Mammalian Sequences Research Articles

The Crystal Structure of Human IgD-Fc Reveals Unexpected Differences With Other Antibody Isotypes.

Of the five human antibody isotypes, the function of IgD is the least well-understood, although various studies point to a role for IgD in mucosal immunity. IgD is also the least well structurally characterized isotype. Until recently, when crystal structures were reported for the IgD Fab, the only structural information available was a model for intact IgD based on solution scattering data. We now report the crystal structure of human IgD-Fc solved at 3.0 Å resolution. Although similar in overall architecture to other human isotypes, IgD-Fc displays markedly different orientations of the Cδ3 domains in the Cδ3 domain dimer and the lowest interface area of all the human isotypes. The nature of the residues that form the dimer interface also differs from those conserved in the other isotypes. By contrast, the interface between the Cδ2 and Cδ3 domains in each chain is the largest among the human isotypes. This interface is characterized by two binding pockets, not seen in other isotypes, and points to a potential role for the Cδ2/Cδ3 interface in stabilizing the IgD-Fc homodimer. We investigated the thermal stability of IgD-Fc, alone and in the context of an intact IgD antibody, and found that IgD-Fc unfolds in a single transition. Human IgD-Fc clearly has unique structural features not seen in the other human isotypes, and comparison with other mammalian IgD sequences suggests that these unique features might be widely conserved.

Histidine-rich calcium-binding protein: a molecular integrator of cardiac excitation-contraction coupling.

During mammalian cardiomyocyte excitation-contraction coupling, Ca2+ influx through voltage-gated Ca2+ channels triggers Ca2+ release from the sarcoplasmic reticulum (SR) through ryanodine receptor channels. This Ca2+-induced Ca2+ release mechanism controls cardiomyocyte contraction and is exquisitely regulated by SR Ca2+ levels. The histidine-rich calcium-binding protein (HRC) and its aspartic acid-rich paralogue aspolin are high-capacity, low-affinity Ca2+-binding proteins. Aspolin also acts as a trimethylamine N-oxide demethylase. At low intraluminal Ca2+ concentrations, HRC binds to the SR Ca2+-ATPase 2, inhibiting its Ca2+-pumping activity. At high intraluminal Ca2+ levels, HRC interacts with triadin to reduce Ca2+ release through ryanodine receptor channels. This Review analyses the evolution of these Ca2+-regulatory proteins, to gain insights into their roles. It reveals that HRC homologues are present in chordates, annelid worms, molluscs, corals and sea anemones. In contrast, triadin appears to be a chordate innovation. Furthermore, HRC is evolving more rapidly than other cardiac excitation-contraction coupling proteins. This positive selection (or relaxed negative selection) occurs along most of the mammalian HRC protein sequence, with the exception being the C-terminal cysteine-rich region, which is undergoing negative selection. The histidine-rich region of HRC might be involved in pH sensing, as an adaptation to air-breathing, endothermic and terrestrial life. In addition, a cysteine-rich pattern within HRC and aspolin is also found in a wide range of iron-sulfur cluster proteins, suggesting roles in redox reactions and metal binding. The polyaspartic regions of aspolins are likely to underlie their trimethylamine N-oxide demethylase activity, which might be mimicked by the acidic regions of HRCs. These potential roles of HRCs and aspolins await experimental verification.

Characterisation of seryl tRNA synthetase (srs-2) in Haemonchus contortus and Teladorsagia circumcincta

The aim of the study was to purify and characterise recombinant proteins with the potential as an anti-parasite vaccine. Full-length cDNAs encoding seryl-tRNA synthetase (srs-2) were cloned from Haemonchus contortus (HcSRS-2) and Teladorsagia circumcincta (TcSRS-2). TcSRS-2 and HcSRS-2 cDNA (1458bp) encoded proteins of 486 amino acids, each of which was present as a single band of about 55 kDa on SDS-PAGE. Multiple alignments of the protein sequences showed homology of 94% between TcSRS-2 and HcSRS-2, 76–93% with SRS-2s of eight nematodes and 68% with Mus musculus SRS-2. The predicted three-dimensional structures revealed an overall structural homology of TcSRS-2 and HcSRS-2, highly conserved binding and catalytic sites, and minor differences in the tautomerase binding site residues in other nematode SRS-2 homologues. A phylogenetic tree was constructed using helminth and mammalian SRS-2 sequences. Soluble C-terminal SRS-2 proteins were expressed in Escherichia coli strain AY2.4 and purified. Recombinant HcSRS-2 assay shows that the recombinant enzyme was active and stable. The Km and Vmax for ATP were 3.9 ± 1.0 μM and 2.7 ± 0.1 μmol min−1 mg−1 protein, respectively. Antibodies in serum and saliva from field-immune, but not nematode-naïve, sheep recognised recombinant HcSRS-2 and TcSRS-2 in enzyme-linked immunosorbent assays. Recognition of the recombinant proteins by antibodies generated by exposure of sheep to the native enzyme indicates similar antigenicity of the two proteins.

