Biology subjects

Macias, S.

Publications and source records attributed to Macias, S..

9 recordsLinked to original sources

bioRxiv2026

Universal donor plasmids for biallelic degron-tagging in mammalian cells and organoids

Degron tags enable rapid, tuneable and reversible control of protein expression, and have become a critical tool for target validation and elucidating biological mechanisms. Tag engineering uses genome editing to generate biallelic gene fusions, though this process remains laborious and requires new donor vectors to be generated for each locus. Here, we have generated a series of universal donor vectors to enable biallelic integration of different degron tags (AID, dTAG, BromoTag, SMASh) using different selection strategies (FACS, drug selection). Using this system, tags can be fused in-frame to endogenous protein C-termini via homology-independent repair following Cas9-induced double strand breaks, as shown here using human lymphoma cells, mouse embryonic stem cells and colonic tumour organoids. This workflow simplifies the genetic engineering of degron tags and is applicable in contexts where homology-dependent repair is challenging.

synthetic biology↗

bioRxiv2026

Identification of cellular double-stranded RNAs in mammalian embryonic stem cells

Antiviral defence mechanisms are typically activated upon sensing virus-derived nucleic acids. During replication, viruses generate double-stranded RNA (dsRNA) intermediates that the innate immune system can sense, triggering several defence pathways. Conversely, mammalian cells avoid accumulating their own endogenous dsRNA to prevent activating these defence mechanisms. However, we demonstrate that mammalian embryonic stem cells (ESCs) accumulate endogenous dsRNA without activating these responses, as they lack all classical dsRNA-mediated antiviral pathways. To identify these endogenous dsRNAs, we have developed a method that includes an antibody-based purification coupled to RNase I treatment to enrich bona fide dsRNAs. The RNase I treatment results in an enrichment on sense/antisense and A-to-I edited transcripts, suggesting that they are true dsRNA in cells. Our refined protocol reveals that transposable elements (TEs), primarily the young elements from the LINE and LTR classes, are the predominant sources of dsRNA in ESCs. This approach will be useful for investigating the role of dsRNA in disease settings, such as autoimmunity or cancer, where endogenous dsRNA accumulation has also been observed.

molecular biology↗

bioRxiv2025

DICER1 syndrome mutations lead to a gain of 3p-miRNA function, HERVH activity and increased metastatic potential

The DICER1 gene is mutated in cancer, including DICER1 syndrome, a rare tumour predisposition syndrome. Cancer-associated hotspot mutations have been reported in both catalytic domains of DICER1 and are predicted to disrupt miRNA biogenesis. To understand how these hotspot mutations contribute to cancer development, we have generated cell lines harbouring single amino acid substitutions within the catalytic RNase IIIa (S1344L) or RNase IIIb (D1709N) domains of the endogenous DICER1 gene. We show that both mutations result in a widespread loss of 5p miRNAs, and, unexpectedly, an increase in 3p passenger strands loading into AGO2. The shared similarities between both mutants can be attributed to the structural proximity of the S1344 residue to the RNase IIIb catalytic centre. Functionally, we found that changes in the repertoire of miRNAs loaded into AGO2 result in altered gene expression, impacting critical pathways for cancer development, including metastatic potential. Additionally, our results indicate that inactivating the processing activity of DICER1 does not result in genomic instability. Instead, mutations cause specific upregulation of human endogenous retrovirus H (HERVH) through miRNA-independent mechanisms, suggesting that both canonical and non-canonical DICER1 functions are important to understand DICER1 syndrome.

molecular biology↗

bioRxiv2025

Control of retrotransposon-driven activation of the interferon response by the double-stranded RNA binding protein DGCR8

The type I interferon (IFN) response is the main innate immune pathway against viruses in mammals. This pathway must be tightly regulated to prevent viral spread while avoiding excessive immune responses. Here, we show that inactivation of the double-stranded RNA (dsRNA)-binding protein DGCR8 unleashes the IFN response in human cells. We demonstrate that DGCR8 restricts the accumulation of endogenous dsRNA originating from protein-coding mRNAs that harbour transposable elements (TEs), primarily Alu. We propose that DGCR8 binding to TE-rich mRNAs is essential to resolve dsRNA structures, and in its absence, accumulated dsRNA signals through the RIG-I-like signalling pathway triggering the IFN response. This mechanism is relevant to conditions where DGCR8 expression levels are altered, including the 22q11.2 deletion syndrome (22qDS). Supporting this, we show that 22qDS-derived cells exhibit an exacerbated type I IFN response which inversely correlated with DGCR8 levels. All these together demonstrate the importance of suppressing endogenous TE-dsRNA accumulation to prevent unwanted immune activation and associated disease pathogenesis.

