Trans regulation in the Ultrabithorax gene of Drosophila: alterations in the promoter enhance transvection

PMCID: PMC556824We report a genetic and molecular study of UbxMX6 and Ubx195rx1, two mutations in the Ultrabithorax (Ubx) locus which appear to have a strong effect on the activity of the homologous Ubx gene. These mutations show the characteristic embryonic and adult phenotypes of Ubx null alleles, and also fail to produce any detectable Ubx product. Yet, genetic and phenotypic analyses involving a large number of trans heterozygous combinations of UbxMX6 and Ubx195rx1 with different classes of Ubx mutations, indicate that they hyperactivate the homologous gene. This effect is induced on wildtype or mutant forms of Ubx, provided that the pairing in the bithorax region is normal, i.e. these mutations have a strong positive effect on transvection. We also show that, unlike all the other known cases of transvection in Ubx, this is not zeste-dependent. Southern analyses indicate that UbxMX6 is a 3.4 kb deletion, and Ubx195rx1 is an approximately 11 kb insertion of foreign DNA, both in the promoter region. We speculate that the region altered in the mutations may have a wildtype function to ensure cis-autonomy of the regulation of Ubx transcription.This work was supported by grants from the DGICYT and the Fundación Ramón Areces.Peer reviewe

Accurate expression quantification from nanopore direct RNA sequencing with NanoCount

Accurately quantifying gene and isoform expression changes is essential to understanding cell functions, differentiation and disease. Sequencing full-length native RNAs using long-read direct RNA sequencing (DRS) has the potential to overcome many limitations of short and long-read sequencing methods that require RNA fragmentation, cDNA synthesis or PCR. However, there are a lack of tools specifically designed for DRS and its ability to identify differential expression in complex organisms is poorly characterised. We developed NanoCount for fast, accurate transcript isoform quantification in DRS and demonstrate it outperforms similar methods. Using synthetic controls and human SH-SY5Y cell differentiation into neuron-like cells, we show that DRS accurately quantifies RNA expression and identifies differential expression of genes and isoforms. Differential expression of 231 genes, 333 isoforms, plus 27 isoform switches were detected between undifferentiated and differentiated SH-SY5Y cells and samples clustered by differentiation state at the gene and isoform level. Genes upregulated in neuron-like cells were associated with neurogenesis. NanoCount quantification of thousands of novel isoforms discovered with DRS likewise enabled identification of their differential expression. Our results demonstrate enhanced DRS isoform quantification with NanoCount and establish the ability of DRS to identify biologically relevant differential expression of genes and isoforms

Nanopore ReCappable sequencing maps SARS-CoV-2 5′ capping sites and provides new insights into the structure of sgRNAs

The SARS-CoV-2 virus has a complex transcriptome characterised by multiple, nested subgenomic RNAsused to express structural and accessory proteins. Long-read sequencing technologies such as nanopore direct RNA sequencing can recover full-length transcripts, greatly simplifying the assembly of structurally complex RNAs. However, these techniques do not detect the 5 ' cap, thus preventing reliable identification and quantification of full-length, coding transcript models. Here we used Nanopore ReCappable Sequencing (NRCeq), a new technique that can identify capped full-length RNAs, to assemble a complete annotation of SARS-CoV-2 sgRNAs and annotate the location of capping sites across the viral genome. We obtained robust estimates of sgRNA expression across cell lines and viral isolates and identified novel canonical and non-canonical sgRNAs, including one that uses a previously un-annotated leader-to-body junction site. The data generated in this work constitute a useful resource for the scientific community and provide important insights into the mechanisms that regulate the transcription of SARS-CoV-2 sgRNAs

RNA modifications detection by comparative Nanopore direct RNA sequencing.

RNA molecules undergo a vast array of chemical post-transcriptional modifications (PTMs) that can affect their structure and interaction properties. In recent years, a growing number of PTMs have been successfully mapped to the transcriptome using experimental approaches relying on high-throughput sequencing. Oxford Nanopore direct-RNA sequencing has been shown to be sensitive to RNA modifications. We developed and validated Nanocompore, a robust analytical framework that identifies modifications from these data. Our strategy compares an RNA sample of interest against a non-modified control sample, not requiring a training set and allowing the use of replicates. We show that Nanocompore can detect different RNA modifications with position accuracy in vitro, and we apply it to profile m6A in vivo in yeast and human RNAs, as well as in targeted non-coding RNAs. We confirm our results with orthogonal methods and provide novel insights on the co-occurrence of multiple modified residues on individual RNA molecules.The Kouzarides laboratory is supported by Cancer Research UK (grant reference RG72100) and core support from the Wellcome Trust (core grant reference WT203144) and Cancer Research UK (grant reference C6946/A24843). PPA was supported by a Borysiewicz Biomedical Sciences postdoctoral fellowship (University of Cambridge) and AL by a COFUND Marie Skłodowska-Curie Actions postdoctoral fellowship (EMBL). FW and TS are supported by the Francis Crick Institute, which receives its core funding from Cancer Research UK (FC001203), the UK Medical Research Council (FC001203), and the Wellcome Trust (FC001203). IB and V Miano are supported by Cancer Research UK (grant reference RG86786) and by the Joseph Mitchell Fund

Extracorporeal Membrane Oxygenation for Severe Acute Respiratory Distress Syndrome associated with COVID-19: An Emulated Target Trial Analysis.