Semi-supervised learning with pseudo-labeling compares favorably with large language models for regulatory sequence prediction.

Predicting molecular processes using deep learning is a promising approach to provide biological insights for non-coding single nucleotide polymorphisms identified in genome-wide association studies. However, most deep learning methods rely on supervised learning, which requires DNA sequences associated with functional data, and whose amount is severely limited by the finite size of the human genome. Conversely, the amount of mammalian DNA sequences is growing exponentially due to ongoing large-scale sequencing projects, but in most cases without functional data. To alleviate the limitations of supervised learning, we propose a novel semi-supervised learning (SSL) based on pseudo-labeling, which allows to exploit unlabeled DNA sequences from numerous genomes during model pre-training. We further improved it incorporating principles from the Noisy Student algorithm to predict the confidence in pseudo-labeled data used for pre-training, which showed improvements for transcription factor with very few binding (very small training data). The approach is very flexible and can be used to train any neural architecture including state-of-the-art models, and shows in most cases strong predictive performance improvements compared to standard supervised learning. Moreover, small models trained by SSL showed similar or better performance than large language model DNABERT2.

Identification of an intrinsically disordered region (IDR) in arginyltransferase 1 (ATE1).

Arginyltransferase 1 (ATE1) catalyzes arginylation, an important post-translational modification (PTM) in eukaryotes that plays a critical role in cellular homeostasis. The disruption of ATE1 function is implicated in mammalian neurodegenerative disorders and cardiovascular maldevelopment, while post-translational arginylation has also been linked to the activities of several important human viruses such as SARS-CoV-2 and HIV. Despite the known significance of ATE1 in mammalian cellular function, past biophysical studies of this enzyme have mainly focused on yeast ATE1, leaving the mechanism of arginylation in mammalian cells unclear. In this study, we sought to structurally and biophysically characterize mouse (Mus musculus) ATE1. Using size-exclusion chromatography (SEC), small angle X-ray scattering (SAXS), and hydrogen deuterium exchange mass spectrometry (HDX-MS), assisted by AlphaFold modeling, we found that mouse ATE1 is structurally more complex than yeast ATE1. Importantly, our data indicate the existence of an intrinsically disordered region (IDR) in all mouse ATE1 splice variants. However, comparative HDX-MS analyses show that yeast ATE1 does not have such an IDR, consistent with prior X-ray, cryo-EM, and SAXS analyses. Furthermore, bioinformatics approaches reveal that mammalian ATE1 sequences, as well as in a large majority of other eukaryotes, contain an IDR-like sequence positioned in proximity to the ATE1 GNAT active-site fold. Computational analysis suggests that the IDR likely facilitates the formation of the complex between ATE1 and tRNAArg, adding a new complexity to ATE1 structure and providing new insights for future studies of ATE1 functions.

Molecular recording using DNA Typewriter.

Recording molecular information to genomic DNA is a powerful means of investigating topics ranging from multicellular development to cancer evolution. With molecular recording based on genome editing, events such as cell divisions and signaling pathway activity drive specific alterations in a cell's DNA, marking the genome with information about a cell's history that can be read out after the fact. Although genome editing has been used for molecular recording, capturing the temporal relationships among recorded events in mammalian cells remains challenging. The DNA Typewriter system overcomes this limitation by leveraging prime editing to facilitate sequential insertions to an engineered genomic region. DNA Typewriter includes three distinct components: DNA Tape as the 'substrate' to which edits accrue in an ordered manner, the prime editor enzyme, and prime editing guide RNAs, which program insertional edits to DNA Tape. In this protocol, we describe general design considerations for DNA Typewriter, step-by-step instructions on how to perform recording experiments by using DNA Typewriter in HEK293T cells, and example scripts for analyzing DNA Typewriter data ( https://doi.org/10.6084/m9.figshare.22728758 ). This protocol covers two main applications of DNA Typewriter: recording sequential transfection events with programmed barcode insertions by using prime editing and recording lineage information during the expansion of a single cell to many. Compared with other methods that are compatible with mammalian cells, DNA Typewriter enables the recording of temporal information with higher recording capacities and can be completed within 4-6 weeks with basic expertise in molecular cloning, mammalian cell culturing and DNA sequencing data analysis.

