Biology subjects

Hidalgo, E.

Publications and source records attributed to Hidalgo, E..

6 recordsLinked to original sources

bioRxiv2026

Evolutionary emergence and preservation of microproteins encoded by upstream ORFs

The analysis of ribosome profiling (Ribo-Seq) data has provided evidence that many eukaryotic mRNAs contain translated upstream or downstream ORFs (uORFs/dORFs), but the biological significance of this translation activity remains, for the most part, unknown. One of the principal limitations has been the lack of Ribo-Seq data from several closely related species, precluding the identification of cases in which translation is phylogenetically conserved. Here, by combining Ribo-Seq data from 100 different experiments, we identify 2,332 translated uORFs and 1,008 translated dORFs in S. cerevisiae, which result in microproteins that tend to be highly hydrophobic or positively charged. To study their phylogenetic conservation, we have generated Nanopore direct RNA sequencing data, together with Ribo-Seq data, from six additional Saccharomyces species, spanning an evolutionary period of around 16 million years. We have identified 195 translated S. cerevisiae uORFs that are also translated in other Saccharomyces species; these uORFs are translated at levels comparable to the main coding sequence and display signatures of purifying selection at the level of the encoded microproteins. In contrast, dORFs are translated at very low levels and they are rarely conserved, suggesting much more limited microprotein functionalization. We have also discovered that uORF translation is associated with the formation of alternative transcript isoforms encompassing the region containing the uORFs but not the main protein coding sequence, implying that some microproteins can be produced independently of the main protein product. This work significantly advances our understanding of how initially pervasive uORF translation can result in new microproteins, providing many new candidates for further functional studies.

genomics↗

bioRxiv2024

Hydroxyurea induces ER stress and cytoplasmic protein aggregation

The endoplasmic reticulum (ER) lumen provides the proper redox environment for disulfide bond formation, which is required for the appropriate folding of proteins that enter the secretory pathway and constitute membranes. Defective protein folding in the ER activates proteostatic mechanisms that are now beginning to be elucidated. Here, we show that hydroxyurea (HU) causes ER stress and triggers a transient perinuclear ER expansion, which leads to the clustering of nuclear pore complexes. This striking phenotype is mimicked by diamide (DIA), a specific thiol stress inductor, and prevented or rapidly reverted by dithiothreitol, a dithiol-reducing agent, suggesting that ER expansion is caused by disulfide stress. ER expansion induced by HU or DIA depends on glutathione (GSH), is Ire1-independent, and is associated with a unique transcriptome program that differs from the canonical unfolding protein response (UPR). The ER luminal expansion accumulates Hsp70 Bip1 chaperone, and it evolves parallel with the appearance of cytoplasmic protein aggregates containing heat stress proteins (HSPs), indicating that both HU and DIA are impinging on protein folding. Thus, our data reveal that HU induces disulfide stress that impinges on protein folding in the cytoplasm and ER.

cell biology↗

bioRxiv2023

A systematic screen identifies Saf5 as a link between splicing and transcription in fission yeast

Splicing is an important step of gene expression regulation in eukaryotes, as there are many mRNA precursors that can be alternatively spliced in different tissues, at different cell cycle phases or under different external stimuli. We have developed several life constructs that allow the quantification of fission yeast splicing in vivo on intact cells, and we have compared their splicing efficiency in a wild type strain and in a prp2-1 (U2AF65) genetic background, showing a clear dependency between Prp2 and a consensus signal at 5 splicing site (5SS). To isolate novel genes involved in regulated splicing, we have crossed the reporter containing the weakest splicing rate with the Schizosaccharomyces pombe knock out collection. Among the candidate genes involved in the regulation of splicing, we have detected strong splicing defects in two of the mutants ({Delta}cwf12 and{Delta} saf5), both of them related to the NineTeen Complex (NTC). We have identified that strains with mutations in cwf12 have inefficient splicing, mainly when the 5SS differs from the consensus. However, although{Delta} saf5 cells also have some dependency on 5SS sequence, we noticed that when one intron of a given pre-mRNA was affected, the rest of the introns of the same pre-mRNA had high probabilities of being also affected. This observation points Saf5 as a link between transcription rate and splicing.

