Alternative Splicing Research Articles

Integration of mRNA and miRNA Analysis Sheds New Light on the Muscle Response to Heat Stress in Spotted Sea Bass (Lateolabrax maculatus)

Temperature is a crucial environmental factor for fish. Elevated temperatures trigger various physiological and molecular responses designed to maintain internal environmental homeostasis and ensure the proper functioning of the organism. In this study, we measured biochemical parameters and performed mRNA–miRNA integrated transcriptomic analysis to characterize changes in gene expression profiles in the muscle tissue of spotted sea bass (Lateolabrax maculatus) under heat stress. The measurement of biochemical parameters revealed that the activities of nine biochemical enzymes (ALP, γ-GT, AST, GLU, CK, ALT, TG, LDH and TC) were significantly affected to varying degrees by elevated temperatures. A total of 1940 overlapping differentially expressed genes (DEGs) were identified among the five comparisons in the muscle tissue after heat stress. Protein–protein interaction (PPI) analysis of DEGs indicated that heat shock protein genes (HSPs) were deeply involved in the response to heat stress. In addition, we detected 462 differential alternative splicing (DAS) events and 618 DAS genes, which are closely associated with sarcomere assembly in muscle, highlighting the role of alternative splicing in thermal response regulation. Moreover, 32 differentially expressed miRNAs (DEMs) were identified in response to heat stress, and 599 DEGs were predicted as potential target genes of those DEMs, generating 846 DEG–DEM negative regulatory pairs potentially associated with thermal response. Function enrichment analysis of the target genes suggested that lipid metabolism-related pathways and genes were regulated by miRNAs. By analyzing PPIs of target genes, we identified 28 key negative regulatory pairs, including 13 miRNAs (such as lma-miR-122, lma-miR-200b-5p and novel-miR-444) and 15 target genes (such as hspa13, dnaja1, and dnajb1a). This study elucidates the molecular mechanisms of response to high-temperature stress and offers valuable information for the selection and breeding of heat-tolerant strains of spotted sea bass.

Establishing a dominant early larval sex-selection strain in the Asian malaria vector Anopheles stephensi

BackgroundGenetic biocontrol interventions targeting mosquito-borne diseases require the release of male mosquitoes exclusively, as only females consume blood and transmit pathogens. Releasing only males eliminates the risk of increasing mosquito bites and spreading pathogens while enabling effective population control. The aim of this study is to develop robust sex-sorting methods for early larval stages in mosquitoes, enabling scalable male-only releases for genetic biocontrol interventions.MethodsTo address the challenge of sex-sorting in the Asian malaria vector Anopheles stephensi, we engineer Sexing Element Produced by Alternative RNA-splicing of a Transgenic Observable Reporter (SEPARATOR). This dominant fluorescent-based method, previously proven effective in Aedes aegypti, exploits sex-specific alternative splicing of a reporter to ensure exclusive male-specific expression early in development. The sex-specific alternative RNA splicing of the doublesex gene was selected as a target for engineering SEPARATOR due to its evolutionary conservation in insects. To expand SEPARATOR’s applicability for genetic sexing, we assessed the cross-species sex-specific RNA splicing activity of the An. gambiae doublesex (AngDsx) splicing module in An. stephensi. Male-specific enhanced green fluorescent protein (EGFP) expression was verified throughout the mosquito life cycle using a fluorescent stereomicroscope.ResultsOur results confirm that SEPARATOR regulates male-specific EGFP expression in An. stephensi and enables reliable positive male selection from the first instar larval stages. Molecular analysis demonstrates that male-specific EGFP expression is dependent on doublesex sex-specific splicing events. Additionally, the splicing module from An. gambiae operates effectively in An. stephensi, demonstrating evolutionary conservation in sex-specific splicing events between these species.ConclusionsSEPARATOR’s independence from sex-chromosome linkage provides resistance to breakage that could be mediated by meiotic recombination and chromosomal rearrangements, making it highly suitable for mass male releases. By enabling precise male selection from the first instar larval stages, SEPARATOR represents a significant advancement that will aid in the genetic biocontrol for Anopheles mosquitoes.Graphical

Nanopore-Based Sequencing of the Full-Length Transcriptome of Male and Female Cleavage-Stage Embryos of the Chinese Mitten Crab (Eriocheir sinensis)