Epitope binning for multiple antibodies simultaneously using mammalian cell display and DNA sequencing

Epitope binning, an approach for grouping antibodies based on epitope similarities, is a critical step in antibody drug discovery. However, conventional methods are complex, involving individual antibody production. Here, we established Epitope Binning-seq, an epitope binning platform for simultaneously analyzing multiple antibodies. In this system, epitope similarity between the query antibodies (qAbs) displayed on antigen-expressing cells and a fluorescently labeled reference antibody (rAb) targeting a desired epitope is analyzed by flow cytometry. The qAbs with epitope similar to the rAb can be identified by next-generation sequencing analysis of fluorescence-negative cells. Sensitivity and reliability of this system are confirmed using rAbs, pertuzumab and trastuzumab, which target human epidermal growth factor receptor 2. Epitope Binning-seq enables simultaneous epitope evaluation of 14 qAbs at various abundances in libraries, grouping them into respective epitope bins. This versatile platform is applicable to diverse antibodies and antigens, potentially expediting the identification of clinically useful antibodies.

Rapid molecular species identification of mammalian scat samples using nanopore adaptive sampling

Rapid molecular species identification of mammalian scat samples using nanopore adaptive sampling

Computational analysis of human medium-chain dehydrogenases/reductases revealing substrate- and coenzyme-binding characteristics

The medium-chain dehydrogenase/reductase (MDR) superfamily has more than 600,000 members in UniProt as of March 2023. As the family has been growing, the proportion of functionally characterized proteins has been falling behind. The aim of this project was to investigate the binding pockets of nine different MDR protein families based on sequence conservation patterns and three-dimensional structures of members within the respective families.A search and analysis methodology was developed. Using this, a total of 2000 eukaryotic MDR sequences belonging to nine different families were identified. The pairwise sequence identities within each of the families were 80–90 % for the mammalian sequences, like the levels observed for alcohol dehydrogenase, another MDR family. Twenty conserved residues were identified in the coenzyme part of the binding site by matching structural and conservation data of all nine protein families. The conserved residues in the substrate part of the binding pocket varied between the nine MDR families, implying divergent functions for the different families. Studying each family separately made it possible to identify multiple conserved residues that are expected to be important for substrate binding or catalysis of the enzymatic reaction.By combining structural data with the conservation of the amino acid residues in each protein, important residues in the binding pocket were identified for each of the nine MDRs. The obtained results add new positions of interest for the binding and activity of the enzyme family as well as fit well to earlier published data. Three distinct types of binding pockets were identified, containing no, one, or two tyrosine residues.

Phylogenetic analysis of mammalian SIP30 sequences indicating accelerated adaptation of functional domain in primates

SIP30, characterized by a coiled-coil functional domain, plays a key role in regulating synaptic vesicle exocytosis and is implicated in neuropathic pain resulting from peripheral nerve injury. Because neuropathic pain is studied in primates (including human), domesticated animals, and rodents, we conducted a phylogenetic analysis of SIP30 in selected species of these three groups of mammals. SIP30 exhibits a high degree of sequence divergence in comparison to its protein binding partners SNAP25 and ZW10, which show broad sequence conservation. Notably, we observed an increased rate of change in the highly conserved coiled-coil domain in the SIP30 protein, specifically within primates. This observation suggests an accelerated adaptation of this functional domain in primate species.