molecular biology↗

bioRxiv2025

Double stranded RNA sensing drives interferon silencing in early development

In early mammalian development, the type I interferon (IFN) response is inactive, only becoming functional after gastrulation. As a result, the totipotent and pluripotent embryonic stages remain highly susceptible to pathogens, including viruses. Here, we show that pluripotent mouse embryonic stem cells (mESCs) suppress the RIG-I-like receptor sensing pathway by silencing the expression of the dsRNA sensor MDA5. We show that this silencing is required to avoid the recognition of dsRNAs from endogenous origin, which only accumulate in mESCs. Reintroducing MDA5 results in recognition of these endogenous dsRNAs, and activation of the IFN response through IRF3. The production of IFN alters the differentiation ability of mESCs, as well as the pluripotency gene expression program, as shown by epigenetic, transcriptomic and proteomic analyses. These findings are conserved in zebrafish, where MDA5 is also expressed at later stages of development. Similarly, zebrafish lack early-stage IFN activation and premature IFN signalling results in developmental defects. Altogether, we conclude that silencing the RIG-I-like receptor pathway during early development is the widely conserved and is required to prevent aberrant immune recognition of endogenous dsRNAs, safeguarding normal development.

cell biology↗

bioRxiv2024

DGCR8 haploinsufficiency leads to primate-specific RNA dysregulation and pluripotency defects

The 22q11.2 deletion syndrome (22qDS) is caused by a microdeletion in chromosome 22, including DGCR8, an essential gene for miRNA production. The contribution of human DGCR8 hemizygosity to the disease is still unclear. In this study, we generated two human pluripotent cell models containing a single functional DGCR8 allele to elucidate its role on 22qDS. DGCR8+/-cells show increased apoptosis as well as self-renewal and differentiation defects in both the naive and primed states. The expression of primate-specific miRNAs was largely affected, due to impaired miRNA processing and chromatin accessibility. DGCR8+/- cells also displayed a pronounced reduction in human endogenous retrovirus class H (HERVH) expression, a primate-specific retroelement essential for pluripotency maintenance. Importantly, the reintroduction of primate-specific miRNAs as well as the miR-371-3 cluster rescued the cellular and molecular phenotypes of DGCR8+/-cells. Our results suggest that DGCR8 is haploinsufficient in humans and that miRNAs and transposable elements may have co-evolved in primates as part of an essential regulatory network to maintain stem cell identity.

molecular biology↗

bioRxiv2019

ILF3 contributes to the establishment of the antiviral type I interferon program

Upon detection of viral infections, cells activate the expression of type I interferons (IFNs) and pro-inflammatory cytokines to control viral dissemination. As part of their antiviral response, cells also trigger the translational shutoff response which prevents translation of viral mRNAs and cellular mRNAs in a non-selective manner. Intriguingly, mRNAs encoding for antiviral factors bypass this translational shutoff, suggesting the presence of additional regulatory mechanisms enabling expression of the self-defence genes. Here, we identified the dsRNA binding protein ILF3 as an essential host factor required for efficient translation of the central antiviral cytokine, IFNB1, and a subset of interferon-stimulated genes. By combining polysome profiling and next-generation sequencing, ILF3 was also found to be necessary to establish the dsRNA-induced transcriptional and translational programs. We propose a central role for the host factor ILF3 in enhancing expression of the antiviral defence mRNAs in cellular conditions where cap-dependent translation is compromised.

molecular biology↗

bioRxiv2018

MicroRNA-deficient embryonic stem cells acquire a functional Interferon response

When mammalian cells detect a viral infection, they initiate a type-I Interferon (IFNs) response as part of their innate immune system. This antiviral mechanism is conserved in virtually all cell types, except for embryonic stem cells (ESCs) and oocytes which are intrinsically incapable of producing IFNs. Despite the importance of the IFN response to fight viral infections, the mechanisms regulating this pathway during pluripotency are still unknown. Here we show that, in the absence of miRNAs, ESCs acquire an active IFN response. Proteomic analysis identified MAVS, a central component of the IFN pathway, to be actively silenced by miRNAs and responsible for suppressing IFN expression in ESCs. Furthermore, we show that knocking out a single miRNA, miR-673, restores the antiviral response in ESCs through MAVS regulation. Our findings suggest that the interaction between miR-673 and MAVS acts as a switch to suppress the antiviral IFN during pluripotency and present genetic approaches to enhance their antiviral immunity.

cell biology↗

bioRxiv2016

Genetic variation and RNA structure regulate microRNA biogenesis

MiRNA biogenesis is highly regulated at the post-transcriptional level; however, the role of sequence and secondary RNA structure in this process has not been extensively studied. A single G to A substitution present in the terminal loop of pri-mir-30c-1 in breast cancer patients had been previously described to result in increased levels of mature miRNA. Here, we report that this genetic variant directly affects Drosha-mediated processing of pri-mir-30c-1 in vitro and in cultured cells. Structural analysis of this variant revealed an altered RNA structure that facilitates the interaction with SRSF3, an SR protein family member that promotes pri-miRNA processing. Our results are compatible with a model whereby a genetic variant in pri-mir-30c-1 leads to a secondary RNA structure rearrangement that facilitates binding of SRSF3 resulting in increased levels of miR-30c. These data highlights that primary sequence determinants and RNA structure are key regulators of miRNA biogenesis.

biochemistry↗