Biology subjects

Seitz, H.

Publications and source records attributed to Seitz, H..

10 recordsLinked to original sources

bioRxiv2026

miR-202 drives medaka fertility by targeting antagonistic Yap-dependent transcriptional regulators tead3b and vgll4b in a sex-dependent manner

How miRNAs can sometimes drive major organism-level phenotypes by targeting a single gene and by triggering limited changes in mRNA levels remains poorly understood, especially in vertebrates. In medaka, the knockout of miR-202-5p, a gonad-specific miRNA in vertebrates, leads to impaired male and female fertility, including drastically reduced egg production and low developmental success. Here we show that miR-202-5p drives gamete formation by targeting antagonist Yap-dependent transcriptional regulators tead3b and vgll4b in a sex-dependent manner. Disruption of the miR-202-5p binding site in the 3UTR of tead3b, but not vgll4b, results in a significant decrease in female fertility. In contrast, disrupting miR-202-5p target site in the 3UTR of vgll4b, but not tead3b, results in impaired male fertility. In females, 3D ovary imaging and RNA-seq analysis of isolated ovarian follicles revealed that disrupting miR-202-5p binding to the 3 UTR of tead3b results in a polycystic ovarian syndrome (PCOS)-like phenotype and the expression of many PCOS-associated marker, including androgen signaling and estrogen metabolism genes. In males, disrupting miR-202-5p binding to the 3UTR of vgll4b triggers severe phenotypes, including reduced sperm motility and abnormal testicular development. No effects on sex ratio were observed, indicating that miR-202-5p drives gamete formation by regulating mechanisms acting down-stream of the sex-determining cascade. The analysis of miR-202-5p target sites in 3 UTRs suggests long-term conservation of antagonistic TEAD and VGLL targeting across vertebrate species, including mammals. Together, our results show that miR-202-5p drives fertility by leveraging antagonistic Yap-dependent transcriptional regulators in a sex dependent manner.

genetics↗

bioRxiv2025

CRISPR-Cas9 repair complexity in Drosophila melanogaster: NHEJ-induced deletions and HDR variability in the bantam microRNA gene

CRISPR-Cas9-mediated gene editing was used to generate specific mutants of the bantam gene in Drosophila melanogaster. To drive non-homologous end joining (NHEJ) and achieve a precise deletion of most of the bantam locus, two guide RNAs targeting sites 90 base pairs apart were expressed in the germline using the UAS/GAL4 system. Thirty lethal and eight viable lines were established and analyzed. One lethal line exhibited the expected 90 bp deletion, while the others carried diverse indels at one or both cleavage sites. Among the viable lines, seven harbored a single-nucleotide deletion that did not disrupt bantam function. Notably, one viable line, band1-44, carried a hypomorphic allele that reduced organismal size without affecting viability. To generate precisely edited bantam variants, CRISPR-Cas9-mediated homology-directed repair (HDR) was used using donor plasmids containing engineered mutations in the miRNA seed region, along with a scarless dsRED fluorescent marker. Approximately 40% of the resulting fluorescent lines were correctly edited, demonstrating the efficiency of this strategy for producing specific bantam variants. The remaining lines exhibited unexpected outcomes, among which partial HDR events, where the dsRED marker was integrated but not the bantam mutations, and full donor plasmid integrations, which led to duplication of the bantam locus. These findings reveal the complexity of CRISPR-Cas9 outcomes, emphasizing the need for thorough screening and characterization of individual candidates in gene-editing experiments. They also provide valuable insights for optimizing genome editing strategies. Article summaryThe authors used Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) gene editing to mutate the bantam microRNA in Drosophila melanogaster. They guided Non-Homologous End Joining to induce a precise 90 bp deletion. It was the rarest occurrence, while small inactivating indels occurred frequently. They employed Homology-Directed Repair using a fluorescent marker to specifically target the bantam seed region. This efficiently produced the intended mutations but also led to unexpected outcomes, including partial sequence replacements and full donor plasmid integrations. These results reveal the complexity of gene editing outcomes and highlight the importance of thorough molecular characterization in genome engineering experiments.

genetics↗

bioRxiv2024

TopBP1 biomolecular condensates: a new therapeutic target in advanced-stage colorectal cancer.

In cancer cells, ATR signaling is crucial to tolerate the intrinsically high damage levels that normally block replication fork progression. Assembly of TopBP1, a multifunctional scaffolding protein, into condensates is required to amplify ATR kinase activity to the levels needed to coordinate the DNA damage response and manage DNA replication stress. Many ATR inhibitors are tested for cancer treatment in clinical trials, but their overall effectiveness is oven compromised by the emergence of resistance and toxicities. In this proof-of-concept study, we propose to disrupt the ATR pathway by targeting TopBP1 condensation. First, we screened a molecule-based library using a previously developed optogenetic approach and identified several TopBP1 condensation inhibitors. Amongst them, AZD2858 disrupted TopBP1 assembly induced by the clinically relevant topoisomerase I inhibitor SN-38, thereby inhibiting the ATR/Chk1 signaling pathway. We found that AZD2858 exerted its effects by disrupting TopBP1 self-interaction and binding to ATR in mammalian cells, and by increasing its chromatin recruitment n cell-free Xenopus laevis egg extracts. Moreover, AZD2858 prevented S-phase checkpoint induction by SN-38, leading to increased DNA damage and apoptosis in a colorectal cancer cell line. Lastly, AZD2858 showed synergistic effect in combination with the FOLFIRI chemotherapy regimen in a spheroid model of colorectal cancer. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=110 SRC="FIGDIR/small/612204v3_ufig1.gif" ALT="Figure 1"> View larger version (61K): org.highwire.dtl.DTLVardef@184d016org.highwire.dtl.DTLVardef@7975f4org.highwire.dtl.DTLVardef@2f22f0org.highwire.dtl.DTLVardef@9ecb11_HPS_FORMAT_FIGEXP M_FIG C_FIG