Immunomodulatory role of vasoactive intestinal peptide and ghrelin in Oncorhynchus mykiss

Neuropeptides are a group of peptides derived from precursor proteins synthesized in neuronal and nonneuronal cells. The classical functions of neuropeptides have been extensively studied in mammals, including neuromodulation in the central nervous system, molecular signaling in the peripheral nervous system, and immunomodulation associated mainly with anti-inflammatory activity. In contrast, in teleosts, studies of the immunomodulatory function of these neuropeptides are limited. In Oncorhynchus mykiss, vasoactive intestinal peptide (VIP) mRNA sequences have not been cloned, and the role of VIP in modulating the immune system has not been studied. Furthermore, in relation to other neuropeptides with possible immunomodulatory function, such as ghrelin, there are also few studies. Therefore, in this work, we performed molecular cloning, identification, and phylogenetic analysis of three VIP precursor sequences (prepro-VIP1, VIP2 and VIP3) in rainbow trout. In addition, the immunomodulatory function of both neuropeptides was evaluated in an in vitro model using the VIP1 sequence identified in this work and a ghrelin sequence already studied in O. mykiss. The results suggest that the prepro-VIP2 sequence has the lowest percentage of identity with respect to the other homologous sequences and is more closely related to mammalian orthologous sequences. VIP1 induces significant expression of both pro-inflammatory (IFN-γ, IL-1β) and anti-inflammatory (IL-10 and TGF-β) cytokines, whereas ghrelin only induces significant expression of proinflammatory cytokines such as IL-6 and TNF-α.

Resequencing of the TMF-1 (TATA Element Modulatory Factor) regulated protein (TRNP1) gene in domestic and wild canids

BackgroundCortical folding is related to the functional organization of the brain. The TMF-1 regulated protein (TRNP1) regulates the expansion and folding of the mammalian cerebral cortex, a process that may have been accelerated by the domestication of dogs. The objectives of this study were to sequence the TRNP1 gene in dogs and related canid species, provide evidence of its expression in dog brain and compare the genetic variation within dogs and across the Canidae. The gene was located in silico to dog chromosome 2. The sequence was experimentally confirmed by amplifying and sequencing the TRNP1 exonic and promoter regions in 72 canids (36 purebred dogs, 20 Gy wolves and wolf-dog hybrids, 10 coyotes, 5 red foxes and 1 Gy fox).ResultsA partial TRNP1 transcript was isolated from several regions in the dog brain. Thirty genetic polymorphisms were found in the Canis sp. with 17 common to both dogs and wolves, and only one unique to dogs. Seven polymorphisms were observed only in coyotes. An additional 9 variants were seen in red foxes. Dogs were the least genetically diverse. Several polymorphisms in the promoter and 3'untranslated region were predicted to alter TRNP1 function by interfering with the binding of transcriptional repressors and miRNAs expressed in neural precursors. A c.259_264 deletion variant that encodes a polyalanine expansion was polymorphic in all species studied except for dogs. A stretch of 15 nucleotides that is found in other mammalian sequences (corresponding to 5 amino acids located between Pro58 and Ala59 in the putative dog protein) was absent from the TRNP1 sequences of all 5 canid species sequenced. Both of these aforementioned coding sequence variations were predicted to affect the formation of alpha helices in the disordered region of the TRNP1 protein.ConclusionsPotentially functionally important polymorphisms in the TRNP1 gene are found within and across various Canis species as well as the red fox, and unique differences in protein structure have evolved and been conserved in the Canidae compared to all other mammalian species.

Metalloproteinases in Restorative Dentistry: An In Silico Study toward an Ideal Animal Model.

In dentistry, various animal models are used to evaluate adhesive systems, dental caries and periodontal diseases. Metalloproteinases (MMPs) are enzymes that degrade collagen in the dentin matrix and are categorized in over 20 different classes. Collagenases and gelatinases are intrinsic constituents of the human dentin organic matrix fibrillar network and are the most abundant MMPs in this tissue. Understanding such enzymes' action on dentin is important in the development of approaches that could reduce dentin degradation and provide restorative procedures with extended longevity. This in silico study is based on dentistry's most used animal models and intends to search for the most suitable, evolutionarily close to Homo sapiens. We were able to retrieve 176,077 mammalian MMP sequences from the UniProt database. These sequences were manually curated through a three-step process. After such, the remaining 3178 sequences were aligned in a multifasta file and phylogenetically reconstructed using the maximum likelihood method. Our study inferred that the animal models most evolutionarily related to Homo sapiens were Orcytolagus cuniculus (MMP-1 and MMP-8), Canis lupus (MMP-13), Rattus norvegicus (MMP-2) and Orcytolagus cuniculus (MMP-9). Further research will be needed for the biological validation of our findings.

BK Channels Function in Nematocyst Discharge from Vibration-Sensitive Cnidocyte Supporting Cell Complexes of the Sea Anemone Diadumene lineata.