The cleavage stage plays a crucial role in embryo development, characterized by a swift surge in cell proliferation alongside the accurate genetic material transmission to offspring. To delve into the characteristics of sex development during the cleavage stage of embryos, we generated the full-length transcriptome of Eriocheir sinensis male and female cleavage-stage embryos using Oxford Nanopore Technologies (ONT). Notably, this investigation represents the first sequencing effort distinguishing between genders in E. sinensis embryos. In the transcriptome structure analysis, male and female cleavage-stage embryos, while not clustered, exhibited a comparable frequency of alternative splicing (AS) occurrences. We also successfully identified 2875 transcription factors (TFs). The quantitative analysis showed the top 150 genes, in which the highly expressed genes in male embryos predominantly related to protein synthesis and metabolism. Further investigation unveiled 500 differentially expressed genes (DEGs), of which 7 male-biased ribosomal protein genes (RPGs) were particularly noteworthy and further confirmed. These analyses suggest that there may be a more active protein synthesis process in male E. sinensis cleavage-stage embryos. Furthermore, among the 2875 identified TFs, we predicted that 18 TFs could regulate the differentially expressed RPGs, with most TFs belonging to the zf-C2H2 and Homeobox families, which are crucial for embryonic development. During the cleavage stage of E. sinensis, the differential RPGs between genders were intricately linked to energy metabolism. We proposed that these RPGs exert regulatory effects on gene expression in E. sinensis, thereby regulating the difference of development between male and females. Our research sheds light on the developmental mechanisms of E. sinensis during the embryo stage and establishes a groundwork for a deeper understanding of sex development in E. sinensis. The results also provide comprehensive full-length transcriptome data for future gene expression and genetic studies in E. sinensis.

XRCC1 is linked to poor prognosis in adenocarcinoma of the esophagogastric junction after radiotherapy: transcriptome and alternative splicing events analysis

XRCC1 is linked to poor prognosis in adenocarcinoma of the esophagogastric junction after radiotherapy: transcriptome and alternative splicing events analysis

IMMU-49. TARGETING ALTERNATIVE SPLICING VARIANTS TO STIMULATE ANTI-TUMOR IMMUNITY IN A NOVEL SYNGENEIC MOUSE MODEL OF DIFFUSE HEMISPHERIC GLIOMA, H3 G34-MUTANT

Abstract The prognosis for pediatric high-grade gliomas associated with mutations in the H3-A3 histone gene is very poor. To investigate whether peptide-pulsed dendritic cells (DC) together with checkpoint blockade might be a potential treatment modality for diffuse hemispheric glioma, H3 G34-mutant (DHG), we have developed a novel mouse model, and utilized a novel bioinformatics pipeline to identify tumor-associated peptide antigens (TAs). We used the RCAS/tv-A system to target the expression of H3/G34R and PDGFb, and to knock out p53 in neural progenitors in transgenic C57BL/6 neonatal mice. Three independent cell lines were obtained that expressed transcripts associated with oligodendrocyte and interneuron lineages, and formed lethal tumors after intracranial implantation. To identify TAs, we implemented a new computational workflow to identify nonapeptides generated from alternative splicing of mRNAs that were highly enriched in the tumor transcriptomes of two DHG patients, and predicted to bind the HLA-A*2:01 class I allele carried by these patients. Three of the candidate peptides were conserved in the mouse and also predicted to bind the H2-Kb MHC I allele carried by C57/BL6 mice. Using our syngeneic mouse model, we showed that DCs pulsed with these peptides together with anti-PD1 mAb provided a significant survival benefit compared with checkpoint inhibitor and DCs pulsed with peptides that were not predicted to bind the H2-Kb allele. Splenocytes isolated from successfully treated mice were analyzed through bulk TCR sequencing, and in comparison with control mice and publicly-accessible TCR libraries, 6 highly-represented TCR clonotypes were identified only in treated mice. In summary, we demonstrate that effective TAs can be extracted from RNAseq data, and can be targeted with DC vaccination to generate a systemic anti-tumor cytotoxic T cell response to eliminate DHG in a novel immunocompetent mouse model.

The Intricate Functional Networks of Pre-mRNA Alternative Splicing in Mammalian Spermatogenesis

Spermatogenesis is a highly coordinated process that requires the precise expression of specific subsets of genes in different types of germ cells, controlled both temporally and spatially. Among these genes, those that can exert an indispensable influence in spermatogenesis via participating in alternative splicing make up the overwhelming majority. mRNA alternative-splicing (AS) events can generate various isoforms with distinct functions from a single DNA sequence, based on specific AS codes. In addition to enhancing the finite diversity of the genome, AS can also regulate the transcription and translation of certain genes by directly binding to their cis-elements or by recruiting trans-elements that interact with consensus motifs. The testis, being one of the most complex tissue transcriptomes, undergoes unparalleled transcriptional and translational activity, supporting the dramatic and dynamic transitions that occur during spermatogenesis. Consequently, AS plays a vital role in producing an extensive array of transcripts and coordinating significant changes throughout this process. In this review, we summarize the intricate functional network of alternative splicing in spermatogenesis based on the integration of current research findings.

