Publications

Human meiosis in oocytes entails an intricate regulation of the transcriptome to support late oocyte growth and early embryo development, both crucial to reproductive success. Currently, little is known about the co- and post-transcriptional mRNA processing mechanisms regulating the last meiotic phases, which contribute to transcriptome complexity and influence translation rates. We analyzed gene expression changes, splicing and pre-mRNA processing in an RNA sequencing set of 40 human oocytes at different meiotic maturation stages, matured both in vivo and in vitro. We found abundant untranslated region (UTR) processing, mostly at the 3' end, of meiosis-related genes between the germinal vesicle (GV) and metaphase II (MII) stages, supported by the differential expression of spliceosome and pre-mRNA processing related genes. Importantly, we found very few differences among GV oocytes across several durations of IVM, as long as they did not reach MII, suggesting an association of RNA processing and successful meiosis transit. Changes in protein isoforms are minor, although specific and consistent for genes involved in chromosome organization and spindle assembly. In conclusion, we reveal a dynamic transcript remodeling during human female meiosis, and show how pre-mRNA processing, specifically 3'UTR shortening, drives a selective translational regulation of transcripts necessary to reach final meiotic maturation.© The Author(s) 2023. Published by Oxford University Press on behalf of European Society of Human Reproduction and Embryology. All rights reserved. For permissions, please email: journals.permissions@oup.com.

JTD Keywords: 3 & prime, alternative splicing, gene expression, meiosis, oocyte competence, program, rna, splicing, untranslated region processing, untranslated regions, 3′ untranslated region processing, 3′ untranslated regions, Alternative splicing, Expression, Gene expression, Human oocytes, Meiosis, Oocyte competence, Splicing

Enteroaggregative Escherichia coli (EAEC) strains are one of the diarrheagenic pathotypes. EAEC strains harbor a virulence plasmid (pAA2) that encodes, among other virulence determinants, the aggR gene. The expression of the AggR protein leads to the expression of several virulence determinants in both plasmids and chromosomes. In this work, we describe a novel mechanism that influences AggR expression. Because of the absence of a Rho-independent terminator in the 3?UTR, aggR transcripts extend far beyond the aggR ORF. These transcripts are prone to PNPase-mediated degradation. Structural alterations in the 3?UTR result in increased aggR transcript stability, leading to increased AggR levels. We therefore investigated the effect of increased AggR levels on EAEC virulence. Upon finding the previously described AggR-dependent virulence factors, we detected novel AggR-regulated genes that may play relevant roles in EAEC virulence. Mutants exhibiting high AggR levels because of structural alterations in the aggR 3?UTR show increased mobility and increased pAA2 conjugation frequency. Furthermore, among the genes exhibiting increased fold change values, we could identify those of metabolic pathways that promote increased degradation of arginine, fatty acids and gamma-aminobutyric acid (GABA), respectively. In this paper, we discuss how the AggR-dependent increase in specific metabolic pathways activity may contribute to EAEC virulence.

JTD Keywords: aggregative adherence, arginine metabolism, biofilm formation, escherichia-coli, gene-expression, messenger-rna, operon, persistent diarrhea, untranslated region, Fimbria-i expression

Gene editing methods are an attractive therapeutic option for Duchenne muscular dystrophy, and they have an immediate application in the generation of research models. To generate myoblast cultures that could be useful in in vitro drug screening, we have optimised a CRISPR/Cas9 gene edition protocol. We have successfully used it in wild type immortalised myoblasts to delete exon 52 of the dystrophin gene, modelling a common Duchenne muscular dystrophy mutation; and in patient’s immortalised cultures we have deleted an inhibitory microRNA target region of the utrophin UTR, leading to utrophin upregulation. We have characterised these cultures by demonstrating, respectively, inhibition of dystrophin expression and overexpression of utrophin, and evaluating the expression of myogenic factors (Myf5 and MyH3) and components of the dystrophin associated glycoprotein complex (α-sarcoglycan and β-dystroglycan). To demonstrate their use in the assessment of DMD treatments, we have performed exon skipping on the DMDΔ52-Model and have used the unedited DMD cultures/ DMD-UTRN-Model combo to assess utrophin overexpression after drug treatment. While the practical use of DMDΔ52-Model is limited to the validation to our gene editing protocol, DMD-UTRN-Model presents a possible therapeutic gene edition target as well as a useful positive control in the screening of utrophin overexpression drugs.

JTD Keywords: expression, in-vitro, mouse model, muscle, mutations, phenotype, quantification, sarcolemma, therapy, 3' untranslated regions, Cells, cultured, Crispr-cas systems, Cytoskeletal proteins, Drug discovery, Dystroglycans, Dystrophin, Gene editing, Hek293 cells, Humans, Muscular dystrophy, duchenne, Myoblasts, Myogenic regulatory factor 5, Primary cell culture, Sarcoglycans, Utrophin, Utrophin up-regulation