AbstractIntegrated chemo- and mechanosensory pathways, along with activated calcium influxes, regulate nematocyst discharge from sea anemone tentacles. Discharge from vibration-sensitive Type A cnidocyte supporting cell complexes use calcium-conducting transient receptor potential V4-like channels. Because calcium influxes often couple with calcium-activated, large-conductance potassium (BK) channels, we hypothesized that BK channels function in nematocyst discharge. To verify this hypothesis, we first tested five selective BK channel blockers on nematocyst-mediated prey killing in Diadumene lineata (aka Haliplanella luciae). All tested BK channel blockers inhibited prey killing at concentrations comparable to their inhibition of vertebrate BK channels. In addition, the BK channel blocker paxilline selectively inhibited prey killing mediated by vibration-sensitive Type A cnidocyte supporting cell complexes. We queried a mammalian BKα amino acid sequence to the Exaiptasia diaphena database, from which we identified a putative anemone, pore-forming BKα subunit sequence. Using the E. diaphena BKα sequence as a template, we assembled a BKα transcript from our assembled D. lineata transcriptome. In addition, the hydra homolog of D. lineata BKα localizes to nematocytes on the hydra single-cell RNA sequencing map. Our findings suggest that D. lineata expresses BK channels that play a role in vibration-sensitive nematocyst discharge from Type A cnidocyte supporting cell complexes. We believe this is the first functional demonstration of BK channels in nonbilaterians. Because stimulated chemoreceptors frequency tune Type A cnidocyte supporting cell complexes to frequencies matching swimming movements of prey via a protein kinase A signaling pathway and protein kinase A generally activates BK channels, we suggest that D. lineata BK channels may participate in protein kinase A-mediated frequency tuning.

Comparative analysis of PB2 residue 627E/K/V in H5 subtypes of avian influenza viruses isolated from birds and mammals.

Avian influenza viruses (AIVs) are naturally found in wild birds, primarily in migratory waterfowl. Although species barriers exist, many AIVs have demonstrated the ability to jump from bird species to mammalian species. A key contributor to this jump is the adaption of the viral RNA polymerase complex to a new host for efficient replication of its RNA genome. The AIV PB2 gene appears to be essential in this conversion, as key residues have been discovered at amino acid position 627 that interact with the host cellular protein, acidic nuclear phosphoprotein 32 family member A (ANP32A). In particular, the conversion of glutamic acid (E) to lysine (K) is frequently observed at this position following isolation in mammals. The focus of this report was to compare the distribution of PB2 627 residues from different lineages and origins of H5 AIV, determine the prevalence between historical and contemporary sequences, and investigate the ratio of amino acids in avian vs. mammalian AIV sequences. Results demonstrate a low prevalence of E627K in H5 non-Goose/Guangdong/1996-lineage (Gs/GD) AIV samples, with a low number of mammalian sequences in general. In contrast, the H5-Gs/GD lineage sequences had an increased prevalence of the E627K mutation and contained more mammalian sequences. An approximate 40% conversion of E to K was observed in human sequences of H5 AIV, suggesting a non-exclusive requirement. Taken together, these results expand our understanding of the distribution of these residues within different subtypes of AIV and aid in our knowledge of PB2 mutations in different species.

Transposable elements in mammalian chromatin organization.

Transposable elements (TEs) are mobile DNA elements that comprise almost 50% of mammalian genomic sequence. TEs are capable of making additional copies of themselves that integrate into new positions in host genomes. This unique property has had an important impact on mammalian genome evolution and on the regulation of gene expression because TE-derived sequences can function as cis-regulatory elements such as enhancers, promoters and silencers. Now, advances in our ability to identify and characterize TEs have revealed that TE-derived sequences also regulate gene expression by both maintaining and shaping 3D genome architecture. Studies are revealing how TEs contribute raw sequence that can give rise to the structures that shape chromatin organization, and thus gene expression, allowing for species-specific genome innovation and evolutionary novelty.