AtDGCR14L contributes to salt‐stress tolerance via regulating pre‐mRNA splicing in Arabidopsis

SUMMARYIn plants, pre‐mRNA alternative splicing has been demonstrated to be a crucial tier that regulates gene expression in response to salt stress. However, the underlying mechanisms remain elusive. Here, we studied the roles of DIGEORGE‐SYNDROME CRITICAL REGION 14‐like (AtDGCR14L) in regulating pre‐mRNA splicing and salt stress tolerance. We discovered that Arabidopsis AtDGCR14L is required for maintaining plant salt stress tolerance and the constitutively spliced and active isoforms of important stress‐ and/or abscisic acid (ABA)‐responsive genes. We also identified the interaction between AtDGCR14L and splicing factor U1‐70k, which needs a highly conserved three amino acid (TWG) motif in DGCR14. Different from wild‐type AtDGCR14L, the overexpression of the TWG‐substituted AtDGCR14L mutant did not change salt stress tolerance or pre‐mRNA splicing of stress/ABA‐responsive genes. Additionally, SWITCH3A (SWI3A) is a core subunit of the SWI/SUCROSE NONFERMENTING (SWI/SNF) chromatin‐remodeling complexes. We found that SWI3A, whose splicing depends on AtDGCR14L, actively enhances salt stress tolerance. These results revealed that AtDGCR14L may play an essential role in crosstalk between plant salt‐stress response and pre‐mRNA splicing mechanisms. We also unveiled the potential role of SWI3A in controlling salt stress tolerance. The TWG motif in the intrinsically disordered region of AtDGCR14L is highly conserved and crucial for DGCR14 functions.

ZmHSFA2B self‐regulatory loop is critical for heat tolerance in maize

SummaryThe growth and development of maize (Zea mays L.) are significantly impeded by prolonged exposure to high temperatures. Heat stress transcription factors (HSFs) play crucial roles in enabling plants to detect and respond to elevated temperatures. However, the genetic mechanisms underlying the responses of HSFs to heat stress in maize remain unclear. Thus, we aimed to investigate the role of ZmHSFA2B in regulating heat tolerance in maize. Here, we report that ZmHSFA2B has two splicing variants, ZmHSFA2B‐I and ZmHSFA2B‐II. ZmHSFA2B‐I encodes full‐length ZmHSFA2B (ZmHSFA2B‐I), whereas ZmHSFA2B‐II encodes a truncated ZmHSFA2B (ZmHSFA2B‐II). Overexpression of ZmHSFA2B‐I improved heat tolerance in maize and Arabidopsis thaliana, but it also resulted in growth retardation as a side effect. RNA‐sequencing and CUT&Tag analyses identified ZmMBR1 as a putative target of ZmHSFA2B‐I. Overexpression of ZmMBR1 also enhanced heat tolerance in Arabidopsis. ZmHSFA2B‐II was primarily synthesized in response to heat stress and competitively interacted with ZmHSFA2B‐I. This interaction consequently reduced the DNA‐binding activities of ZmHSFA2B‐I homodimers to the promoter of ZmMBR1. Subsequent investigations indicate that ZmHSFA2B‐II limits the transactivation and tempers the function of ZmHSFA2B‐I, thereby reducing the adverse effects of excessive ZmHSFA2B‐I accumulation. Based on these observations, we propose that the alternative splicing of ZmHSFA2B generates a self‐regulatory loop that fine‐tunes heat stress response in maize.

