Co-transcriptional splicing facilitates transcription of gigantic genes.

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.

RNA polymerase II-mediated rDNA transcription mediates rDNA copy number expansion in Drosophila.

Ribosomal DNA (rDNA), which encodes ribosomal RNA, is an essential but unstable genomic element due to its tandemly repeated nature. rDNA's repetitive nature causes spontaneous intrachromatid recombination, leading to copy number (CN) reduction, which must be counteracted by a mechanism that recovers CN to sustain cells' viability. Akin to telomere maintenance, rDNA maintenance is particularly important in cell types that proliferate for an extended time period, most notably in the germline that passes the genome through generations. In Drosophila, the process of rDNA CN recovery, known as 'rDNA magnification', has been studied extensively. rDNA magnification is mediated by unequal sister chromatid exchange (USCE), which generates a sister chromatid that gains the rDNA CN by stealing copies from its sister. However, much remains elusive regarding how germ cells sense rDNA CN to decide when to initiate magnification, and how germ cells balance between the need to generate DNA double-strand breaks (DSBs) to trigger USCE vs. avoiding harmful DSBs. Recently, we identified an rDNA-binding Zinc-finger protein Indra as a factor required for rDNA magnification, however, the underlying mechanism of action remains unknown. Here we show that Indra is a negative regulator of rDNA magnification, balancing the need of rDNA magnification and repression of dangerous DSBs. Mechanistically, we show that Indra is a repressor of RNA polymerase II (Pol II)-dependent transcription of rDNA: Under low rDNA CN conditions, Indra protein amount is downregulated, leading to Pol II-mediated transcription of rDNA. This results in the expression of rDNA-specific retrotransposon, R2, which we have shown to facilitate rDNA magnification via generation of DBSs at rDNA. We propose that differential use of Pol I and Pol II plays a critical role in regulating rDNA CN expansion only when it is necessary.

Running the gauntlet: challenges to genome integrity in spermiogenesis.

Species' continuity depends on gametogenesis to produce the only cell types that can transmit genetic information across generations. Spermiogenesis, which encompasses post-meiotic, haploid stages of male gametogenesis, is a process that leads to the formation of sperm cells well-known for their motility. Spermiogenesis faces three major challenges. First, after two rounds of meiotic divisions, the genome lacks repair templates (no sister chromatids, no homologous chromosomes), making it incredibly vulnerable to any genomic insults over an extended time (typically days-weeks). Second, the sperm genome becomes transcriptionally silent, making it difficult to respond to new perturbations as spermiogenesis progresses. Third, the histone-to-protamine transition, which is essential to package the sperm genome, counterintuitively involves DNA break formation. How spermiogenesis handles these challenges remains poorly understood. In this review, we discuss each challenge and their intersection with the biology of protamines. Finally, we discuss the implication of protamines in the process of evolution.

Insulin signaling regulates R2 retrotransposon expression to orchestrate transgenerational rDNA copy number maintenance.

Preserving a large number of essential yet highly unstable ribosomal DNA (rDNA) repeats is critical for the germline to perpetuate the genome through generations. Spontaneous rDNA loss must be countered by rDNA copy number (CN) expansion. Germline rDNA CN expansion is best understood in Drosophila melanogaster, which relies on unequal sister chromatid exchange (USCE) initiated by DNA breaks at rDNA. The rDNA-specific retrotransposon R2 responsible for USCE-inducing DNA breaks is typically expressed only when rDNA CN is low to minimize the danger of DNA breaks; however, the underlying mechanism of R2 regulation remains unclear. Here we identify the insulin receptor (InR) as a major repressor of R2 expression, limiting unnecessary R2 activity. Through single-cell RNA sequencing we find that male germline stem cells (GSCs), the major cell type that undergoes rDNA CN expansion, have reduced InR expression when rDNA CN is low. Reduced InR activity in turn leads to R2 expression and CN expansion. We further find that dietary manipulation alters R2 expression and rDNA CN expansion activity. This work reveals that the insulin pathway integrates rDNA CN surveying with environmental sensing, revealing a potential mechanism by which diet exerts heritable changes to genomic content.