Phosphorylation sites are evolutionary checkpoints against liquid–solid transition in protein condensates

Assemblies of multivalent RNA-binding protein fused in sarcoma (FUS) can exist in the functional liquid-like state as well as less dynamic and potentially toxic amyloid- and hydrogel-like states. How could then cells form liquid-like condensates while avoiding their transformation to amyloids? Here, we show how posttranslational phosphorylation can provide a "handle" that prevents liquid-solid transition of intracellular condensates containing FUS. Using residue-specific coarse-grained simulations, for 85 different mammalian FUS sequences, we show how the number of phosphorylation sites and their spatial arrangement affect intracluster dynamics preventing conversion to amyloids. All atom simulations further confirm that phosphorylation can effectively reduce the β-sheet propensity in amyloid-prone fragments of FUS. A detailed evolutionary analysis shows that mammalian FUS PLDs are enriched in amyloid-prone stretches compared to control neutrally evolved sequences, suggesting that mammalian FUS proteins evolved to self-assemble. However, in stark contrast to proteins that do not phase-separate for their function, mammalian sequences have phosphosites in close proximity to these amyloid-prone regions. These results suggest that evolution uses amyloid-prone sequences in prion-like domains to enhance phase separation of condensate proteins while enriching phosphorylation sites in close proximity to safeguard against liquid-solid transitions.

Semi-supervised learning improves regulatory sequence prediction with unlabeled sequences

MotivationGenome-wide association studies have systematically identified thousands of single nucleotide polymorphisms (SNPs) associated with complex genetic diseases. However, the majority of those SNPs were found in non-coding genomic regions, preventing the understanding of the underlying causal mechanism. Predicting molecular processes based on the DNA sequence represents a promising approach to understand the role of those non-coding SNPs. Over the past years, deep learning was successfully applied to regulatory sequence prediction using supervised learning. Supervised learning required DNA sequences associated with functional data for training, whose amount is strongly limited by the finite size of the human genome. Conversely, the amount of mammalian DNA sequences is exponentially increasing due to ongoing large sequencing projects, but without functional data in most cases.ResultsTo alleviate the limitations of supervised learning, we propose a paradigm shift with semi-supervised learning, which does not only exploit labeled sequences (e.g. human genome with ChIP-seq experiment), but also unlabeled sequences available in much larger amounts (e.g. from other species without ChIP-seq experiment, such as chimpanzee). Our approach is flexible and can be plugged into any neural architecture including shallow and deep networks, and shows strong predictive performance improvements compared to supervised learning in most cases (up to 70%).Availability and implementationhttps://forgemia.inra.fr/raphael.mourad/deepgnn.

Modulation of plant acetyl-CoA synthetase activity by post-translational lysine acetylation.

Acetyl-CoA synthetase (ACS) is one of several enzymes that generate the key metabolic intermediate, acetyl-CoA. In microbes and mammals ACS activity is regulated by the post-translational acetylation of a key lysine residue. ACS in plant cells is part of a two-enzyme system that maintains acetate homeostasis, but its post-translational regulation is unknown. This study demonstrates that the plant ACS activity can be regulated by the acetylation of a specific lysine residue that is positioned in a homologous position as the microbial and mammalian ACS sequences that regulates ACS activity, occurring in the middle of a conserved motif, near the carboxyl-end of the protein. The inhibitory effect of the acetylation of residue Lys-622 of the Arabidopsis ACS was demonstrated by site-directed mutagenesis of this residue, including its genetic substitution with the non-canonical N-ε-acetyl-lysine residue. This latter modification lowered the catalytic efficiency of the enzyme by a factor of more than 500-fold. Michaelis-Menten kinetic analysis of the mutant enzyme indicates that this acetylation affects the first half-reaction of the ACS catalyzed reaction, namely, the formation of the acetyl adenylate enzyme intermediate. The post-translational acetylation of the plant ACS could affect acetate flux in the plastids and overall acetate homeostasis.

Amniotes co-opt intrinsic genetic instability to protect germ-line genome integrity

Unlike PIWI-interacting RNA (piRNA) in other species that mostly target transposable elements (TEs), >80% of piRNAs in adult mammalian testes lack obvious targets. However, mammalian piRNA sequences and piRNA-producing loci evolve more rapidly than the rest of the genome for unknown reasons. Here, through comparative studies of chickens, ducks, mice, and humans, as well as long-read nanopore sequencing on diverse chicken breeds, we find that piRNA loci across amniotes experience: (1) a high local mutation rate of structural variations (SVs, mutations ≥ 50 bp in size); (2) positive selection to suppress young and actively mobilizing TEs commencing at the pachytene stage of meiosis during germ cell development; and (3) negative selection to purge deleterious SV hotspots. Our results indicate that genetic instability at pachytene piRNA loci, while producing certain pathogenic SVs, also protects genome integrity against TE mobilization by driving the formation of rapid-evolving piRNA sequences.