Biology subjects

Haase, A. D.

Publications and source records attributed to Haase, A. D..

8 recordsLinked to original sources

bioRxiv2026

Nuclear speckles comprise hierarchical nanoscale interaction networks that shape molecular dynamics

Biomolecular condensates organize essential cellular processes, but how their internal architecture regulates molecular behavior in living cells remains poorly understood. Here, we use MINFLUX nanoscopy for quantitative single-molecule imaging and live-cell tracking to resolve the organization and dynamics of specific macromolecules within nuclear speckles. Rather than behaving as uniform liquid-like compartments, nuclear speckles are hierarchically organized into dynamic nanoscale interaction networks. Proteins and RNAs assemble into molecular clusters that generate transient interaction nanodomains, where individual molecules alternate between diffusion and confinement while exploring higher-order networks. Physiological and genetic perturbations selectively remodel molecular clustering and nanodomain organization, and these changes are not consistently coupled to changes in the abundance of the measured components. Together, these findings reveal a multiscale organization of nuclear speckles in which molecular clustering, transient confinement, and dynamic exchange represent distinct but interconnected features of condensate architecture in living cells.

cell biology↗

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↗

bioRxiv2024

A deficiency screen identifies genomic regions critical for sperm head-tail connection

A stable connection between the sperm head and tail is critical for fertility in species with flagellated sperm. The head-tail coupling apparatus (HTCA) serves as the critical link between the nucleus (head) and the axoneme (tail) via the centriole. To identify regions of the Drosophila melanogaster genome that contain genetic elements that influence HTCA formation, we undertook a two part screen using the Drosophila deficiency (Df) kit. For this screen, we utilized a sensitized genetic background that overexpresses the pericentriolar material regulatory protein Pericentrin-Like Protein (PLP). We had previously shown that PLP overexpression (PLPOE) disrupts the head-tail connection in some spermatids, but not to a degree sufficient to reduce fertility. In the first step of the screen we tested for Dfs that in combination with PLPOE cause a reduction in fertility. We ultimately identified 11 regions of the genome that showed an enhanced fertility defect when combined with PLP overexpression. In the second step of the screen we tested these Dfs for their ability to enhance the HTCA defect caused by PLPOE, finding six. We then tested smaller Dfs to narrow the region of the genome that contained these enhancers. To further analyze the regions of the genome removed by these Dfs, we examined the expression patterns of the genes within these Dfs in publicly available datasets of RNAseq of Drosophila tissues and snRNAseq of Drosophila testes. In total, our analysis suggests that some of these Dfs may contain a single gene that might influence HTCA formation and / or fertility, while others appear to be regions of the genome especially rich in testis-expressed genes that might affect the HTCA because of complex, multi-gene interactions. Article SummaryWe perform a genetic enhancer deficiency screen to uncover genomic regions required for proper sperm head-tail connection. We identified 6 regions and provide insight into these regions using publicly available RNA sequence data. Our data reveal that these regions are exceptionally rich in testes specific genes. Further analysis using small deficiencies resulted in two classes of enhancers: one class likely enhances head-tail connection by disrupting multiple genes, while the second class might house a single gene responsible for the reduction in fertility.

genetics↗

bioRxiv2022

Transposase N-terminal phosphorylation and asymmetric transposon ends inhibit piggyBac transposition in mammalian cells

Mechanistic regulation of DNA transposon systems in mammalian cells remains poorly understood. Using modeling, biochemical, and cell-based assays, we sought to extend the recent cryoEM structural insight into the piggyBac transpososome to evaluate the previously unexplained role of the transposase N-terminus, the need for asymmetric transposon ends, and the complexity of transposase tetramer formation for transposition in mammalian cells. We found that N-terminal phosphorylation by casein kinase II inhibits transposase-DNA interaction and designed deletion of this phosphorylated domain releases inhibition thereby enhancing activity. We also found that the N-terminal domain promotes transposase dimerization in the absence of transposon DNA. N-terminal deletion enables transposition of symmetric transposon ends that was previously not achievable with piggyBac. The complex transposase tetramer needed for transposition of asymmetric transposon ends can be overcome via appending a second transposase C-terminal domain in combination with symmetric transposon ends overcoming the negative regulation by asymmetric ends. Our results demonstrate that N-terminal transposase phosphorylation and the requirement for asymmetric transposon ends both negatively regulate piggyBac transposons in mammalian cells. These novel insights into mechanism and structure of the piggyBac transposase expand its potential use for genomic applications.