The implications of satellite DNA instability on cellular function and evolution.

Abundant tandemly repeated satellite DNA is present in most eukaryotic genomes. Previous limitations including a pervasive view that it was uninteresting junk DNA, combined with challenges in studying it, are starting to dissolve - and recent studies have found important functions for satellite DNAs. The observed rapid evolution and implied instability of satellite DNA now has important significance for their functions and maintenance within the genome. In this review, we discuss the processes that lead to satellite DNA copy number instability, and the importance of mechanisms to manage the potential negative effects of instability. Satellite DNA is vulnerable to challenges during replication and repair, since it forms difficult-to-process secondary structures and its homology within tandem arrays can result in various types of recombination. Satellite DNA instability may be managed by DNA or chromatin-binding proteins ensuring proper nuclear localization and repair, or by proteins that process aberrant structures that satellite DNAs tend to form. We also discuss the pattern of satellite DNA mutations from recent mutation accumulation (MA) studies that have tracked changes in satellite DNA for up to 1000 generations with minimal selection. Finally, we highlight examples of satellite evolution from studies that have characterized satellites across millions of years of Drosophila fruit fly evolution, and discuss possible ways that selection might act on the satellite DNA composition.

rDNA magnification is a unique feature of germline stem cells.

Ribosomal DNA (rDNA) encodes ribosomal RNA and exists as tandem repeats of hundreds of copies in the eukaryotic genome to meet the high demand of ribosome biogenesis. Tandemly repeated DNA elements are inherently unstable; thus, mechanisms must exist to maintain rDNA copy number (CN), in particular in the germline that continues through generations. A phenomenon called rDNA magnification was discovered over 50 y ago in Drosophila as a process that recovers the rDNA CN on chromosomes that harbor minimal CN. Our recent studies indicated that rDNA magnification is the mechanism to maintain rDNA CN under physiological conditions to counteract spontaneous CN loss that occurs during aging. Our previous studies that explored the mechanism of rDNA magnification implied that asymmetric division of germline stem cells (GSCs) may be particularly suited to achieve rDNA magnification. However, it remains elusive whether GSCs are the unique cell type that undergoes rDNA magnification or differentiating germ cells are also capable of magnification. In this study, we provide empirical evidence that suggests that rDNA magnification operates uniquely in GSCs, but not in differentiating germ cells. We further provide computer simulation that suggests that rDNA magnification is only achievable through asymmetric GSC divisions. We propose that despite known plasticity and transcriptomic similarity between GSCs and differentiating germ cells, GSCs' unique ability to divide asymmetrically serves a critical role of maintaining rDNA CN through generations, supporting germline immortality.

A maternally programmed intergenerational mechanism enables male offspring to make piRNAs from Y-linked precursor RNAs in Drosophila.

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.

Asymmetric Stem Cell Division and Germline Immortality.

Germ cells are the only cell type that is capable of transmitting genetic information to the next generation, which has enabled the continuation of multicellular life for the last 1.5 billion years. Surprisingly little is known about the mechanisms supporting the germline's remarkable ability to continue in this eternal cycle, termed germline immortality. Even unicellular organisms age at a cellular level, demonstrating that cellular aging is inevitable. Extensive studies in yeast have established the framework of how asymmetric cell division and gametogenesis may contribute to the resetting of cellular age. This review examines the mechanisms of germline immortality-how germline cells reset the aging of cells-drawing a parallel between yeast and multicellular organisms.

The retrotransposon R2 maintains Drosophila ribosomal DNA repeats.

