Biology subjects

Vidigal, J. A.

Publications and source records attributed to Vidigal, J. A..

7 recordsLinked to original sources

bioRxiv2025

miRISC inhibition causes mitotic defects and synergizes with genotoxic agents in cancers

Although individual microRNAs (miRNAs) can have tumorigenic or tumor-suppressive properties, their overall role in cancer remains controversial. Here, we show that cancer tissues and cell lines are characterized by preferential accumulation of the high-molecular-weight miRNA-induced silencing complex (HMWR), the functionally active form of the effector complex responsible for miRNA-mediated gene repression. Experimentally induced disassembly of the HMWR impairs the growth of human tumor xenografts and of autochthonous tumors in mouse models of human cancer in vivo. Furthermore, disassembly of the HMWR increases chromosome mis-segregation, which synergized with genotoxic agents to potentiate cancer cell vulnerability and improve therapeutic response. These findings suggest pharmacologic inhibition of HMWR as a novel anti-cancer strategy.

cancer biology↗

bioRxiv2025

AGO2 slicing of a domesticated retrotransposon is necessary for normal vasculature development

Argonaute-mediated RNA slicing is an ancient genome-defense mechanism linked to transposon repression. Although RNAi is no longer a major repeat-control pathway in mammals, AGO2 has retained catalytic activity, and this activity is essential for postnatal viability. The physiological targets that explain this requirement have remained unknown. Here we show that mammalian AGO2 slicing is required to restrain Rtl1, an imprinted domesticated retrotransposon whose cleavage is directed by miRNAs from the miR-433[~]127 cluster. Loss of AGO2 catalysis derepresses Rtl1 and causes endothelial defects and lethal neonatal muscle failure. Mechanistically, RTL1 is an oligomerization-prone retrotransposon-derived protein whose excess expression in muscle induces a non-aggregate proteostasis-stress state characterized by increased proteasome-dependent activity, reduced nascent protein synthesis, and depletion of mitochondrial, calcium-handling, and contractile gene programs. Thus, an essential function of mammalian AGO2 catalysis is to restrain a domesticated transposon-derived protein whose uncontrolled expression compromises endothelial and skeletal muscle function. These findings reveal how an ancestral transposon-Argonaute regulatory interaction has been repurposed to support mammalian development and survival.

developmental biology↗

bioRxiv2024

CRISPR-based dissection of miRNA binding sites using isogenic cell lines is hampered by pervasive noise.

Non-coding regulatory sequences play essential roles in adjusting gene output to cellular needs and are thus critical to animal development and health. Numerous such sequences have been identified in mammalian genomes ranging from transcription factors binding motifs to recognition sites for RNA-binding proteins and non-coding RNAs. The advent of CRISPR has raised the possibility of assigning functionality to individual endogenous regulatory sites by facilitating the generation of isogenic cell lines that differ by a defined set of genetic modifications. Here we investigate the usefulness of this approach to assign function to individual miRNA binding sites. We find that the process of generating isogenic pairs of mammalian cell lines with CRISPR-mediated mutations introduces extensive molecular and phenotypic variability between biological replicates making any attempt of assigning function to the binding site essentially impossible. Our work highlights an important consideration when employing CRISPR editing to characterize non-coding regulatory sequences in cell lines and calls for the development and adoption of alternative strategies to address this question in the future.