RATIONALE: Whether COVID patients may benefit from extracorporeal membrane oxygenation (ECMO) compared with conventional invasive mechanical ventilation (IMV) remains unknown. OBJECTIVES: To estimate the effect of ECMO on 90-Day mortality vs IMV only Methods: Among 4,244 critically ill adult patients with COVID-19 included in a multicenter cohort study, we emulated a target trial comparing the treatment strategies of initiating ECMO vs. no ECMO within 7 days of IMV in patients with severe acute respiratory distress syndrome (PaO2/FiO2 <80 or PaCO2 ≥60 mmHg). We controlled for confounding using a multivariable Cox model based on predefined variables. MAIN RESULTS: 1,235 patients met the full eligibility criteria for the emulated trial, among whom 164 patients initiated ECMO. The ECMO strategy had a higher survival probability at Day-7 from the onset of eligibility criteria (87% vs 83%, risk difference: 4%, 95% CI 0;9%) which decreased during follow-up (survival at Day-90: 63% vs 65%, risk difference: -2%, 95% CI -10;5%). However, ECMO was associated with higher survival when performed in high-volume ECMO centers or in regions where a specific ECMO network organization was set up to handle high demand, and when initiated within the first 4 days of MV and in profoundly hypoxemic patients. CONCLUSIONS: In an emulated trial based on a nationwide COVID-19 cohort, we found differential survival over time of an ECMO compared with a no-ECMO strategy. However, ECMO was consistently associated with better outcomes when performed in high-volume centers and in regions with ECMO capacities specifically organized to handle high demand. This article is open access and distributed under the terms of the Creative Commons Attribution Non-Commercial No Derivatives License 4.0 (http://creativecommons.org/licenses/by-nc-nd/4.0/)

Identification of miRNAs Involved in Reprogramming Acinar Cells into Insulin Producing Cells

Reprogramming acinar cells into insulin producing cells using adenoviral (Ad)-mediated delivery of Pdx1, Ngn3 and MafA (PNM) is an innovative approach for the treatment of diabetes. Here, we aimed to investigate the molecular mechanisms involved in this process and in particular, the role of microRNAs. To this end, we performed a comparative study of acinar-to-β cell reprogramming efficiency in the rat acinar cell line AR42J and its subclone B13 after transduction with Ad-PNM. B13 cells were more efficiently reprogrammed than AR42J cells, which was demonstrated by a strong activation of β cell markers (Ins1, Ins2, IAPP, NeuroD1 and Pax4). miRNome panels were used to analyze differentially expressed miRNAs in acinar cells under four experimental conditions (i) non-transduced AR42J cells, (ii) non-transduced B13 cells, (iii) B13 cells transduced with Ad-GFP vectors and (iv) B13 cells transduced with Ad-PNM vectors. A total of 59 miRNAs were found to be differentially expressed between non-transduced AR42J and B13 cells. Specifically, the miR-200 family was completely repressed in B13 cells, suggesting that these cells exist in a less differentiated state than AR42J cells and as a consequence they present a greater plasticity. Adenoviral transduction per se induced dedifferentiation of acinar cells and 11 miRNAs were putatively involved in this process, whereas 8 miRNAs were found to be associated with PNM expression. Of note, Ad-PNM reprogrammed B13 cells presented the same levels of miR-137-3p, miR-135a-5p, miR-204-5p and miR-210-3p of those detected in islets, highlighting their role in the process. In conclusion, this study led to the identification of miRNAs that might be of compelling importance to improve acinar-to-β cell conversion for the future treatment of diabetes

Eocene to Oligocene upper plate tectonic response to Apennine subduction

Phases and patterns of uplift and subsidence characterise the tectonic evolution over subduction zones at the Myr time scale. The dynamics of the subducting plate influences upper plate deformation at crustal level. Crustal deformation, in turn, causes vertical motion through isostatic response of thinned or thickened crust. Consequently, the influence of mantle-driven processes needs to be discriminated from the influence of more local isostatic adjustment. In this work we investigate the upper plate tectonic response to changing subduction dynamics in the western Mediterranean from Mid Eocene to end Oligocene. The surface effects of the push-to-pull mode switch of the subduction system are shown on palinspastically restored paleogeographic maps. Regional distribution of uplift and subsidence and compression-extension at the Apennine-Alps-Pyrenees intersection are integrated in a plate kinematic framework using the free software GPlates. A new restoration at crustal scale of the Corsica-Sardinian block from Eocene to present is proposed using published geophysical and geological data, onshore and offshore. The main result is that the Corsica-Sardinia block needs to be divided in threes blocks to restore the crustal thickness to 30 km at 35 Ma. The position of these blocks during the Pyrenean orogeny is then clarified. New structural and stratigraphic data from Sardinia, coupled with available thermochronological data from basement and Tertiary sediments are in agreement with this restoration and shed light on the Eocene to Oligocene poorly constrained period for Sardinia. The rheological role of Triassic layers during compression and extension, the onset of the Pyrenean foreland subsidence at Lower Eocene and the Late Eocene-Oligocene stratigraphic response to the onset of back-arc extension are elucidated with field data.This study is part of the >Orogen> research program funded by Total, BRGM (Bureau de Recherches Géologiques et Minières), and CNRS (Centre National de la Recherche Scientifique)