RESUMO
Although introns are typically tens to thousands of nucleotides, there are notable exceptions. In flies as well as humans, a small number of genes contain introns that are more than 1000 times larger than typical introns, exceeding hundreds of kilobases (kb) to megabases (Mb). It remains unknown why gigantic introns exist and how cells overcome the challenges associated with their transcription and RNA processing. The Drosophila Y chromosome contains some of the largest genes identified to date: multiple genes exceed 4Mb, with introns accounting for over 99% of the gene span. Here we demonstrate that co-transcriptional splicing of these gigantic Y-linked genes is important to ensure successful transcription: perturbation of splicing led to the attenuation of transcription, leading to a failure to produce mature mRNA. Cytologically, defective splicing of the Y-linked gigantic genes resulted in disorganization of transcripts within the nucleus suggestive of entanglement of transcripts, likely resulting from unspliced long RNAs. We propose that co-transcriptional splicing maintains the length of nascent transcripts of gigantic genes under a critical threshold, preventing their entanglement and ensuring proper gene expression. Our study reveals a novel biological significance of co-transcriptional splicing.
Assuntos
Drosophila melanogaster , Íntrons , Splicing de RNA , Transcrição Gênica , Splicing de RNA/genética , Animais , Íntrons/genética , Drosophila melanogaster/genética , Cromossomo Y/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila/genética , Masculino , HumanosRESUMO
In animals, PIWI-interacting RNAs (piRNAs) direct PIWI proteins to silence complementary targets such as transposons. In Drosophila and other species with a maternally specified germline, piRNAs deposited in the egg initiate piRNA biogenesis in the progeny. However, Y chromosome loci cannot participate in such a chain of intergenerational inheritance. How then can the biogenesis of Y-linked piRNAs be initiated? Here, using Suppressor of Stellate (Su(Ste)), a Y-linked Drosophila melanogaster piRNA locus as a model, we show that Su(Ste) piRNAs are made in the early male germline via 5'-to-3' phased piRNA biogenesis initiated by maternally deposited 1360/Hoppel transposon piRNAs. Notably, deposition of Su(Ste) piRNAs from XXY mothers obviates the need for phased piRNA biogenesis in sons. Together, our study uncovers a developmentally programmed, intergenerational mechanism that allows fly mothers to protect their sons using a Y-linked piRNA locus.
Assuntos
Proteínas de Drosophila , Drosophila , Animais , Drosophila/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , RNA de Interação com Piwi , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Proteínas Argonautas/genéticaRESUMO
Researchers have used RNA in situ hybridization to detect the presence of RNA in cells and tissues for approximately 50 years. The recent development of a method capable of visualizing a single RNA molecule by utilizing tiled fluorescently labeled oligonucleotide probes that together produce a diffraction-limited spot has greatly increased the resolution of this technique, allowing for the precise determination of subcellular RNA localization and relative abundance. Here, we present our method for single molecule RNA fluorescence in situ hybridization (smFISH) in whole mount Drosophila testes and discuss how we have utilized this method to better understand the expression pattern of the highly unusual Y-linked genes.
Assuntos
Drosophila , Testículo , Animais , Masculino , Drosophila/genética , Hibridização in Situ Fluorescente/métodos , Testículo/química , RNA/genética , RNA/análise , Expressão GênicaRESUMO
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.
Assuntos
Células-Tronco Adultas , Testículo , Animais , Masculino , Testículo/metabolismo , Drosophila , RNA-Seq , SêmenRESUMO
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.
Assuntos
Drosophila melanogaster , Sêmen , Animais , Masculino , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Sêmen/metabolismo , Drosophila/metabolismo , Espermatogênese/genética , Fator de Iniciação 4E em Eucariotos/genética , Fator de Iniciação 4E em Eucariotos/metabolismoRESUMO
Satellite DNAs are arrays of tandem repeats found in the eukaryotic genome. They are mainly found in pericentromeric heterochromatin and have been believed to be mostly inert, leading satellite DNAs to be erroneously regarded as junk. Recent studies have started to elucidate the function of satellite DNA, yet little is known about the peculiar case where satellite DNA is found within the introns of protein coding genes, resulting in incredibly large introns, a phenomenon termed intron gigantism. Studies in Drosophila demonstrated that satellite DNA-containing introns are transcribed with the gene and require specialized mechanisms to overcome the burdens imposed by the extremely long stretches of repetitive DNA. Whether intron gigantism confers any benefit or serves any functional purpose for cells and/or organisms remains elusive. Here we review our current understanding of intron gigantism: where it is found, the challenges it imposes, how it is regulated and what purpose it may serve.
Assuntos
DNA Satélite , Gigantismo , Animais , DNA Satélite/genética , Drosophila/genética , Gigantismo/genética , Heterocromatina/genética , ÍntronsRESUMO
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.
Assuntos
Cílios/metabolismo , Citoplasma/metabolismo , Drosophila melanogaster/metabolismo , RNA Mensageiro/metabolismo , Espermatogênese/fisiologia , Animais , Dineínas do Axonema/metabolismo , Axonema/metabolismo , Proteínas de Transporte/metabolismo , DNA Helicases/metabolismo , Proteínas de Drosophila/metabolismo , MasculinoRESUMO
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.
Assuntos
DNA Satélite/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Íntrons , Espermatócitos/metabolismo , Espermatogênese/genética , Animais , DNA Satélite/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/citologia , Drosophila melanogaster/crescimento & desenvolvimento , Drosophila melanogaster/metabolismo , Éxons , Regulação da Expressão Gênica no Desenvolvimento , Masculino , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Transdução de Sinais , Espermatócitos/citologia , Espermatócitos/crescimento & desenvolvimento , Transcrição Gênica , Cromossomo Y/químicaRESUMO
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.
Assuntos
Divisão Celular Assimétrica , Animais , Diferenciação Celular , Linhagem da Célula , Microambiente Celular , Humanos , Modelos Biológicos , Células-Tronco/citologiaRESUMO
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.