Biology subjects

Munoz, M. J.

Publications and source records attributed to Munoz, M. J..

5 recordsLinked to original sources

bioRxiv2024

Enhanced RNA-targeting CRISPR-Cas technology in zebrafish

CRISPR-Cas13 systems are widely used in basic and applied sciences. However, its application has recently generated controversy due to collateral activity in mammalian cells and mouse models. Moreover, its efficiency could be improved in vivo. Here, we optimized transient formulations as ribonucleoprotein complexes or mRNA-gRNA combinations to enhance the CRISPR-RfxCas13d system in zebrafish. We i) used chemically modified gRNAs to allow more penetrant loss-of-function phenotypes, ii) improved nuclear RNA-targeting, and iii) compared different computational models and determined the most accurate to predict gRNA activity in vivo. Furthermore, we demonstrated that transient CRISPR-RfxCas13d can effectively deplete endogenous mRNAs in zebrafish embryos without inducing collateral effects, except when targeting extremely abundant and ectopic RNAs. Finally, we implemented alternative RNA-targeting CRISPR-Cas systems with reduced or absent collateral activity. Altogether, these findings contribute to CRISPR-Cas technology optimization for RNA targeting in zebrafish through transient approaches and assist in the progression of in vivo applications.

developmental biology↗

bioRxiv2024

RNA Polymerase II Degradation Triggered by DNA Repair Occurs In Trans and Independently of how the Lesion is Recognised

In response to DNA damage, RPB1, the catalytic subunit of RNA Polymerase II (RNAPII), is degraded by the ubiquitin-proteasome system. Degradation models only consider transcriptionally engaged molecules, where a stalled RNAPII complex functions as a lesion recognition factor and its RPB1 subunit is proposed to be subsequently degraded to facilitate access of core Nucleotide Excision Repair (NER) factors. This Transcription Coupled repair is complemented by the Global Genome repair (GG-NER) system, where lesions are recognized by the XPE and XPC factors. Here we show that RPB1 degradation is controlled in trans by a pathway that depends on NER activity, irrespectively of whether the lesion is recognized by RNAPII itself or by GG-NER factors. Incomplete lesion repair due to absence of any core NER factor enhances RPB1 degradation, indicating that the signal controlling RPB1 abundance is started by lesion recognition and continues until DNA repair is completed. Consistent with an in trans mechanism, damage-induced RPB1 degradation is not restricted to active nor phosphorylated RPB1 molecules and depends on Cullin-RING ubiquitin ligases. These findings uncover a repair-dependent mechanism controlling RPB1 levels and provide a rationale for the control of gene expression under stress, where more damage implies more repair and less RPB1 levels, hence restricting RNAPII activity.

molecular biology↗

bioRxiv2020

Serial co-expression analysis of host factors from SARS-CoV viruses highly converges with former high-throughput screenings and proposes key regulators and co-option of cellular pathways

The current genomics era is bringing an unprecedented growth in the amount of gene expression data, only comparable to the exponential growth of sequences in databases during the last decades. This data now allows the design of secondary analyses that take advantage of this information to create new knowledge through specific computational approaches. One of these feasible analyses is the evaluation of the expression level for a gene through a series of different conditions or cell types. Based on this idea, we have developed ASACO, Automatic and Serial Analysis of CO-expression, which performs expression profiles for a given gene along hundreds of normalized and heterogeneous transcriptomics experiments and discover other genes that show either a similar or an inverse behavior. It might help to discover co-regulated genes, and even common transcriptional regulators in any biological model, including human diseases or microbial infections. The present SARS-CoV-2 pandemic is an opportunity to test this novel approach due to the wealth of data that is being generated, which could be used for validating results. In addition, new cell mechanisms identified could become new therapeutic targets. Thus, we have identified 35 host factors in the literature putatively involved in the infectious cycle of SARS-CoV and/or SARS-CoV-2 and searched for genes tightly co-expressed with them. We have found around 1900 co-expressed genes whose assigned functions are strongly related to viral cycles. Moreover, this set of genes heavily overlap with those identified by former laboratory high-throughput screenings (with p-value near 0). Some of these genes aim to cellular structures such as the stress granules, which could be essential for the virus replication and thereby could constitute potential targets in the current fight against the virus. Additionally, our results reveal a series of common transcription regulators, involved in immune and inflammatory responses, that might be key virus targets to induce the coordinated expression of SARS-CoV-2 host factors. All of this proves that ASACO can discover gene co-regulation networks with potential for proposing new genes, pathways and regulators participating in particular biological systems. HighlightsO_LIASACO identifies regulatory associations of genes using public transcriptomics data. C_LIO_LIASACO highlights new cell functions likely involved in the infection of coronavirus. C_LIO_LIComparison with high-throughput screenings validates candidates proposed by ASACO. C_LIO_LIGenes co-expressed with hosts genes used by SARS-CoV-2 are related to stress granules. C_LI

