Biology subjects

Meister, G.

Publications and source records attributed to Meister, G..

10 recordsLinked to original sources

bioRxiv2025

Pachytene piRNAs define a conserved program of meiotic gene regulation

PIWI-interacting RNAs (piRNAs) safeguard genome integrity and fertility across animals and in humans1-3. In mammals, pre-pachytene piRNAs execute the ancestral program of transposon silencing, whereas a second, mammalian-specific class functions during meiosis4. These pachytene piRNAs are indispensable for spermatogenesis3, yet their sequence diversity, rapid evolution, and lack of obvious complementarity have long obscured their primary targets and mode of action5. Here, we show that groups of pachytene piRNAs converge on single mRNAs with near-perfect complementarity, evoking the specificity and efficacy of classical RNA interference6. These mRNA-targeting piRNAs originate from pseudogene fragments within discrete piRNA clusters, establishing an unexpected one-to-one relationship: a given cluster encodes thousands of piRNAs that collectively silence a single gene. Deleting such piRNA clusters or their embedded pseudogene fragments, eliminates the gene-targeting piRNAs, derepresses the target mRNA, and causes spermatogenic defects. Comparative genomics reveals a simple, unifying logic, whereby rapidly evolving cluster-target pairs coexist with a conserved, primate-specific regulon maintained over tens of millions of years. These findings provide a mechanistic framework for pachytene piRNA action, repositioning them as precise gene regulators during meiosis and highlighting a uniquely preserved piRNA-target pair in primates.

developmental biology↗

bioRxiv2025

In Vivo AGO-APP For Cell-Type- and Compartment-Specific miRNA Profiling in the Mouse Brain

AGO-APP through the expression of the T6B peptide permits the isolation of Ago-bound miRNAs. Here we present the generation and characterization of two transgenic mouse lines allowing to perform AGO-APP in vivo. First, we generated mice for CRE-dependent expression of T6B in the cytoplasm. Using this line we performed Ago-APP in olfactory bulb (OB) inhibitory interneurons and cerebral cortex excitatory neurons. Bioinformatic analysis validated the high reproducibility of the approach. It also demonstrated that, despite global miRNome conservation between the two cell types, a set of miRNAs including the miR-200 family and the miR-183/96/182 cluster, is massively enriched in OB interneurons which is consistent with previous observations. In the second mouse line T6B is fused to the postsynaptic protein PSD95. Isolation of T6B-PSD95 fractions from OB and cortical neurons identified specific sets of post-synapse enriched miRNAs. Gene ontology analyses confirmed that these miRNAs preferentially target mRNAs related to synaptic functions. MotivationCollectively, miRNAs are required for all biological processes in eukaryotes. However, due to a lack of appropriate methodological tools, the actual mode of action of individual miRNAs in a given cell is currently poorly understood. In this article, we describe a novel approach to study the specific expression of miRNAs in vivo, at the level of cell type and in a specific cell compartment.

neuroscience↗

bioRxiv2023

Darwin's "neuters" and the evolution of the sex continuum in a superorganism

Ant castes are an amazing example of phenotypic plasticity. In worker-destined embryos of the ant Cardiocondyla obscurior, the default female developmental trajectory is interrupted even before the gonadal precursor cells acquire a sexual identity. miRNA and mRNA expression in embryos reveal three distinct phenotypic entities: males, females, and "neuters", as Darwin coined the worker caste in "On the Origin of Species". Based on these results we propose that haplodiploidy, in conjunction with insect sex determination, allows for the expression of a third dimension on the sex continuum, thus facilitating the evolution of individuals which develop traits their parents do not have. One sentence summarySex and caste differentiation begin during the same embryonic developmental window in the ant Cardiocondyla obscurior.

evolutionary biology↗

bioRxiv2023

In vivo AGO-APP identifies a module of microRNAs cooperatively controlling exit from neural stem cell state

MicroRNAs (miRNAs) are essential regulators of all developmental processes. Their function is particularly important during neurogenesis, when the production of large numbers of neurons from a limited number of neural stem cells depends on the precise control of determination, proliferation and differentiation. However, miRNA regulation of target mRNAs is highly promiscuous, one miRNA can target many mRNAs and vice versa, raising the question of how specificity is achieved to elicit a precise regulatory response. Here we introduce AGO-APP, a novel approach to purify Argonaute-bound miRNAs directly from cells and tissues in vivo, to isolate actively inhibiting miRNAs from different neural cell populations in the larval Drosophila central nervous system. We identify a defined group of miRNAs that redundantly target all iconic genes known to control the transition from neuroblasts to neurons. In vivo functional studies demonstrate that knockdown of individual miRNAs does not induce detectable cellular phenotypes. However, simultaneous knockdown of multiple miRNAs leads to precocious stem cell differentiation, demonstrating functional interdependence. Thus, miRNAs cooperate within a regulatory module to specify the targeted gene network.

