Integrative analyses of TEDDY Omics data reveal lipid metabolism abnormalities, increased intracellular ROS and heightened inflammation prior to autoimmunity for type 1 diabetes

27 páginas, 6 figuras, Contiene material suplementarioBackground: The Environmental Determinants of Diabetes in the Young (TEDDY) is a prospective birth cohort designed to study type 1 diabetes (T1D) by following children with high genetic risk. An integrative multi-omics approach was used to evaluate islet autoimmunity etiology, identify disease biomarkers, and understand progression over time. Results: We identify a multi-omics signature that was predictive of islet autoimmunity (IA) as early as 1 year before seroconversion. At this time, abnormalities in lipid metabolism, decreased capacity for nutrient absorption, and intracellular ROS accumulation are detected in children progressing towards IA. Additionally, extracellular matrix remodeling, inflammation, cytotoxicity, angiogenesis, and increased activity of antigen-presenting cells are observed, which may contribute to beta cell destruction. Our results indicate that altered molecular homeostasis is present in IA-developing children months before the actual detection of islet autoantibodies, which opens an interesting window of opportunity for therapeutic intervention. Conclusions: The approach employed herein for assessment of the TEDDY cohort showcases the utilization of multi-omics data for the modeling of complex, multifactorial diseases, like T1D.This study was supported by grant 2015PG-T1D050 provided by the Leona M. and Harry B. Helmsley Charitable Trust. The TEDDY Study is funded by U01 DK63829, U01 DK63861, U01 DK63821, U01 DK63865, U01 DK63863, U01 DK63836, U01 DK63790, UC4 DK63829, UC4 DK63861, UC4 DK63821, UC4 DK63865, UC4 DK63863, UC4 DK63836, UC4 DK95300, UC4 DK100238, UC4 DK106955, UC4 DK112243, UC4 DK117483, and Contract No. HHSN267200700014C from the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK), National Institute of Allergy and Infectious Diseases (NIAID), National Institute of Child Health and Human Development (NICHD), National Institute of Environmental Health Sciences (NIEHS), Centers for Disease Control and Prevention (CDC), and JDRF. This work was supported in part by the NIH/NCATS Clinical and Translational Science Awards to the University of Florida (UL1 TR000064) and the University of Colorado (UL1 TR001082).Peer reviewe