genetics↗

bioRxiv2024

The small non-coding RNA Vaultrc5 is dispensable to mouse development

Vault RNAs (vRNAs) are evolutionarily conserved small non-coding RNAs transcribed by RNA polymerase lll. Initially described as components of the vault particle, they have since also been described as noncanonical miRNA precursors and as riboregulators of autophagy. As central molecules in these processes, vRNAs have been attributed numerous biological roles including regulation of cell proliferation and survival, response to viral infections, drug resistance, and animal development. Yet, their impact to mammalian physiology remains largely unexplored. To study vault RNAs in vivo, we generated a mouse line with a conditional Vaultrc5 loss of function allele. Because Vaultrc5 is the sole murine vRNA, this allele enables the characterization of the physiological requirements of this conserved class of small regulatory RNAs in mammals. Using this strain, we show that mice constitutively null for Vaultrc5 are viable and histologically normal but have a slight reduction in platelet counts pointing to a potential role for vRNAs in hematopoiesis. This work paves the way for further in vivo characterizations of this abundant but mysterious RNA molecule. Specifically, it enables the study of the biological consequences of constitutive or lineage-specific Vaultrc5 deletion and of the physiological requirements for an intact Vaultrc5 during normal hematopoiesis or in response to cellular stresses such as oncogene expression, viral infection, or drug treatment.

genetics↗

bioRxiv2022

AGO2 silences mobile transposons in the nucleus of quiescent cells

Argonaute 2 (AGO2) is a cytoplasmic component of the miRNA pathway with essential roles in development and disease. Yet, little is known about its regulation in vivo. Here, we show that in quiescent mouse splenocytes, AGO2 localizes almost exclusively to the nucleus. AGO2 subcellular localization is modulated by the Pi3K-AKT-mTOR pathway, a well-established regulator of quiescence. Signaling through this pathway in proliferating cells promotes AGO2 cytoplasmic accumulation, at least in part by stimulating the expression of TNRC6, an essential AGO2 binding partner in the miRNA pathway. In quiescent cells where mTOR signaling is low, AGO2 accumulates in the nucleus where it binds to young mobile transposons co-transcriptionally to repress their expression via its catalytic domain. Our data point to an essential but previously unrecognized nuclear role for AGO2 during quiescence as part of a genome-defense system against young mobile elements and provide evidence of RNA interference in the soma of mammals.

molecular biology↗

bioRxiv2021

Inducible and reversible inhibition of miRNA-mediated gene repression in vivo

Although virtually all gene networks are predicted to be controlled by miRNAs, the contribution of this important layer of gene regulation to tissue homeostasis in adult animals remains unclear. Gain and loss of function experiments have provided key insights into the specific function of individual miRNAs, but effective genetic tools to study the functional consequences of global inhibition of miRNA activity in vivo are lacking. Here we report the generation and characterization of a genetically engineered mouse strain in which miRNA-mediated gene repression can be reversibly inhibited without affecting miRNA biogenesis or abundance. We demonstrate the usefulness of this strategy by investigating the consequences of acute inhibition of miRNA function in adult animals. We find that different tissues and organs respond differently to global loss of miRNA function. While miRNA-mediated gene repression is essential for the homeostasis of the heart and the skeletal muscle, it is largely dispensable in the majority of other organs. Even in tissues where it is not required for homeostasis, such as the intestine and hematopoietic system, miRNA activity can become essential during regeneration following acute injury. These data support a model where many metazoan tissues primarily rely on miRNA function to respond to potentially pathogenic events.

molecular biology↗

bioRxiv2019

Correction of Off-Targeting in CRISPR Screens Uncovers Genetic Dependencies in Melanoma Cells

Off-target cleavage by Cas9 can confound measurements of cell proliferation/viability in CRISPR assays by eliciting a DNA-damage response that includes cell cycle arrest1-3. This gene-independent toxicity has been documented in large scale assays2-4 and shown to be a source of false-positives when libraries are populated by promiscuous guide RNAs (gRNAs)7. To address this, we developed CSC, a computational method to correct for the effect of specificity on gRNA depletion. We applied CSC to screening data from the Cancer Dependency Map and show that it significantly improves the specificity of CRISPR-Cas9 essentiality screens while preserving known gene essentialities even for genes targeted by highly pro-miscuous guides. We packaged CSC in a Python software to allow its seamless integration into current CRISPR analysis pipelines and improve the sensitivity of essentiality screens for repetitive genomic loci.

genomics↗