molecular biology↗

bioRxiv2022

Perturbed fatty-acid metabolism is linked to localized chromatin hyperacetylation, increased stress-response gene expression and resistance to oxidative stress

Oxidative stress is associated with cardiovascular and neurodegenerative diseases, diabetes, cancer, psychiatric disorders and aging. In order to counteract, eliminate and/or adapt to the sources of stress, cells possess elaborate stress-response mechanisms, which also operate at the level of regulating transcription. Interestingly, it is becoming apparent that the metabolic state of the cell and certain metabolites can directly control the epigenetic information and gene expression. In the fission yeast Schizosaccharomyces pombe, the conserved Sty1 stress-activated protein kinase cascade is the main pathway responding to most types of stresses, and regulates the transcription of hundreds of genes via the Atf1 transcription factor. Here we report that fission yeast cells defective in fatty acid synthesis (cbf11, mga2 and ACC/cut6 mutants) show increased expression of a subset of stress-response genes. This altered gene expression depends on Sty1, and the Gcn5 and Mst1 histone acetyltransferases, is associated with increased acetylation of histone H3 at lysine 9 in the corresponding gene promoters, and results in increased cellular resistance to oxidative stress. Since both fatty-acid synthesis and histone acetylation compete for the same substrate, acetyl-CoA, we propose that changes in lipid metabolism can regulate the chromatin and transcription of specific stress-response genes, which in turn might help cells to maintain redox homeostasis.

molecular biology↗

bioRxiv2021

Stress-dependent inhibition of cell polarity through unbalancing the GEF/GAP regulation of Cdc42

Cdc42 rules cell polarity and growth in fission yeast. It is negatively and positively regulated by GTPase-activating proteins (GAPs) and by Guanine nucleotide Exchange factors (GEFs), respectively. Active Cdc42-GTP localizes to the poles, where it associates with numerous proteins constituting the polarity module. However, little is known about its down-regulation. We describe here that oxidative stress causes Sty1 kinase-dependent Cdc42 inactivation at cell poles. Both the amount of active Cdc42 at poles and cell length inversely correlate with Sty1 activity, explaining the elongated morphology of{Delta} sty1 cells. We have created stress-blinded cell poles by either eliminating two Cdc42 GAPs or through the constitutive tethering of a GEF to the cell tips, and biochemically demonstrate that Rga3 is a direct substrate of Sty1. We propose that stress-activated Sty1 promotes GTP hydrolysis and prevents GEF activity at the cell tips, thus leading to the inhibition of Cdc42 and polarized growth cessation.

cell biology↗

bioRxiv2017

Cdk9, Spt5 and histone H2B mono-ubiquitylation cooperate to ensure antisense suppression by the Clr6-CII/Rpd3S HDAC complex

Cyclin-dependent kinase 9 (Cdk9) and histone H2B monoubiquitylation (H2Bub1) are both implicated in elongation by RNA polymerase II (RNAPII). In fission yeast, Cdk9 and H2Bub1 regulate each other through a feedback loop involving phosphorylation of the elongation factor Spt5. Conversely, genetic interactions suggest opposing functions of H2Bub1 and Cdk9 through an Spt5-independent pathway. To understand these interactions, we performed RNA-seq analysis after H2Bub1 loss, Cdk9 inhibition, or both. Either Cdk9 inhibition or H2Bub1 loss increased levels of antisense transcription initiating within coding regions of distinct subsets of genes; ablation of both pathways led to antisense derepression affecting over half the genome. Cdk9 and H2Bub1 cooperate to suppress antisense transcription by promoting function of the Clr6-CII histone deacetylase (HDAC) complex. H2Bub1 plays a second role, in opposition to Clr6-CII, to promote sense transcription in subtelomeric regions. Therefore, functional genomics revealed both collaborative and antagonistic functions of H2Bub1 and Cdk9.

molecular biology↗