BCAS2 and hnRNPH1 orchestrate alternative splicing for DNA double-strand break repair and synapsis in meiotic prophase I

Understanding the intricacies of homologous recombination during meiosis is crucial for reproductive biology. However, the role of alternative splicing (AS) in DNA double-strand breaks (DSBs) repair and synapsis remains elusive. In this study, we investigated the impact of conditional knockout (cKO) of the splicing factor gene Bcas2 in mouse germ cells, revealing impaired DSBs repair and synapsis, resulting in non-obstructive azoospermia (NOA). Employing crosslinking immunoprecipitation and sequencing (CLIP-seq), we globally mapped BCAS2 binding sites in the testis, uncovering its predominant association with 5’ splice sites (5’SS) of introns and a preference for GA-rich regions. Notably, BCAS2 exhibited direct binding and regulatory influence on Trp53bp1 (codes for 53BP1) and Six6os1 through AS, unveiling novel insights into DSBs repair and synapsis during meiotic prophase I. Furthermore, the interaction between BCAS2, hnRNPH1, and SRSF3 was discovered to orchestrate Trp53bp1 expression via AS, underscoring its role in meiotic prophase I DSBs repair. In summary, our findings delineate the indispensable role of BCAS2-mediated post-transcriptional regulation in DSBs repair and synapsis during male meiosis. This study provides a comprehensive framework for unraveling the molecular mechanisms governing the post-transcriptional network in male meiosis, contributing to the broader understanding of reproductive biology.

A second RUBCN variant associated with epileptic encephalopathy and neurodevelopmental delay

ABSTRACTThe RUBCN gene encodes a widely expressed protein called Rubicon, the main function of which is to negatively regulate macroautophagy. A single homozygous pathogenic variant of the RUBCN gene has been reported to date in two unrelated consanguineous Saudi families with spinocerebellar ataxia autosomal recessive 15 (OMIM#613516). This variant is responsible for the deletion of the highly conserved Rubicon Homology (RH) domain, which is important for the colocalization of Rubicon with Rab7 in the late endosome. In this work, we describe a female patient with childhood‐onset epileptic encephalopathy and neurodevelopmental delay carrying a novel homozygous variant in RUBCN (NM_014687.3: c.2126 + 1G>A). A functional study of the RNA revealed that this variant completely abolishes the consensus donor site at the exon 14/intron 14 junction, resulting in the absence of expression of the reference transcript. Two alternative transcripts were expressed: a major transcript resulting from activation of an alternative exonic splice site and a minor transcript with skipping of exon 14. The two alternative transcripts lead to a shift in the reading frame introducing a premature stop codon. The resulting truncated protein lacks the RH domain, which may lead to defective endosomal trafficking as previously described. To our best knowledge, this is the first report of an impairment of RUBCN caused by a splice variant.

A WFS1 variant disrupting acceptor splice site uncovers the impact of alternative splicing on beta cell apoptosis in a patient with Wolfram syndrome

A WFS1 variant disrupting acceptor splice site uncovers the impact of alternative splicing on beta cell apoptosis in a patient with Wolfram syndrome

Alternative production of pro-death Bax∆2 protein via ribosomal frameshift in Alzheimer’s disease

Pro-death Bax family member, Bax∆2, forms protein aggregates in Alzheimer’s disease neurons and causes stress granule-mediated neuronal death. Production of Bax∆2 originated from two events: alternative splicing of Bax exon 2 and a microsatellite mutation (a deletion from poly guanines, G8 to G7). Each event alone leads to a reading frameshift and premature termination. While Bax exon 2 alternative splicing is common in Alzheimer’s brains, the G7 mutation is not, which is inconsistent with the high Bax∆2 protein levels detected in clinical samples. Here, we report an alternative mechanism to produce Bax∆2 protein in the absence of the G7 mutation. Using dual-tag systems, we showed that a ribosomal frameshift (RFS) can compensate the lack of G7 mutation in translating Bax∆2 protein. Intriguingly, the microsatellite poly G repeat is neither essential nor the site for the RFS. However, disruption of the poly G sequence impaired the RFS, potentially due to alteration of the local RNA structure. Using immunoprecipitation-mass spectrometry, we pinpointed the RFS site at 15 base pairs upstream of the microsatellite. These results uncover an alternative mechanism for generating functional Bax∆2-subfamily isoforms, highlighting the production plasticity of Bax family isoforms and unveiling potential new therapeutic targets for Alzheimer’s disease.