Ribosomal DNA (rDNA) loci contain hundreds of tandemly repeated copies of ribosomal RNA genes needed to support cellular viability. This repetitiveness makes it highly susceptible to copy number (CN) loss due to intrachromatid recombination between rDNA copies, threatening multigenerational maintenance of rDNA. How this threat is counteracted to avoid extinction of the lineage has remained unclear. Here, we show that the rDNA-specific retrotransposon R2 is essential for restorative rDNA CN expansion to maintain rDNA loci in the Drosophila male germline. The depletion of R2 led to defective rDNA CN maintenance, causing a decline in fecundity over generations and eventual extinction. We find that double-stranded DNA breaks created by the R2 endonuclease, a feature of R2's rDNA-specific retrotransposition, initiate the process of rDNA CN recovery, which relies on homology-dependent repair of the DNA break at rDNA copies. This study reveals that an active retrotransposon provides an essential function for its host, contrary to transposable elements' reputation as entirely selfish. These findings suggest that benefiting host fitness can be an effective selective advantage for transposable elements to offset their threat to the host, which may contribute to retrotransposons' widespread success throughout taxa.

Analysis of Gene Expression Patterns and RNA Localization by Fluorescence in Situ Hybridization in Whole Mount Drosophila Testes.

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.

Derepression of Y-linked multicopy protamine-like genes interferes with sperm nuclear compaction in D. melanogaster.

Across species, sperm maturation involves the dramatic reconfiguration of chromatin into highly compact nuclei that enhance hydrodynamic ability and ensure paternal genomic integrity. This process is mediated by the replacement of histones by sperm nuclear basic proteins, also referred to as protamines. In humans, a carefully balanced dosage between two known protamine genes is required for optimal fertility. However, it remains unknown how their proper balance is regulated and how defects in balance may lead to compromised fertility. Here, we show that a nucleolar protein, modulo, a homolog of nucleolin, mediates the histone-to-protamine transition during Drosophila spermatogenesis. We find that modulo mutants display nuclear compaction defects during late spermatogenesis due to decreased expression of autosomal protamine genes (including Mst77F) and derepression of Y-linked multicopy Mst77F homologs (Mst77Y), leading to the mutant's known sterility. Overexpression of Mst77Y in a wild-type background is sufficient to cause nuclear compaction defects, similar to modulo mutant, indicating that Mst77Y is a dominant-negative variant interfering with the process of histone-to-protamine transition. Interestingly, ectopic overexpression of Mst77Y caused decompaction of X-bearing spermatids nuclei more frequently than Y-bearing spermatid nuclei, although this did not greatly affect the sex ratio of offspring. We further show that modulo regulates these protamine genes at the step of transcript polyadenylation. We conclude that the regulation of protamines mediated by modulo, ensuring the expression of functional ones while repressing dominant-negative ones, is critical for male fertility.

Fusome topology and inheritance during insect gametogenesis.

From insects to mammals, oocytes and sperm develop within germline cysts comprising cells connected by intercellular bridges (ICBs). In numerous insects, formation of the cyst is accompanied by growth of the fusome-a membranous organelle that permeates the cyst. Fusome composition and function are best understood in Drosophila melanogaster: during oogenesis, the fusome dictates cyst topology and size and facilitates oocyte selection, while during spermatogenesis, the fusome synchronizes the cyst's response to DNA damage. Despite its distinct and sex-specific roles during insect gametogenesis, elucidating fusome growth and inheritance in females and its structure and connectivity in males has remained challenging. Here, we take advantage of advances in three-dimensional (3D) confocal microscopy and computational image processing tools to reconstruct the topology, growth, and distribution of the fusome in both sexes. In females, our experimental findings inform a theoretical model for fusome assembly and inheritance and suggest that oocyte selection proceeds through an 'equivalency with a bias' mechanism. In males, we find that cell divisions can deviate from the maximally branched pattern observed in females, leading to greater topological variability. Our work consolidates existing disjointed experimental observations and contributes a readily generalizable computational approach for quantitative studies of gametogenesis within and across species.