Estudio de isoformas del gen SUS1 en Saccharomyces cerevisiae

[EN] The main objective of this project is the study and characterization of different isoforms of the SUS1 gene in Saccharomyces cerevisiae. Elimination of introns in eukaryotes is a co/post transcriptional process which occurs in the nucleus of eukaryotic cells, by means of which sequences called ¿introns¿ are removed from a molecule of messenger RNA (mRNA), while the ¿exons¿, which code for proteins, are kept in the molecule. This process of splicing is strongly regulated, being one of the main mechanisms that play an important role in gene expression regulation and protein diversity. Furthermore, there is a process called ¿alternative splicing¿, which taking advantage of combining the different exonic and intronic sequences of a gene, allows the production of different proteins from a single gene. In particular during the development of this project, SUS1 gene will be studied, whose protein is involved at different steps of the gene expression pathway. Sus1 is necessary for histone H2B deubiquitination, mRNA export and gene gating, being part of the protein complexes SAGA and TREX-2 respectively. Moreover, interesting observations also suggest a link with the cytoplasmatic mRNP fate, being related to structures called P-bodies. However, the main feature of said gene is the fact that it has two introns, since just 5% of the total of genes of S. cerevisiae present introns, of which less than 2% have the two that can be found in SUS1 gene. It also worth noting that secondary structure plays a very important role in splicing regulation. Besides, by means of a transcriptomic analysis of this organism, a circular variant of the RNA of the second exon of SUS1 has been found (cE2RNA), being its function still unknown. This project will try to characterize this circular RNA molecule (cE2RNA) and study its possible biological function. In order to address this, these main goals will be undertaken: 1- The confirmation of the circular RNA (cE2RNA). 2- A strain containing a mutated sequence of exon 2 of SUS1 will be obtained (cE2mutRNA) so as to check its effect on the formation of the circular RNA and the implications that this change in the sequence might have in the functions of Sus1 protein (growth, histone ubiquitination, intron retention and mRNA localization). 3- The intronic sequences of SUS1 will be eliminated to check their implication in forming the circular RNA (cE2RNA). 4- An overexpression of circular RNA (both normal and mutated sequence) will be induced to check effects on the phenotype. With these studies, we aim to achieve a better comprehension of the importance of the secondary structure of the RNA as well as the function of Sus1 in gene expression.[ES] El objetivo principal del presente proyecto es el estudio y caracterización de diferentes isoformas del gen SUS1 en la levadura Saccharomyces cerevisiae. La eliminación de los intrones es un proceso co/post-transcripcional que ocurre en el núcleo de las células eucariotas, mediante el cual se eliminan de la molécula de ARN mensajero (ARNm) unas secuencias denominadas ¿intrones¿, manteniéndose los ¿exones¿ que son codificantes en la molécula. Este es un proceso de splicing está fuertemente regulado, siendo uno de los mecanismos implicados en la regulación de la expresión génica y en la diversidad de proteínas. Además, existe un proceso denominado ¿splicing alternativo¿, mediante el cual, al combinarse los distintos exones e intrones de un mismo gen, pueden generarse múltiples proteínas diferentes a partir de un único gen. Concretamente, durante el desarrollo de este proyecto, se estudiará el gen SUS1, cuya proteína está implicada en diferentes pasos de la ruta de expresión génica. Sus1 es necesaria para la desubicuitinación de la histona H2B, el exporte de ARN mensajeros y el acercamiento de los genes hacia el complejo del poro nuclear, siendo parte de los complejos proteicos SAGA y TREX-2 respectivamente. Además, observaciones sugieren un vínculo con el destino de las ribonucleoporteínas mensajeras (mRNP) en el citoplasma, estando relacionada con unas estructuras denominadas cuerpos-P. No obstante, la principal característica de este gen es que presenta dos intrones, pues solamente el 5% de los genes de S. cerevisiae presenta algún intrón, y de éstos menos del 2% presentan los dos que se pueden encontrar en SUS1. Es importante también remarcar que la estructura secundaria de éstos juega un papel muy importante en la regulación del splicing. Además, mediante análisis transcriptómico de este organismo, se ha podido localizar la presencia de una variante circular del ARN del segundo exón de SUS1 (cE2ARN), pero su función es hasta el momento desconocida. En este proyecto, se tratará de caracterizar la molécula del ARN circular (cE2ARN) y estudiar su posible función biológica. Para conseguir esto, se tratará de llevar a cabo los siguientes objetivos: 1- Confirmación del ARN circular (cE2ARN). 2- Se obtendrá una cepa con la secuencia del segundo exón de SUS1 mutada para ver su efecto en la formación del ARN circular y, comprobar las implicaciones que este cambio en la secuencia puede tener en diferentes funciones de la proteína Sus1 (crecimiento, ubicuitinización de histonas, retención de intrones y localización de ARNm). 3- Se eliminarán las secuencias intrónicas de SUS1 para comprobar su implicación en la formación del ARN circular (cE2ARN). 4- Se inducirá la sobreexpresión del ARN circular, tanto secuencia original como mutada, para comprobar posibles efectos en el fenotipo. Mediante estos estudios, se pretende lograr una mayor comprensión de la importancia de la estructura secundaria del ARN así como la función de Sus1 en la expresión génica.Serrano Quílez, J. (2015). Estudio de isoformas del gen SUS1 en Saccharomyces cerevisiae. http://hdl.handle.net/10251/56266.TFG

Sharing Marks: H3K4 Methylation and H2B Ubiquitination as Features of Meiotic Recombination and Transcription

© 2020 by the authors.Meiosis is a specialized cell division that gives raise to four haploid gametes from a single diploid cell. During meiosis, homologous recombination is crucial to ensure genetic diversity and guarantee accurate chromosome segregation. Both the formation of programmed meiotic DNA double-strand breaks (DSBs) and their repair using homologous chromosomes are essential and highly regulated pathways. Similar to other processes that take place in the context of chromatin, histone posttranslational modifications (PTMs) constitute one of the major mechanisms to regulate meiotic recombination. In this review, we focus on specific PTMs occurring in histone tails as driving forces of different molecular events, including meiotic recombination and transcription. In particular, we concentrate on the influence of H3K4me3, H2BK123ub, and their corresponding molecular machineries that write, read, and erase these histone marks. The Spp1 subunit within the Complex of Proteins Associated with Set1 (COMPASS) is a critical regulator of H3K4me3-dependent meiotic DSB formation. On the other hand, the PAF1c (RNA polymerase II associated factor 1 complex) drives the ubiquitination of H2BK123 by Rad6-Bre1. We also discuss emerging evidence obtained by cryo-electron microscopy (EM) structure determination that has provided new insights into how the “cross-talk” between these two marks is accomplished.This research was funded by MICIIN, Spain, grant number PGC2018-099872-B-I00. The APC was funded by MICIIN (PGC2018-099872-B-I00). J.S-Q. was funded by the FPU (Formación de Profesorado Universitario) programme (MICIIN, FPU15/03862).Peer reviewe

