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Publications (46)
PIWI-interacting RNAs (piRNAs) play a critical role in safeguarding genome integrity in germ cells, ensuring fertility. Here, we provide a protocol for processing piRNA sequencing data, identifying piRNA-rich genomic regions as piRNA clusters, and preparing these clusters for downstream analyses. We describe steps for assembling piRNA cluster regio...
Background
East African cichlid fishes have diversified in an explosive fashion, but the (epi)genetic basis of the phenotypic diversity of these fishes remains largely unknown. Although transposable elements (TEs) have been associated with phenotypic variation in cichlids, little is known about their transcriptional activity and epigenetic silencin...
Members of the piggyBac superfamily of DNA transposons are widely distributed in host genomes ranging from insects to mammals. The human genome has retained five piggyBac-derived genes as domesticated elements although they are no longer mobile. Here, we have investigated the transposition properties of piggyBat from Myotis lucifugus, the only know...
The Sperm Neck provides a stable connection between the sperm head and tail, which is critical for fertility in species with flagellated sperm. Within the Sperm Neck, the Head–Tail Coupling Apparatus serves as the critical link between the nucleus (head) and the axoneme (tail) via the centriole. To identify regions of the Drosophila melanogaster ge...
PIWI-interacting RNAs (piRNA) suppress selfish genetic elements and are essential for germ cell biology in animals. They also play critical roles in regeneration in planaria, regulate gene expression in adult mammalian testes, and participate in antiviral defense in mosquitoes. Inspired by a recent workshop on PIWI proteins and piRNAs, this comment...
PIWI-interacting RNAs (piRNAs) play a crucial role in safeguarding genome integrity by silencing mobile genetic elements. From flies to humans, piRNAs originate from long single-stranded precursors encoded by genomic piRNA clusters. How piRNA clusters form to adapt to genomic invaders and evolve to maintain protection remain key outstanding questio...
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 influen...
PIWI-interacting RNAs (piRNAs) are responsible for preventing the movement of transposable elements in germ cells and protect the integrity of germline genomes. In this review, we examine the common elements of piRNA-guided silencing as well as the differences observed between species. We have categorized the mechanisms of piRNA biogenesis and func...
DNA transposon systems are widely used in mammalian cells for genetic modification experiments, but their regulation remains poorly understood. We used biochemical and cell-based assays together with AlphaFold modeling and rational protein redesign to evaluate aspects of piggyBac transposition including the previously unexplained role of the transp...
The harnessing of the CRISPR‐Cas9 system allows for quick and inexpensive genome editing in tissue culture models. Traditional CRISPR‐Cas9 genome editing techniques rely on the ability of single progenitor cells to expand into new pools in a process known as clonal expansion. This is a significant technical challenge that is difficult to overcome f...
PIWI proteins and their associated PIWI-interacting RNAs (piRNAs) constitute a small RNA-based adaptive immune system that restricts the deleterious activity of mobile genetic elements to protect genome integrity. Self/nonself discrimination is at the very core of successful defence and relies on complementary base-pairing in RNA-guided immunity. H...
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...
Embryonic stem cells (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 catalog gene expression, histone modifications, chromatin conformation, and accessibility transitions in ESC populations and...
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...
PIWI-interacting RNAs (piRNAs) guard germline genomes against the deleterious action of mobile genetic elements. PiRNAs use extensive base-pairing to recognize their targets and variable 3’ends could change the specificity and efficacy of piRNA silencing. Here, we identify conserved rules that ensure the generation of a single major piRNA 3’end in...
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 acq...
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...
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)...
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)...
Crosslinking and immunoprecipitation (CLIP) methods are powerful techniques to interrogate direct protein-RNA interactions and dissect posttranscriptional gene regulatory networks. One widely used CLIP variant is photoactivatable ribonucleoside enhanced CLIP (PAR-CLIP) that involves in vivo labeling of nascent RNAs with the photoreactive nucleoside...
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...
Key to CRISPR-Cas adaptive immunity is maintaining an ongoing record of invading nucleic acids, a process carried out by the Cas1-Cas2 complex that integrates short segments of foreign genetic material (spacers) into the CRISPR locus. It is hypothesized that Cas1 evolved from casposases, a novel class of transposases. We show here that the Methanos...
PIWI-interacting RNAs (piRNAs) are at the center of a small RNA-based immune system that defends genomes against the deleterious action of mobile genetic elements (transposons). PiRNAs are highly variable in sequence with extensive targeting potential. Their diversity is restricted by their preference to start with a Uridine (U) at the 5′ most posi...
Oncogenic RAS (H-RAS(V12)) induces premature senescence in primary cells by triggering production of reactive oxygen species (ROS), but the molecular role of ROS in senescence remains elusive. We investigated whether inhibition of protein tyrosine phosphatases by ROS contributed to H-RAS(V12)-induced senescence. We identified protein tyrosine phosp...
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Argonautes are the central protein component in small RNA silencing pathways. Of the four human Argonautes (hAgo1-hAgo4) only hAgo2 is an active slicer. We determined the structure of hAgo1 bound to endogenous copurified RNAs to 1.75 Å resolution and hAgo1 loaded with let-7 microRNA to 2.1 Å. Both structures are strikingly similar to the structures...
PIWI-family proteins and their associated small RNAs (piRNAs) act in an evolutionarily conserved innate immune mechanism to provide essential protection for germ-cell genomes against the activity of mobile genetic elements. piRNA populations comprise a molecular definition of transposons, which permits them to distinguish transposons from host gene...
Argonaute proteins lie at the heart of the RNA-induced silencing complex (RISC), wherein they use small RNA guides to recognize targets. Initial insight into the architecture of Argonautes came from studies of prokaryotic proteins, revealing a crescent-shaped base made up of the amino-terminal, PAZ, middle, and PIWI domains. The recently reported c...
Combining RNAi in cultured cells and analysis of mutant animals, we probed the roles of known Piwi-interacting RNA (piRNA) pathway components in the initiation and effector phases of transposon silencing. Squash associated physically with Piwi, and reductions in its expression led to modest transposon derepression without effects on piRNAs, consist...
Consistent with the role of microRNAs (miRNAs) in down-regulating gene expression by reducing the translation and/or stability of target messenger RNAs, the levels of specific miRNAs are important for correct embryonic development and have been linked to several forms of cancer. However, the regulatory mechanisms by which primary miRNAs (pri-miRNAs...
Dicer is a key enzyme involved in RNA interference (RNAi) and microRNA (miRNA) pathways. It is required for biogenesis of miRNAs and small interfering RNAs (siRNAs), and also has a role in the effector steps of RNA silencing. Apart from Argonautes, no proteins are known to associate with Dicer in mammalian cells. In this work, we describe the ident...
The mammalian SIN3 complex consists of histone deacetylases (HDAC1, HDAC2), several known proteins (SAP30, N-CoR) and as yet unidentified proteins. Here we show that the mouse tetradecanoyl phorbol acetate induced sequence 7 (TIS7) protein is a novel transcriptional co-repressor that can associate with the SIN3 complex. We have identified tis7 as a...