cancer biology↗

bioRxiv2023

Looking for a needle in a haystack: de novo phenotypic target identification reveals Hippo pathway-mediated miR-202 regulation of egg production

Understanding microRNA (miRNA) functions has been hampered by major difficulties in identifying their biological target(s). Currently, the main limitation is the lack of a suitable strategy to identify biologically relevant targets among a high number of putative targets. Here we provide a proof of concept of successful de novo (i.e., without prior knowledge of its identity) miRNA phenotypic target (i.e., target whose de-repression contributes to the phenotypic outcomes) identification from RNA-seq data. Using the medaka mir-202 knock-out (KO) model in which inactivation leads to a major organism-level reproductive phenotype, including reduced egg production, we introduced novel criteria including limited fold-change in KO and low interindividual variability in gene expression to reduce the list of 2,853 putative targets to a short list of 5. We selected tead3b, a member of the evolutionarily-conserved Hippo pathway, known to regulate ovarian functions, due to its remarkably strong and evolutionarily conserved binding affinity for miR-202-5p. Deleting the miR-202-5p binding site in the 3 UTR of tead3b, but not of other Hippo pathway members sav1 and vgll4b, triggered a reduced egg production phenotype. This is one of the few successful examples of de novo functional assignment of a miRNA phenotypic target in vivo in vertebrates.

genomics↗

bioRxiv2022

Evaluation of microRNA variant maturation prior to genome edition

* AbstractAssessment of the functionality of individual microRNA/target sites is a crucial issue. Genome editing techniques should theoretically permit a fine functional exploration of such interactions, allowing the mutation of microRNAs or individual binding sites in a complete in vivo setting, therefore abrogating or restoring individual interactions on demand. A major limitation to this experimental strategy is the influence of microRNA sequence on its accumulation level, which introduces a confounding effect when assessing phenotypic rescue by compensatorily mutated microRNA and target site. Here we describe a simple assay to identify microRNA variants most likely to accumulate at wild-type levels even though their sequence has been mutated. In this assay, quantification of a reporter construct in cultured cells predicts the efficiency of an early biogenesis step, the Drosha-dependent cleavage of microRNA precursors, which appears to be a major determinant of microRNA accumulation in our variant collection. This system allowed the generation of a mutant Drosophila strain expressing a bantam microRNA variant at wild-type levels.

molecular biology↗

bioRxiv2022

Biochemistry-informed design selects potent siRNAs against SARS-CoV-2

RNA interference (RNAi) offers an efficient way to repress genes of interest, and it is widely used in research settings. Clinical applications emerged more recently, with 5 approved siRNAs (the RNA guides of the RNAi effector complex) against human diseases. The development of siRNAs against the SARS-CoV-2 virus could therefore provide the basis of novel COVID-19 treatments, while being easily adaptable to future variants or to other, unrelated viruses. Because the biochemistry of RNAi is very precisely described, it is now possible to design siRNAs with high predicted activity and specificity using only computational tools. While previous siRNA design algorithms tended to rely on simplistic strategies (raising fully complementary siRNAs against targets of interest), our approach uses the most up-to-date mechanistic description of RNAi to allow mismatches at tolerable positions and to force them at beneficial positions, while optimizing siRNA duplex asymmetry. Our pipeline proposes 8 siRNAs against SARS-CoV-2, and ex vivo assessment confirms the high antiviral activity of 6 out of 8 siRNAs, also achieving excellent variant coverage (with several 3-siRNA combinations recognizing each correctly-sequenced variant as of September 2022). Our approach is easily generalizable to other viruses as long as a variant genome database is available. With siRNA delivery procedures being currently improved, RNAi could therefore become an efficient and versatile antiviral therapeutic strategy.

