Retrotransposon-Derived Capsid Genes PNMA1 and PNMA4 Maintain Reproductive Capacity

Endocrinology Geriatrics Obstetrics and Gynecology Bioengineering Biophysics Cell Biology Biochemistry Genetics Life Sciences Medicine
retrotransposon capsid genes PNMA1 PNMA4 reproductive capacity gonadal tissue evolution

Mechanistic Study on How Retrotransposon-derived Capsid Genes PNMA1 and PNMA4 Maintain Reproductive Capacity — A New Research Review from Nature Aging

I. Research Background and Scientific Significance

Nearly half of the DNA in the human and mammalian genome is made up of retrotransposons, sequences that originally existed as “parasitic molecules” in the genome, inserting themselves into the host genome through an RNA intermediary. Although most retrotransposons have become inactive due to silencing or functional mutations, scientists have gradually realized that some retrotransposons can be “domesticated” and gradually evolve into new genes that confer adaptive advantages to their host.

The PNMA (Paraneoplastic Ma) family is a representative member of such “domesticated products.” Its origin can be traced back at least 100 million years to ancient vertebrate Metaviridae retrotransposons. The PNMA family contains multiple protein-coding genes with capsid (capsid, virus shell-like) structural domains. Although the proteins associated with PNMA1 and PNMA4 originate from “viruses,” increasing attention has been drawn to their evolutionary conservation, roles in reproductive development regulation, and interactions with a range of RNA-binding regulatory proteins (such as DAZL, DAZ, PUM1/2). Representatives such as PNMA1 and PNMA4 are not only found in the human protein-coding genome, but multiple homologous genes are also present in model organisms like mice.

The “domestication” of large numbers of retrotransposons has provided an evolutionary stage for hosts to acquire new genes and functions, yet how these genes are unearthed, regulated, and whether they play key roles in reproductive aging and the maintenance of fertility remain scientific questions this study seeks to explore. The central questions posed by the research team are: Are PNMA1 and PNMA4 indispensable factors for maintaining reproductive function and extending reproductive lifespan in mammals? Is their mechanism related to the molecular assembly of capsid-like structures and signal transduction? These questions are at the forefront of issues such as intercellular signaling, the molecular evolutionary mechanisms of new gene functions, and mammalian reproductive health.

II. Paper Source, Authors, and Publication Information

This study, titled “the retrotransposon-derived capsid genes pnma1 and pnma4 maintain reproductive capacity,” was completed jointly by many authors including Thomas W. P. Wood, William S. Henriques, Harrison B. Cullen, et al., from world-renowned research institutions and laboratories such as Columbia University, Yale University, Rutgers University, and Montana State University. The corresponding author is Luke E. Berchowitz (email: leb2210@cumc.columbia.edu).

The paper was published in the May 2025 issue of Nature Aging, Volume 5, pages 765–779. DOI: https://doi.org/10.1038/s43587-025-00852-y. The study was supported by multiple grants and shared experimental platforms, reflecting a prominent feature of international and interdisciplinary collaboration.

III. Research Workflow and Experimental Design Details

This project is a systematic and original laboratory-based and analytical research undertaking, applying human tissue sample analysis, genome-wide association studies (GWAS), transgenic mouse models, tissue cytology, molecular biochemistry, and proteomics, among other multidisciplinary approaches, to reveal the reproductive functions of PNMA1 and PNMA4 from molecular, cellular, individual, and population levels. The research is logically divided into six major modules:

1. Gene Expression Profiling and Tissue Specificity Analysis

a) Single-cell Sequencing and Data Integration

  • Subjects: Human ovaries and testes, including stratification by age (20s group & 50s group); mouse ovaries at different reproductive stages.
  • Methods: Using single-cell/single-nucleus RNA sequencing to analyze donated ovarian and testicular tissues (refer to data sources and sample sizes: a total of 13 ovarian donors, 8 testicular donors, and 8 mice) for PNMA1-5 expression, focusing on PNMA1 and PNMA4.
  • Results:
    • PNMA1 and PNMA4 are highly expressed in human ovaries/testes and mouse oocytes and spermatocytes, with expression levels dropping significantly with age or reproductive aging stages.
    • In ovarian tissue, expression is mainly in granulosa cells, stromal cells, and smooth muscle (supporting) cell populations, with a higher proportion in the younger group.