developmental biology↗

bioRxiv2022

A rapid protocol for ribosome profiling of low input samples

Ribosome profiling provides quantitative, comprehensive, and high-resolution snapshots of cellular translation by the high-throughput sequencing of short mRNA fragments that are protected from nucleolytic digestion by ribosomes. While the overall principle is simple, the workflow of ribosome profiling experiments is complex and challenging, and typically requires large amounts of sample, limiting its broad applicability. Here, we present a new protocol for ultra-rapid ribosome profiling from low-input samples. It features a robust strategy for sequencing library preparation within one day that employs solid phase purification of reaction intermediates, allowing to reduce the input to as little as 0.1 pmol of RNA. Hence, it is particularly suited for the analyses of small samples or targeted ribosome profiling. Its high sensitivity and its ease of implementation will foster the generation of higher quality data from small samples, which opens new opportunities in applying ribosome profiling.

systems 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↗

bioRxiv2020

A nuclear role for ARGONAUTE-2 in regulation of neuronal alternative polyadenylation

Argonaute 2 (AGO2), the effector protein partner of microRNAs (miRNAs) in the cytoplasmic RNA induced silencing complex, is further involved in nuclear RNA processing. However, a role for AGO2 in regulation of alternative polyadenylation was not yet demonstrated. Here, we reveal unexpected abundance of AGO2 in mouse neuronal nuclei and characterize nuclear AGO2 interactors by mass spectrometry. We discover that AGO2 broadly regulated alternative polyadenylation (APA) in neuronal cells. Specifically, we demonstrate how two isoforms of Ret mRNA, which encodes a receptor tyrosine kinase are regulated by AGO2-depenent APA, affecting downstream GDNF signaling in primary motor neurons.

cell biology↗

bioRxiv2019

Low miRNA abundance disables microRNA pathway in mammalian oocytes

MicroRNAs (miRNAs) are ubiquitous small RNAs, which guide post-transcriptional gene repression in countless biological processes. However, the miRNA pathway in mouse oocytes appears inactive and is dispensable for development. We report that miRNAs do not accumulate like maternal mRNAs during oocyte growth. The most abundant miRNAs total tens of thousands of molecules in fully-grown oocytes, a number similar to that observed in much smaller fibroblasts. The lack of miRNA accumulation acts like miRNA knock-down, where miRNAs can engage their targets but are not abundant enough to produce significant silencing effect. Injection of 100,000 miRNAs was sufficient to restore reporter repression in oocytes, confirming that miRNA inactivity primarily stems from low miRNA abundance and not from an active repression of the miRNA pathway per se. Similar situation was observed in rat, hamster, porcine, and bovine oocytes arguing that miRNA inactivity is not a mouse-specific adaptation but a common mammalian oocyte feature.

molecular biology↗

bioRxiv2019

AGO1x prevents dsRNA-induced interferon signaling to promote breast cancer cell proliferation

Initially reported for viral RNA, elongation of polypeptide chains beyond the stop codon (translational readthrough (TR)) also occurs on eukaryotic transcripts. TR diversifies the proteome and can modulate protein levels 1-6. Here we report that AGO1x, a conserved TR isoform of Argonaute 1, is generated in highly proliferative breast cancer cells, where it curbs accumulation of double stranded RNAs, the induction of the interferon response and apoptosis. In contrast to other mammalian Argonaute protein family members with primarily cytoplasmic functions, AGO1x localizes to the nucleus, in the vicinity of nucleoli. We identify a novel interaction of AGO1x with the Polyribonucleotide Nucleotidyltransferase 1, depletion of either protein leading to dsRNA accumulation and impaired cell proliferation. Our study thus uncovers a novel function of an Argonaute protein outside of the miRNA effector pathway, in buffering dsRNA-induced interferon responses. As AGO1x expression is tightly linked to breast cancer cell proliferation, our study suggests a new direction for limiting tumor growth.

cell biology↗

bioRxiv2018

Reconstitution of mammalian Cleavage Factor II involved in 3’ processing of mRNA precursors

Cleavage factor II (CF II) is a poorly characterized component of the multi-protein complex catalyzing 3 cleavage and polyadenylation of mammalian mRNA precursors. We have reconstituted CF II as a heterodimer of hPcf11 and hClp1. The heterodimer is active in partially reconstituted cleavage reactions, whereas hClp1 by itself is not. Pcf11 moderately stimulates the RNA 5 kinase activity of hClp1; the kinase activity is dispensable for RNA cleavage. CF II binds RNA with nanomolar affinity. Binding is mediated mostly by the two zinc fingers in the C-terminal region of hPcf11. RNA is bound without pronounced sequence-specificity, but extended G-rich sequences appear to be preferred. We discuss the possibility that CF II contributes to the recognition of cleavage/polyadenylation substrates through interaction with G-rich far-downstream sequence elements.

biochemistry↗