molecular biology↗

bioRxiv2021

miR-34a is not a candidate tumor suppressor

While several microRNAs (miRNAs) have been proposed to act as tumor suppressors, a consensual definition of tumor suppressing miRNAs is still missing. Similarly to coding genes, we propose that tumor suppressor miRNAs must show evidence of genetic or epigenetic inactivation in cancers, and exhibit an anti-tumorigenic (e.g., anti-proliferative) activity under endogenous expression levels. Here we observe that this definition excludes the most extensively studied tumor suppressor candidate miRNA, miR-34a. In analyzable cancer types, miR-34a does not appear to be down-regulated in primary tumors relatively to normal adjacent tissues. Deletion of miR-34a is occasionally found in human cancers, but it does not seem to be driven by an anti-tumorigenic activity of the miRNA, since it is not observed upon smaller, miR-34a-specific alterations. Its anti-proliferative action was observed upon large, supra-physiological transfection of synthetic miR-34a in cultured cells, and our data indicates that endogenous miR-34a levels do not have such an effect. Our results therefore argue against a general tumor suppressive function for miR-34a, providing an explanation to the lack of efficiency of synthetic miR-34a administration against solid tumors.

genetics↗

bioRxiv2018

Bioinorganic Angiogenesis

Causing a large diameter blood vessel to sprout branches and a capillary network on demand to create a new angiosome is key to harnessing to potential of regenerative medicine and advancing reconstructive surgery. Currently this can only be achieved by connecting a vein graft to an artery by microsurgery, the arteriovenous loop technique (AVL). The arterial blood pressure in the thin-walled vein is thought to drive remodelling to create branches, however the surgical complexity limits the application of the technique. In this study we demonstrate that unexpectedly, a vessel density of luminal branches in excess of that achieved by the surgical AVL approach can be induced simply by placing a vein in contact with a microporous calcium phosphate. Only osteoinductive biomaterials have been reported previously, this is thought to be the first report of an angio-inductive material. Pilot studies indicated that the material type greatly affected the degree of luminal vascularization. Material contact with the vein is not a requirement for luminal angiogenesis of the vein and together these findings point to a bioinorganic effect, wherein the degradation of the material both releases a stimulatory ionic milieu and creates space for the developing angiosome.

bioengineering↗

bioRxiv2018

A novel approach reveals that HLA class 1 single antigen bead-signatures provide a means of high-accuracy pre-transplant risk assessment of acute cellular rejection

Acute cellular rejection (ACR) is associated with complications after kidney transplantation, such as graft dysfunction and graft loss. Early risk assessment is therefore critical for the improvement of transplantation outcomes. In this work, we retrospectively analyzed a pre-transplant HLA antigen bead assay data set that was acquired by the e:KID consortium as part of a systems medicine approach. The data set included single antigen bead (SAB) reactivity profiles of 52 low-risk graft recipients (negative complement dependent cytotoxicity crossmatch, PRA<30%) who showed detectable pre-transplant anti-HLA 1 antibodies. To assess whether the reactivity profiles provide a means for ACR risk assessment, we established a novel approach which differs from standard approaches in two aspects: the use of quantitative continuous data and the use of a multiparameter classification method. Remarkably, it achieved significant prediction of the 38 graft recipients who experienced ACR with a balanced accuracy of 82.7% (sensitivity=76.5%, specificity= 88.9%). The resultant classifier achieved one of the highest prediction accuracies in the literature for pre-transplant risk assessment of ACR. Importantly, it can facilitate risk assessment in non-sensitized patients who lack donor-specific antibodies. As the classifier is based on continuous data and includes weak signals, our results emphasize that not only strong but also weak binding interactions of antibodies and HLA 1 antigens contain predictive information.

immunology↗

bioRxiv2018

Functional lability of RNA-dependent RNA polymerases in animals

RNA interference (RNAi) requires RNA-dependent RNA polymerases (RdRPs) in many eukaryotes, and RNAi amplification constitutes the only known function for eukaryotic RdRPs. Yet in animals, classical model organisms can elicit RNAi without possessing RdRPs, and only nematode RNAi was shown to require RdRPs. Here we show that RdRP genes are much more common in animals than previously thought, even in insects, where they had been assumed not to exist. RdRP genes were present in the ancestors of numerous clades, and they were subsequently lost at a high frequency. In order to probe the function of RdRPs in a deuterostome (the cephalochordate Branchiostoma lanceolatum), we performed high-throughput analyses of small RNAs from various Branchiostoma developmental stages. Our results show that Branchiostoma RdRPs do not appear to participate in RNAi: we did not detect any candidate small RNA population exhibiting classical siRNA length or sequence features. Our results show that RdRPs have been independently lost in dozens of animal clades, and even in a clade where they have been conserved (cephalochordates) their function in RNAi amplification is not preserved. Such a dramatic functional variability reveals an unexpected plasticity in RNA silencing pathways.\n\nAuthor summaryRNA interference (RNAi) is a conserved gene regulation system in eukaryotes. In non-animal eukaryotes, it necessitates RNA-dependent RNA polymerases (\"RdRPs\"). Among animals, only nematodes appear to require RdRPs for RNAi. Yet additional animal clades have RdRPs and it is assumed that they participate in RNAi. Here, we find that RdRPs are much more common in animals than previously thought, but their genes were independently lost in many lineages. Focusing on a species with RdRP genes (a cephalochordate), we found that it does not use them for RNAi. While RNAi is the only known function for eukaryotic RdRPs, our results suggest additional roles. Eukaryotic RdRPs thus have a complex evolutionary history in animals, with frequent independent losses and apparent functional diversification.

evolutionary biology↗