b) Molecular Regulatory Exploration

  • Experiment: Investigating the regulatory effects of DAZL (a translationally regulating RNA-binding protein) on PNMA1 and PNMA4.
  • Method: Co-transfection of PNMA1, PNMA4, and DAZL in HEK293T cells, with detection of protein and mRNA levels.
  • Observation: DAZL promotes PNMA1 protein expression while inhibiting PNMA4 protein expression, with no effect on mRNA levels, indicating DAZL acts at a post-translational level.

c) Evolution and Sequence Conservation Analysis

  • Method: Comparison of major mammalian lineages, analysis of nonsynonymous/synonymous substitution ratio (dN/dS) to verify evolutionary selective pressure.
  • Results: PNMA1 is widely conserved among eutherians and under purifying selection; PNMA4 has undergone pseudogenization in some species but still shows significant conservation.

2. Functional Validation of Genes: CRISPR Transgenic Mouse Model Construction and Phenotypic Analysis

a) Construction of Knockout Models

  • Method: Use of specific sgRNA and CRISPR-Cas9 to knock out PNMA1 and PNMA4, obtaining single and double gene knockout mice.
  • Samples: Several wild-type, PNMA1-/-, PNMA4-/-, and PNMA1-/-PNMA4-/- double knockout mice (n=4–10 per group, double-blind setup).
  • Genotype Verification: PCR, Sanger sequencing, etc.

b) Baseline Phenotype Evaluation

  • Experiment: Neurobehavioral tests to ensure knockout does not affect the brain (Y-maze, memory, anxiety, etc.).
  • Finding: No significant neurobehavioral abnormalities.

c) Dynamic Monitoring of Fertility

  • Subjects: Knockouts and controls, paired with fertility tester mice starting at 2 months of age, followed until 12 months.
  • Experimental Process: Changing mating partners every two weeks, counting litters and offspring.
  • Data: Knockout groups showed a significant decline in fertility from 3 months old; by 6 months, only 25–50% as many offspring as controls, with more severe impact in double knockouts.

d) Gonadal and Cellular Level Detection

  • Analysis: Measurement of testes/ovary weights, sperm counts, hormone levels, histological sectioning, TUNEL apoptosis staining, immunofluorescent labeling, and chromosome segregation imaging.
  • Data:
    • Male knockout mice: testicular atrophy, steep decline in sperm count, some seminiferous tubules completely devoid of germ cells, and a drastic drop in serum testosterone from 6 months old.
    • Females: Juveniles nearly normal, but with ages, ovarian atrophy, decline in antral follicles, and increased follicular cysts; no obvious meiotic defects detected.
    • At the cellular level: Increased germ cell apoptosis in knockouts (TUNEL positive); no abnormality in PLZF-positive undifferentiated spermatogonia, indicating the knockout mainly affects germ cell survival rather than generation.

3. Functional Validation and Dynamics of Capsid-like Structures

a) Expression of Recombinant Protein and Structural Identification

  • Subjects: Expression and purification of recombinant mouse PNMA1/PNMA4 proteins in E. coli.
  • Method: Custom His-tagged protein purification, high-resolution Superose 6 size exclusion chromatography.
  • Special Instruments: Negative stain transmission electron microscopy (TEM), CRYOSPARC 2D structure classification.
  • Results: Confirmation that PNMA1 forms capsid-like structures of 16–21nm in diameter, and PNMA4 forms 36–51nm structures, with varying degrees of self-assembly.

b) Cellular Secretion and In Vitro Transport

  • Experiment: Expression of PNMA1/PNMA4 in human HEK293T cells; after culture, supernatants are run through ultrafiltration (100 kDa MWCO) and ultracentrifugation, with stepwise fractionation and immunoblot detection.
  • Finding: Some PNMA1/PNMA4 assemblies migrate to the extracellular environment along with extracellular vesicles.

c) Screening for Large-molecule Complexes from Tissue Origin

  • Sampling: Testes from 3-month-old wild-type and PNMA4-/- mice, ground, lysed, and fractionated by double-layer sucrose cushion ultracentrifugation.
  • Subsequent Processing: Further iodixanol gradient fractionation, immunoblot differentiation, immunoprecipitation to isolate capsid particles.
  • RNA Cargo Identification: Immunoprecipitation extracts PNMA4-enriched mRNA, with qPCR and RNA-seq quantification: capsids are highly enriched for their own mRNA, indicating a self-delivery mechanism.