Gene Expression and Alternative Splicing Analysis in a Large-Scale Multiple Sclerosis Study

Gene Expression and Alternative Splicing Analysis in a Large-Scale Multiple Sclerosis Study

Functional implications of the exon 9 splice insert in GluK1 kainate receptors

Kainate receptors are key modulators of synaptic transmission and plasticity in the central nervous system. Different kainate receptor isoforms with distinct spatiotemporal expressions have been identified in the brain. The GluK1-1 splice variant receptors, which are abundant in the adult brain, have an extra fifteen amino acids inserted in the amino-terminal domain (ATD) of the receptor resulting from alternative splicing of exon 9. However, the functional implications of this post-transcriptional modification are not yet clear. We employed a multi-pronged approach using cryogenic electron microscopy, electrophysiology, and other biophysical and biochemical tools to understand the structural and functional impact of this splice insert in the extracellular domain of GluK1 receptors. Our study reveals that the splice insert alters the key gating properties of GluK1 receptors and their modulation by the cognate auxiliary Neuropilin and tolloid-like (Neto) proteins 1 and 2. Mutational analysis identified the role of crucial splice residues that influence receptor properties and their modulation. Furthermore, the cryoEM structure of the variant shows that the presence of exon 9 in GluK1 does not affect the receptor architecture or domain arrangement in the desensitized state. Our study thus provides the first detailed structural and functional characterization of GluK1-1a receptors, highlighting the role of the splice insert in modulating receptor properties and their modulation.

Functional implications of the exon 9 splice insert in GluK1 kainate receptors.

Kainate receptors are key modulators of synaptic transmission and plasticity in the central nervous system. Different kainate receptor isoforms with distinct spatiotemporal expressions have been identified in the brain. The GluK1-1 splice variant receptors, which are abundant in the adult brain, have an extra fifteen amino acids inserted in the amino-terminal domain (ATD) of the receptor resulting from alternative splicing of exon 9. However, the functional implications of this post-transcriptional modification are not yet clear. We employed a multi-pronged approach using cryogenic electron microscopy, electrophysiology, and other biophysical and biochemical tools to understand the structural and functional impact of this splice insert in the extracellular domain of GluK1 receptors. Our study reveals that the splice insert alters the key gating properties of GluK1 receptors and their modulation by the cognate auxiliary Neuropilin and tolloid-like (Neto) proteins 1 and 2. Mutational analysis identified the role of crucial splice residues that influence receptor properties and their modulation. Furthermore, the cryoEM structure of the variant shows that the presence of exon 9 in GluK1 does not affect the receptor architecture or domain arrangement in the desensitized state. Our study thus provides the first detailed structural and functional characterization of GluK1-1a receptors, highlighting the role of the splice insert in modulating receptor properties and their modulation.

The Spatial-Temporal Alternative Splicing Profile Reveals the Functional Diversity of FXR1 Isoforms in Myogenesis.

Alternative splicing (AS) is a fundamental mechanism contributing to proteome diversity, yet its comprehensive landscape and regulatory dynamics during skeletal muscle development remain largely unexplored. Here, the temporal AS profiles are investigated during myogenesis in five vertebrates, conducting comprehensive profiling across 27 developmental stages in skeletal muscle and encompassing ten tissues in adult pigs. The analysis reveals a pervasive and evolutionarily conserved pattern of alternative exon usage throughout myogenic differentiation, with hundreds of skipped exons (SEs) showing developmental regulation, particularly within skeletal muscle. Notably, this study identifies a muscle-specific SE (exon 15) within the Fxr1 gene, whose AS generates two dynamically expressed isoforms with distinct functions: the isoform without exon 15 (Fxr1E15 -) regulates myoblasts proliferation, while the isoform incorporating exon 15 (Fxr1E15+) promotes myogenic differentiation and fusion. Transcriptome analysis suggests that specifically knocking-down Fxr1E15+ isoform in myoblasts modulates differentiation by influencing gene expression and splicing of specific targets. The increased inclusion of exon 15 during differentiation is mediated by the binding of Rbm24 to the intron. Furthermore, in vivo experiments indicate that the Fxr1E15+ isoform facilitates muscle regeneration. Collectively, these findings provide a comprehensive resource for AS studies in skeletal muscle development, underscoring the diverse functions and regulatory mechanisms governing distinct Fxr1 isoforms in myogenesis.

RBPWorld for exploring functions and disease associations of RNA-binding proteins across species.

