on the branch point. Using UV cross-linking and immunoprecipitation experiments with site-specific labeled RNA in PTB-depleted nuclear extracts, we found that the decrease in PTB was correlated with an increase in U2AF65. In addition, competition experiments showed that PTB is able to displace the binding of U2AF65 on the polypyrimidine tract. Our results strongly support a model whereby PTB competes with U2AF65 for binding to the polypyrimidine tract

On the local geometry of the pet reconstruction problem

International audienceIntrinsic uncertainties related to the ill-posedness of the PET reconstruction problem are investigated in this work. These uncertainties could lead to artifacts in the reconstructed images in particular when Deep Learning approaches are employed. We propose a framework enabling to define a distinguishability measure between PET images and propose a local analysis of such a measure in a neighborhood of a reference image. This approach is numerically tested on a synthetic experiment using a 3D brain PET phantom derived from a measured [18F]-FDG exam with 100 anatomofunctional regions extracted. Our analysis allows us to highlight the key factors impacting the detectability of variations and to exhibit concrete examples of clinically meaningful directions along which variations may not be detectable. In addition, we quantitatively analyze the role played by the injected radiotracer dose and show that low-dose scenarios are particularly prone to the presence of reconstruction artifacts

Short intron-derived ncRNAs

International audienceIntrons represent almost half of the human genome, although they are eliminated from transcripts through RNA splicing. Yet, different classes of non-canonical miRNAs have been proposed to originate directly from intron splicing. Here, we considered the alternative splicing of introns as an interesting source of miRNAs, compatible with a developmental switch. We report computational prediction of new Short Intron-Derived ncRNAs (SID), defined as precursors of smaller ncRNAs like miRNAs and snoR-NAs produced directly by splicing, and tested their dependence on each key factor in canonical or alternative miRNAs biogenesis (Drosha, DGCR8, DBR1, snRNP70, U2AF65, PRP8, Dicer, Ago2). We found that about half of predicted SID rely on debranch-ing of the excised intron-lariat by the enzyme DBR1, as proposed for mirtrons. However, we identified new classes of SID for which miRNAs biogenesis may rely on intermingling between canonical and alternative pathways. We validated selected SID as putative miR-NAs precursors and identified new endogenous miR-NAs produced by non-canonical pathways, including one hosted in the first intron of SRA (Steroid Receptor RNA activator). Consistent with increased SRA intron retention during myogenic differentiation, release of SRA intron and its associated mature miRNA decreased in cells from healthy subjects but not from myotonic dystrophy patients with splicing defects

Characterization of SRp46, a Novel Human SR Splicing Factor Encoded by a PR264/SC35 Retropseudogene

The highly conserved SR family contains a growing number of phosphoproteins acting as both essential and alternative splicing factors. In this study, we have cloned human genomic and cDNA sequences encoding a novel SR protein designated SRp46. Nucleotide sequence analyses have revealed that the SRp46 gene corresponds to an expressed PR264/SC35 retropseudogene. As a result of mutations and amplifications, the SRp46 protein significantly differs from the PR264/SC35 factor, mainly at the level of its RS domain. Northern and Western blot analyses have established that SRp46 sequences are expressed at different levels in several human cell lines and normal tissues, as well as in simian cells. In contrast, sequences homologous to SRp46 are not present in mice. In vitro splicing studies indicate that the human SRp46 recombinant protein functions as an essential splicing factor in complementing a HeLa cell S100 extract deficient in SR proteins. In addition, complementation analyses performed with β-globin or adenovirus E1A transcripts and different splicing-deficient extracts have revealed that SRp46 does not display the same activity as PR264/SC35. These results demonstrate, for the first time, that an SR splicing factor, which represents a novel member of the SR family, is encoded by a functional retropseudogene