molecular biology↗

bioRxiv2022

Transcriptomics, Regulatory Syntax, and Enhancer Identification in Heterogenous Populations of Mesoderm-Induced ESCs at Single-Cell Resolution

ESCs can adopt lineage-specific gene expression programs by stepwise exposure to defined factors, resulting in the generation of functional cell types. Bulk and single cell-based assays were employed to catalogue gene expression, histone modifications, chromatin conformation, and accessibility transitions in ESC populations and individual cells acquiring a presomitic mesoderm fate and undergoing further lineage specification. These assays identified cis-regulatory regions and transcription factors presiding gene expression programs occurring at defined ESC transitions and revealed the presence of heterogeneous cell populations within discrete ESC developmental stages. The datasets were employed to identify previously unappreciated genomic elements directing the initial activation of Pax7 and myogenic and neurogenic gene expression programs. This study provides a resource for the discovery of genomic and transcriptional features of pluripotent, mesoderm-induced ESCs, and ESCs-derived cell lineages.

developmental biology↗

bioRxiv2021

Hierarchical length and sequence preferences establish a single major piRNA 3'-end

PIWI-interacting RNAs (piRNAs) guard germline genomes against the deleterious action of retroviruses and other mobile genetic elements. How piRNAs faithfully discriminate between self and non-self to restrict all mobile elements while sparing essential genes remains a key outstanding question in genome biology. PiRNAs use extensive base-pairing to recognize their targets and variable 3ends could change the specificity and efficacy of piRNA silencing. Here, we identify conserved rules that ensure the generation of a single major piRNA 3end in flies and mice. Our data suggest that the PIWI proteins initially define a short interval on pre-piRNAs that grants access to the ZUC-processor complex. Within this Goldilocks zone, the preference of the ZUC-processor to cut in front of a Uridine determines the ultimate processing site. We observe a mouse-specific roadblock that relocates the Goldilocks zone and generates an opportunity for consecutive trimming by PNLDC1. Our data reveal a conserved hierarchy between length and sequence preferences that controls the piRNA sequence space. The unanticipated precision of 3end formation bolsters the emerging understanding that the functional piRNA sequence space is tightly controlled to ensure effective defense. Key findingsO_LIEvery piRNA has a single major 3end C_LIO_LILength preference trumps sequence preference during 3end formation. C_LIO_LIRelocation of the Goldilocks zone generates opportunity for trimming of mouse piRNAs. C_LI

developmental biology↗

bioRxiv2021

Cellular abundance shapes function in piRNA-guided genome defense

Defense against genome invaders universally relies on RNA-guided immunity. Prokaryotic CRISPR/Cas and eukaryotic RNA interference pathways recognize targets by complementary base-pairing, which places the sequences of their guide RNAs at the center of self/nonself discrimination. Here, we explore the sequence space of PIWI-interacting RNAs (piRNAs), the genome defense of animals, and establish functional priority among individual sequences for the first time. Our results reveal that only the topmost abundant piRNAs are commonly present in every cell, while rare sequences generate cell-to-cell diversity in flies and mice. We identify a skewed distribution of sequence abundance as a hallmark of piRNA populations and show that quantitative differences of more than a thousand-fold are established by conserved mechanisms of biogenesis. Finally, our genomics analyses and direct reporter assays reveal that abundance determines function in piRNA-guided genome defense. Taken together, we identify an effective sequence space and untangle two classes of piRNAs that differ in complexity and function. The first class represents the topmost abundant sequences and drives silencing of genomic parasites. The second class sparsely covers an enormous sequence space. These rare piRNAs cannot function in every cell, every individual or every generation but create diversity with potential for adaptation in the ongoing arms race with genome invaders.

developmental biology↗

bioRxiv2020

Functional tagging of endogenous proteins and rapid selection of cell pools (Rapid generation of endogenously tagged piwi in ovarian somatic sheath cells.)

The combination of genome-editing and epitope tagging provides a powerful strategy to study proteins with high affinity and specificity while preserving their physiological expression patterns. However, stably modifying endogenous genes in cells that do not allow for clonal selection has been challenging. Here, we present a simple and fast strategy to generate stable, endogenously tagged alleles in a non-transformed cell culture model. At the example of piwi in Drosophila ovarian somatic sheath cells, we show that this strategy enables the generation of an N-terminally tagged protein that emulates the expression level and subcellular localization of the wild type protein and forms functional Piwi-piRNA complexes. We present a concise workflow to establish modified cells, characterize the edited allele and probe the function of the tagged protein.

cell biology↗