RNA-binding proteins (RBPs) play key roles in a wide range of physiological and pathological processes. To facilitate the investigation of RBP functions and disease associations, we updated the EuRBPDB and renamed it as RBPWorld (http://research.gzsys.org.cn/rbpworld/#/home). Leveraging 998 RNA-binding domains (RBDs) and 87 RNA-binding Proteome (RBPome) datasets, we successfully identified 1 393 413 RBPs from 445 species, including 3030 human RBPs (hRBPs). RBPWorld includes primary RNA targets of diverse hRBPs, as well as potential downstream regulatory pathways and alternative splicing patterns governed by various hRBPs. These insights were derived from analyses of 1515 crosslinking immunoprecipitation-seq datasets and 616 RNA-seq datasets from cells with hRBP gene knockdown or knockout. Furthermore, we systematically identified 929 RBPs with multi-functions, including acting as metabolic enzymes and transcription factors. RBPWorld includes 838 disease-associated hRBPs and 970 hRBPs that interact with 12 disease-causing RNA viruses. This provision allows users to explore the regulatory roles of hRBPs within the context of diseases. Finally, we developed an intuitive interface for RBPWorld, facilitating users easily access all the included data. We believe that RBPWorld will be a valuable resource in advancing our understanding of the biological roles of RBPs across different species.

Alternative splicing is coupled to gene expression in a subset of variably expressed genes

Numerous factors regulate alternative splicing of human genes at a co-transcriptional level. However, how alternative splicing depends on the regulation of gene expression is poorly understood. We leveraged data from the Genotype-Tissue Expression (GTEx) project to show a significant association of gene expression and splicing for 6874 (4.9%) of 141,043 exons in 1106 (13.3%) of 8314 genes with substantially variable expression in nine GTEx tissues. About half of these exons demonstrate higher inclusion with higher gene expression, and half demonstrate higher exclusion, with the observed direction of coupling being highly consistent across different tissues and in external datasets. The exons differ with respect to multiple characteristics and are enriched for hundreds of isoform-specific Gene Ontology annotations suggesting an important regulatory mechanism. Notably, splicing-expression coupling of exons with roles in JUN and MAP kinase signalling could play an important role during cell division.

Transcriptome and Expression Analysis of Glycerol Biosynthesis-Related Genes in Glenea cantor Fabricius (Cerambycidae: Lamiinae)

Glenea cantor Fabricius (Cerambycidae: Lamiinae) is an important pest that damages kapok trees in Southeast Asia with a wide adaptability to temperature. Glycerol is a protectant and energy source for insects in low-temperature environments. However, glycerol biosynthesis-related genes at the molecular level are limited in G. cantor. In this study, the supercooling points and freezing points at different stages were measured, and the cold hardiness of male and female pupae significantly differed. Moreover, a full-length transcriptome of G. cantor was established; glycerol kinase (GK) and glycerol-3-phosphate dehydrogenase (GPDH) genes, which are related to glycerol metabolism, were identified, with a special focus on their expression profiles. A total of 24,476 isoforms stemmed from the full-length transcriptome, along with 568 lncRNAs, 56 transcription factor (TF) families, and 1467 alternative splicing (AS) events. The KEGG pathway enrichment analysis revealed that the isoforms associated with AS were enriched primarily in glycerolipid and glycerophospholipid metabolism. In total, three GK genes and one GPDH gene were identified, and GcGK1 and GcGK3 presented differential sex expression during the pupal stage, which may play a role in thermal adaptability. This study provides a valuable transcriptional database of G. cantor and helps to elucidate the function of glycerol in the thermal adaptation mechanism of longhorn beetles.

Female germline expression of OVO transcription factor bridges Drosophila generations.

OVO is required for female germ cell viability but has no known function in the male germline in Drosophila. ovo is autoregulated by two antagonistic isoforms, OVO-A and OVO-B. All ovo- alleles were created as partial revertants of the antimorphic ovoD1 allele. Creation of new targeted alleles in an ovo+ background indicated that disrupting the germline-specific exon extension of ovo-B leads to an arrested egg chamber phenotype, rather than germ cell death. RNA-seq analysis, including >1K full length cDNAs, indicates that ovo has several unannotated splice variations in the extended exon and a minor population of ovo-B transcripts have an alternative splice. This indicates that classical ovo alleles such as ovoD1rv23, are not truly null for ovo, and are likely to be weak antimorphs. To generate bonafide nulls, we deleted the ovo-A and ovo-B promoters showing that only ovo-B is required for female germ cell viability and there is an early and continual developmental requirement for ovo-B in the female germline. To visualize OVO expression and localization, we endogenously tagged ovo and found nuclear OVO in all differentiating female germ cells throughout oogenesis in adults. We also found that OVO is maternally deposited into the embryo, where it showed nuclear localization in newly formed pole cells. Maternal OVO persisted in embryonic germ cells until zygotic OVO expression was detectable, suggesting that there is continuous nuclear OVO expression in the female germline in the transition from one generation to the next.