An exon three-way junction structure modulates splicing and degradation of the SUS1 yeast pre-mRNA

14 páginas, material suplementario y 6 figuras en material suplementarioThe SUS1 gene of Saccharomyces cerevisiae is unusual as it contains two introns and undergoes alternative splicing, retaining one or both introns depending on growth conditions. The exon located between the two introns can be skipped during splicing and has been detected in circular form. This exon (E2) has also been found to influence the splicing of the flanking introns, an unusual situation in budding yeast where splicing mainly relies on intron recognition. Using SHAPE (selective 2'-hydroxyl acylation analyzed by primer extension), NMR spectroscopy, gel electrophoresis and UV thermal denaturation experiments combined with computational predictions, we show that E2 of SUS1 comprises a conserved double-helical stem topped by a three-way junction. One of the hairpins emerging from the junction exhibited significant thermal stability and was capped by a purine-rich loop structurally related to the substrate loop of the VS ribozyme. Cellular assays revealed that three mutants containing altered E2 structures had impaired SUS1 expression, and that a compensatory mutation restoring the conserved stem recovered expression to wild-type levels. Semi-quantitative RT-PCR measurements paralleled these results, and revealed that mutations in E2 altered splicing and transcript degradation processes. Thus, exon structure plays an important role in SUS1 RNA metabolism.This work has been supported by MINECO of Spain and FEDER funds (BFU2011-23418 and BFU2014-57636-P to S.R.-N, and BFU-2012-30770 and BFU2015-65103-R to J.G.), by Generalitat Valenciana of Spain 29 (PROMETEO/2013/061, PROMETEO/2016/091, ACOMP/2014/061 and ACOMP/2015/096 to S.R-N., ACOMP/2014/056 to J.G., Santiago Grisolía fellowship to A.AQ and FPU15/03862 fellowship to J.S.-Q ), and by Universidad Católica de Valencia (2017-114-001 to J.G.).Peer reviewe

A role for Mog1 in H2Bub1 and H3K4me3 regulation affecting RNAPII transcription and mRNA export

17 páginas, 12 figuras.Monoubiquitination of histone H2B (to H2Bub1) is required for downstream events including histone H3 methylation, transcription, and mRNA export. The mechanisms and players regulating these events have not yet been completely delineated. Here, we show that the conserved Ran-binding protein Mog1 is required to sustain normal levels of H2Bub1 and H3K4me3 in Saccharomyces cerevisiae Mog1 is needed for gene body recruitment of Rad6, Bre1, and Rtf1 that are involved in H2B ubiquitination and genetically interacts with these factors. We provide evidence that the absence of MOG1 impacts on cellular processes such as transcription, DNA replication, and mRNA export, which are linked to H2Bub1. Importantly, the mRNA export defect in mog1Δ strains is exacerbated by the absence of factors that decrease H2Bub1 levels. Consistent with a role in sustaining H2Bub and H3K4me3 levels, Mog1 co-precipitates with components that participate in these modifications such as Bre1, Rtf1, and the COMPASS-associated factors Shg1 and Sdc1. These results reveal a novel role for Mog1 in H2B ubiquitination, transcription, and mRNA biogenesis.PO-C, JS-Q and CN-C were supported by the FPI, FPU and PROMETEO (BES-2012058587, FPU15/03862, PROMETEO 2016/093). This study was supported by funds to SR-N from the Spanish MINECO (BFU2011-23418, BFU2014-57636) and the Generalitat Valenciana (PROMETEO 2012/061 and ACOMP2014/061), and to JEP-O (BFU2016-77728-C3-3-P, PROMETEOII 2015/006).Peer reviewe