Intron gigantism, where genes contain megabase-sized introns, is observed across species, yet little is known about its purpose or regulation. Here we identify a unique gene expression program utilized for the proper expression of genes with intron gigantism. We find that two Drosophila genes with intron gigantism, kl-3 and kl-5 , are transcribed in a spatiotemporal manner over the course of spermatocyte differentiation, which spans ~90 hours. The introns of these genes contain megabases of simple satellite DNA repeats that comprise over 99% of the gene loci, and these satellite-DNA containing introns are transcribed. We identify two RNA-binding proteins that specifically localize to kl-3 and kl-5 transcripts and are needed for the successful transcription or processing of these genes. We propose that genes with intron gigantism require a unique gene expression program, which may serve as a platform to regulate gene expression during cellular differentiation.

Proper differentiation of sperm from germline stem cells, essential for production of the next generation, requires dramatic changes in gene expression that drive remodeling of almost all cellular components, from chromatin to organelles to cell shape itself. Here, we provide a single nucleus and single cell RNA-seq resource covering all of spermatogenesis in Drosophila starting from in-depth analysis of adult testis single nucleus RNA-seq (snRNA-seq) data from the Fly Cell Atlas (FCA) study. With over 44,000 nuclei and 6000 cells analyzed, the data provide identification of rare cell types, mapping of intermediate steps in differentiation, and the potential to identify new factors impacting fertility or controlling differentiation of germline and supporting somatic cells. We justify assignment of key germline and somatic cell types using combinations of known markers, in situ hybridization, and analysis of extant protein traps. Comparison of single cell and single nucleus datasets proved particularly revealing of dynamic developmental transitions in germline differentiation. To complement the web-based portals for data analysis hosted by the FCA, we provide datasets compatible with commonly used software such as Seurat and Monocle. The foundation provided here will enable communities studying spermatogenesis to interrogate the datasets to identify candidate genes to test for function in vivo.

Many adult stem cells divide asymmetrically, generating one stem cell (self-renewal) and one differentiating cell. Balancing self-renewal and differentiation is critical for sustaining tissue homeostasis throughout the life of an organism. Failure to execute asymmetric stem cell division can have profound impacts on tissue homeostasis, resulting in tissue degeneration or hyperplasia/tumorigenic overgrowth. Recent studies have expanded our understanding of both the extracellular and intracellular mechanisms that regulate, reinforce and ensure an asymmetric outcome following stem cell division. In this review, we discuss newly discovered aspects of asymmetric stem cell division that, in concert with well-established mechanisms, contribute to balancing self-renewal and differentiation.

Cytoplasmic cilia, a specialized type of cilia in which the axoneme resides within the cytoplasm rather than within the ciliary compartment, are proposed to allow for the efficient assembly of very long cilia. Despite being found diversely in male gametes (e.g., Plasmodium falciparum microgametocytes and human and Drosophila melanogaster sperm), very little is known about cytoplasmic cilia assembly. Here, we show that a novel RNP granule containing the mRNAs for axonemal dynein motor proteins becomes highly polarized to the distal end of the cilia during cytoplasmic ciliogenesis in Drosophila sperm. This allows for the incorporation of these axonemal dyneins into the axoneme directly from the cytoplasm, possibly by localizing translation. We found that this RNP granule contains the proteins Reptin and Pontin, loss of which perturbs granule formation and prevents incorporation of the axonemal dyneins, leading to sterility. We propose that cytoplasmic cilia assembly requires the precise localization of mRNAs encoding key axonemal constituents, allowing these proteins to incorporate efficiently into the axoneme.

DNA damage by ultraviolet (UV) light poses a risk for mutagenesis and a potential hindrance for cell cycle progression. Cells cope with UV-induced DNA damage through two general strategies to repair the damaged nucleotides and to promote cell cycle progression in the presence of UV-damaged DNA. Defining the genetic pathways and understanding how they function together to enable effective tolerance to UV remains an important area of research. The structural maintenance of chromosomes (SMC) proteins form distinct complexes that maintain genome stability during chromosome segregation, homologous recombination, and DNA replication. Using a forward genetic screen, we identified two alleles of smc-5 that exacerbate UV sensitivity in Caenorhabditis elegans. Germ cells of smc-5-defective animals show reduced proliferation, sensitivity to perturbed replication, chromatin bridge formation, and accumulation of RAD-51 foci that indicate the activation of homologous recombination at DNA double-strand breaks. Mutations in the translesion synthesis polymerase polh-1 act synergistically with smc-5 mutations in provoking genome instability after UV-induced DNA damage. In contrast, the DNA damage accumulation and sensitivity of smc-5 mutant strains to replication impediments are suppressed by mutations in the C. elegans BRCA1/BARD1 homologs, brc-1 and brd-1. We propose that SMC-5/6 promotes replication fork stability and facilitates recombination-dependent repair when the BRC-1/BRD-1 complex initiates homologous recombination at stalled replication forks. Our data suggest that BRC-1/BRD-1 can both promote and antagonize genome stability depending on whether homologous recombination is initiated during DNA double-strand break repair or during replication stalling.

No abstract

Drosophila sperm development is characterized by extensive post-transcriptional regulation whereby thousands of transcripts are preserved for translation during later stages. A key step in translation initiation is the binding of eukaryotic initiation factor 4E (eIF4E) to the 5’ mRNA cap. In addition to canonical eIF4E-1, Drosophila has multiple eIF4E paralogs, including four (eIF4E-3, -4, -5, and -7) that are highly expressed in the testis. Among these, only eIF4E-3 has been characterized genetically. Here, using CRISPR/Cas9 mutagenesis, we determined that eIF4E-5 is essential for male fertility. eIF4E-5 protein localizes to the distal ends of elongated spermatid cysts, and eIF4E-5 mutants exhibit defects during post-meiotic stages, including a mild defect in spermatid cyst polarization. eIF4E-5 mutants also have a fully penetrant defect in individualization, resulting in failure to produce mature sperm. Indeed, our data indicate that eIF4E-5 regulates non-apoptotic caspase activity during individualization by promoting local accumulation of the E3 ubiquitin ligase inhibitor Soti. Our results further extend the diversity of non-canonical eIF4Es that carry out distinct spatiotemporal roles during spermatogenesis.

Proper differentiation of sperm from germline stem cells, essential for production of the next generation, requires dramatic changes in gene expression that drive remodeling of almost all cellular components, from chromatin to organelles to cell shape itself. Here we provide a single nucleus and single cell RNA-seq resource covering all of spermatogenesis in Drosophila starting from in-depth analysis of adult testis single nucleus RNA-seq (snRNA-seq) data from the Fly Cell Atlas (FCA) study (Li et al., 2022). With over 44,000 nuclei and 6,000 cells analyzed, the data provide identification of rare cell types, mapping of intermediate steps in differentiation, and the potential to identify new factors impacting fertility or controlling differentiation of germline and supporting somatic cells. We justify assignment of key germline and somatic cell types using combinations of known markers, in situ hybridization, and analysis of extant protein traps. Comparison of single cell and single nucleus datasets proved particularly revealing of dynamic developmental transitions in germline differentiation. To complement the web-based portals for data analysis hosted by the FCA, we provide datasets compatible with commonly used software such as Seurat and Monocle. The foundation provided here will enable communities studying spermatogenesis to interrogate the datasets to identify candidate genes to test for function in vivo.