bioinformatics↗

bioRxiv2019

Steroid hormones sulfatase inactivation extends lifespan and ameliorates age-related diseases

Aging and fertility are two interconnected processes. From invertebrates to mammals, absence of the germline increases longevity by a still not fully understood mechanism. We find that loss of function of sul-2, the Caenorhabditis elegans steroid sulfatase (STS), raises the pool of sulfated steroid hormones and increases longevity. This increased longevity requires factors involved in germline-mediated longevity (daf-16, daf-12, kri-1, tcer-1 and daf-36 genes) and is not additive to the longevity of germline-less mutants. Noteworthy, sul-2 mutations do not affect fertility. Thus, STS inactivation affects the germline signalling process regulating longevity. Interestingly, sul-2 is only expressed in sensory neurons, suggesting a regulation of germline longevity by environmental cues. We also demonstrate that treatment with the specific STS inhibitor STX64, reproduces the longevity phenotype of sul-2 mutants. Remarkably, STS inhibition by either mutation or drug treatment ameliorates protein aggregation diseases in C. elegans models of Parkinson, Huntington and Alzheimer, as well as Alzheimer disease in a mammalian model. These results open the possibility of reallocating steroid sulfatase inhibitors for the treatment of aging and aging related diseases.

developmental biology↗

bioRxiv2017

Combined flow cytometry and high throughput image analysis for the study of essential genes in Caenorhabditis elegans

BackgroundThe advancement in automated image based microscopy platforms coupled with high throughput liquid workflows has facilitated the design of large scale screens utilizing multicellular model organisms such as Caenorhabditis elegans to identify genetic interactions, therapeutic drugs or disease modifiers. However, the analysis of essential genes has lagged behind because lethal or sterile mutations pose a bottleneck for high throughput approaches.\n\nResultsIn C. elegans, non-conditional lethal mutations can be maintained in heterozygosis using chromosome balancers, commonly labelled with GFP in the pharynx. Moreover gene-expression is typically monitored by the use of fluorescent reporters marked with the same fluorophore. Therefore, the separation of the different populations of animals at early larval stages represents a challenge. Here, we develop a sorting strategy capable of selecting homozygous mutants carrying a GFP stress reporter from GFP-balanced animals at early larval stages. Because sorting is not completely error-free, we develop an automated high-throughput image-analysis protocol that identifies and discards animals carrying the chromosome balancer. We demonstrate the experimental usefulness of combining sorting of homozygous lethal mutants and automated image-analysis in a functional genomic RNAi screen for genes that genetically interact with mitochondrial prohibitin (PHB). Lack of PHB results in embryonic lethality, while, homozygous PHB deletion mutants develop into sterile adults due to maternal contribution and strongly induce the mitochondrial unfolded protein response (UPRmt). In a chromosome-wide RNAi screen for C. elegans genes having human orthologues, we uncover both, known and new PHB genetic interactors affecting the UPRmt and growth.\n\nConclusionsA systematic way to analyse genetic interactions of essential genes in multicellular organisms is lacking. The method presented here allows the study of balanced lethal mutations in a high-throughput manner and can be easily adapted depending on the users requirements. Therefore, it will serve as a useful resource for the C. elegans community for probing new biological aspects of essential nematode genes as well as the generation of more comprehensive genetic networks.

genetics↗