Emergent dynamics of adult stem cell lineages from single nucleus and single cell RNA-Seq of Drosophila testes.

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.

Ribosomal DNA Instability as a Potential Cause of Karyotype Evolution.

Karyotype refers to the configuration of the genome into a set of chromosomes. The karyotype difference between species is expected to impede various biological processes, such as chromosome segregation and meiotic chromosome pairing, potentially contributing to incompatibility. Karyotypes can rapidly change between closely related species and even among populations of the same species. However, the forces driving karyotype evolution are poorly understood. Here we describe a unique karyotype of a Drosophila melanogaster strain isolated from the Seychelles archipelago. This strain has lost the ribosomal DNA (rDNA) locus on the X chromosome. Because the Y chromosome is the only other rDNA-bearing chromosome, all females carry at least one Y chromosome as the source of rDNA. Interestingly, we found that the strain also carries a truncated Y chromosome (YS) that is stably maintained in the population despite its inability to support male fertility. Our modeling and cytological analysis suggest that the Y chromosome has a larger negative impact on female fitness than the YS chromosome. Moreover, we generated an independent strain that lacks X rDNA and has a karyotype of XXY females and XY males. This strain quickly evolved multiple karyotypes: two new truncated Y chromosomes (similar to YS), as well as two independent X chromosome fusions that contain the Y-derived rDNA fragment, eliminating females' dependence on the Y chromosome. Considering that Robertsonian fusions frequently occur at rDNA loci in humans, we propose that rDNA loci instability may be one of driving forces of karyotype evolution.

Nonrandom sister chromatid segregation mediates rDNA copy number maintenance in Drosophila.

Although considered to be exact copies of each other, sister chromatids can segregate nonrandomly in some cases. For example, sister chromatids of the X and Y chromosomes segregate nonrandomly during asymmetric division of male germline stem cells (GSCs) in Drosophila melanogaster. Here, we demonstrate that the ribosomal DNA (rDNA) loci, which are located on the X and Y chromosomes, and an rDNA binding protein Indra are required for nonrandom sister chromatid segregation (NRSS). We provide the evidence that NRSS, following unequal sister chromatid exchange, is a mechanism by which GSCs recover rDNA copy number, counteracting the spontaneous copy number loss that occurs during aging. Our study reveals an unexpected role for NRSS in maintaining germline immortality through maintenance of a vulnerable genomic element, rDNA.

The regulation and potential functions of intronic satellite DNA.

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.

Stem cell niche signaling goes both ways.

Stem cell niches are well-characterized factories of signaling information, but niche cells themselves also rely on their neighbors for fate maintenance. In this issue of Developmental Cell, Herrera et al. reveal bi-directional communication between Drosophila testis niche "hub" cells and somatic cyst stem cells.

Defective Satellite DNA Clustering into Chromocenters Underlies Hybrid Incompatibility in Drosophila.

Although rapid evolution of pericentromeric satellite DNA repeats is theorized to promote hybrid incompatibility (HI) (Yunis and Yasmineh 1971; Henikoff et al. 2001; Ferree and Barbash 2009; Sawamura 2012; Jagannathan and Yamashita 2017), how divergent repeats affect hybrid cells remains poorly understood. Recently, we demonstrated that sequence-specific DNA-binding proteins cluster satellite DNA from multiple chromosomes into "chromocenters," thereby bundling chromosomes to maintain the entire genome in a single nucleus (Jagannathan et al. 2018, 2019). Here, we show that ineffective clustering of divergent satellite DNA in the cells of Drosophila hybrids results in chromocenter disruption, associated micronuclei formation, and tissue atrophy. We further demonstrate that previously identified HI factors trigger chromocenter disruption and micronuclei in hybrids, linking their function to a conserved cellular process. Together, we propose a unifying framework that explains how the widely observed satellite DNA divergence between closely related species can cause reproductive isolation.