4. Population-level Human Genome-wide Association Studies (GWAS)

  • Method: Open Targets Genetics database, Variant-to-Gene (V2G) / Locus-to-Gene (L2G) algorithms, examining significant GWAS-associated variation for PNMA1/4 genes related to sex hormones, pubertal indices, and more.
  • Results:
    • PNMA1: Six loci associated with changes in testosterone/sex hormone binding globulin (SHBG) levels.
    • PNMA4: Two variants associated with age of menarche and timing of male puberty, respectively.
  • Significance: Suggests that the reproductive phenotypes validated in mice can also be traced to genetic bases in humans, confirming population relevance.

IV. Main Conclusions and Research Significance

1. Scientific Conclusions

  • PNMA1 and PNMA4 are “domesticated genes” derived from retrotransposons, highly expressed in human and mouse gametes and their supporting cells, with expression declining with age.
  • The proteins encoded by the two genes can self-organize into capsid-like macromolecular structures, capable of encapsulating and delivering their own mRNA to extracellular or nearby cells.
  • Mice with knockout of PNMA1/PNMA4 present with premature reproductive aging: gonadal atrophy, increased apoptosis, interruption of gamete generation, ultimately leading to significantly decreased fertility.
  • GWAS data show that polymorphisms in PNMA1/PNMA4 in the general population are closely related to sex hormones and newly described physiological indicators.
  • The study reveals a closed loop from “virus – gene – fertility,” from origin to molecular mechanisms to human functional biology, providing a new paradigm for understanding the adaptive collaboration between retrotransposons and host genomes.

2. Applied Significance

  • The PNMA capsid’s ability to encapsulate its own mRNA and potentially deliver molecules provides a natural medium for intercellular communication and signal transduction in reproductive cells, showing promise as a new generation of RNA delivery systems (such as fertility assistance, gene therapy, and vaccine delivery).
  • PNMA4 is not linked to autoimmune side effects, making it a promising target for the future development of treatments for subfertility/infertility and assisted reproduction.

3. Research Highlights and Innovations

  • For the first time, the core function of domesticated retrotransposon genes in mammalian reproductive aging is systematically validated, overturning the traditional view that “viral remnants are useless.”
  • The mechanism by which the PNMA1/4 capsid-like structures mediate self mRNA delivery introduces an entirely new biological concept of a “self-delivery communication system” at the molecular level.
  • Multilayered, multi-species experimental validation combined with human genetic association analysis provides strong support for animal-to-human translational significance.
  • Innovative methods (such as multi-step density fractionation, organizational capsid immunoprecipitation, and RNA-seq cargo analysis) benefit research on similar molecules.

V. Additional Valuable Information

  • The research team also analyzed the pivotal communications between mouse and human granulosa cells and oocytes, the role of interstitial “bridges,” and extracellular vesicles in the signal exchange among germ cells, proposing that the PNMA capsid likely acts as an auxiliary signal transmission factor alongside these structures.
  • The paper envisions the engineering of PNMA1/4 for clinical treatment and points out that optimizing large-scale purification and cargo identification of the capsid is key to achieving therapeutic translation.

VI. Summary

This landmark original work published in Nature Aging not only reveals the primitive function of domesticated retrotransposon genes in maintaining mammalian reproduction, but also proposes a new paradigm for molecular communication and signal regulation based on virus capsid structures that naturally carry and deliver mRNA. By tying together abundant data from protein structure, germ cell development, tissue morphology, and even human population genetics, this study greatly enriches the theoretical and practical foundations of mammalian (including human) reproductive health, molecular evolution, and innovative drug delivery. The “self-delivery capsid” mechanism of PNMA1 and PNMA4 provides a new type of molecular tool and theoretical model with great application potential for reproductive medicine, gene therapy, vaccine research and other diverse fields in the future.