Ribosome biogenesis drives cell growth and proliferation, but mechanisms that modulate this process within specific lineages remain poorly understood. Here, we identify a Drosophila RNA polymerase I ...(Pol I) regulatory complex composed of Under-developed (Udd), TAF1B, and a TAF1C-like factor. Disruption of udd or TAF1B results in reduced ovarian germline stem cell (GSC) proliferation. Female GSCs display high levels of ribosomal RNA (rRNA) transcription, and Udd becomes enriched in GSCs relative to their differentiating daughters. Increasing Pol I transcription delays differentiation, whereas reducing rRNA production induces both morphological changes that accompany multicellular cyst formation and specific decreased expression of the bone morphogenetic protein (BMP) pathway component Mad. These findings demonstrate that modulating rRNA synthesis fosters changes in the cell fate, growth, and proliferation of female Drosophila GSCs and their daughters.
Single-stranded circular RNAs (circRNAs), generated through 'backsplicing', occur more extensively than initially anticipated. The possible functions of the vast majority of circRNAs remain unknown. ...Virus-derived circRNAs have recently been described in gamma-herpesviruses. We report that oncogenic human papillomaviruses (HPVs) generate circRNAs, some of which encompass the E7 oncogene (circE7). HPV16 circE7 is detectable by both inverse RT-PCR and northern blotting of HPV16-transformed cells. CircE7 is N
-methyladenosine (m
A) modified, preferentially localized to the cytoplasm, associated with polysomes, and translated to produce E7 oncoprotein. Specific disruption of circE7 in CaSki cervical carcinoma cells reduces E7 protein levels and inhibits cancer cell growth both in vitro and in tumor xenografts. CircE7 is present in TCGA RNA-Seq data from HPV-positive cancers and in cell lines with only episomal HPVs. These results provide evidence that virus-derived, protein-encoding circular RNAs are biologically functional and linked to the transforming properties of some HPV.
Information exchange between different cells makes multicellular life possible. Signaling between cells can occur over long distances, as in the case of hormone signaling, or it can take place over ...short distances between immediately juxtaposed neighbors, as in the case of stem cell-niche signaling. The ability of signal-sending and -receiving cells to communicate with one another in a specific manner is of paramount importance in the proper development and function of tissues. Growing evidence indicates that different cellular protrusions help to achieve specificity in signaling that occurs between distinct cell types. Here, we focus on new roles for cellular protrusions in cell-to-cell communication, drawing special attention to how stem cells use specialized extensions to promote reception of self-renewing signals emanating from the niche.
Emerging evidence suggests that ribosome heterogeneity may have important functional consequences in the translation of specific mRNAs within different cell types and under various conditions. ...Ribosome heterogeneity comes in many forms, including post-translational modification of ribosome proteins (RPs), absence of specific RPs and inclusion of different RP paralogs. The Drosophila genome encodes two RpS5 paralogs: RpS5a and RpS5b. While RpS5a is ubiquitously expressed, RpS5b exhibits enriched expression in the reproductive system. Deletion of RpS5b results in female sterility marked by developmental arrest of egg chambers at stages 7-8, disruption of vitellogenesis and posterior follicle cell (PFC) hyperplasia. While transgenic rescue experiments suggest functional redundancy between RpS5a and RpS5b, molecular, biochemical and ribo-seq experiments indicate that RpS5b mutants display increased rRNA transcription and RP production, accompanied by increased protein synthesis. Loss of RpS5b results in microtubule-based defects and in mislocalization of Delta and Mindbomb1, leading to failure of Notch pathway activation in PFCs. Together, our results indicate that germ cell-specific expression of RpS5b promotes proper egg chamber development by ensuring the homeostasis of functional ribosomes.
RNA-binding Fox (Rbfox) proteins have well-established roles in regulating alternative splicing, but specific Rbfox isoforms lack nuclear localization signals and accumulate in the cytoplasm. The ...potential splicing-independent functions of these proteins remain unknown. Here we demonstrate that cytoplasmic Drosophila Rbfox1 regulates germ cell development and represses the translation of mRNAs containing (U)GCAUG elements within their 3′UTRs. During germline cyst differentiation, Rbfox1 targets pumilio mRNA for destabilization and translational silencing, thereby promoting germ cell development. Mis-expression of pumilio results in the formation of germline tumors, which contain cysts that break down and dedifferentiate back to single, mitotically active cells. Together, these results reveal that cytoplasmic Rbfox family members regulate the translation of specific target mRNAs. In the Drosophila ovary, this activity provides a genetic barrier that prevents germ cells from reverting back to an earlier developmental state. The finding that Rbfox proteins regulate mRNA translation has implications for Rbfox-related diseases.
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•Drosophila cytoplasmic Rbfox1 regulates ovarian germ cell differentiation•Rbfox1 regulates mRNA translation in a 3′UTR-dependent manner•Rbfox1 represses the translation of pumilio mRNA•Ectopic expression of pumilio or loss of Rbfox1 blocks germ cell differentiation
Nuclear Rbfox proteins regulate alternative splicing. Carreira-Rosario et al. now provide evidence that cytoplasmic Rbfox proteins regulate the translation of specific mRNAs, including the Drosophila germ cell maintenance factor pumilio. Loss of Rbfox1 in the Drosophila ovary leads to an expansion of Pumilio protein expression, disrupting normal germ cell differentiation.
The regulation of mRNA translation, both globally and at the level of individual transcripts, plays a central role in the development and function of germ cells across species. Genetic studies using ...flies, worms, zebrafish and mice have highlighted the importance of specific RNA binding proteins in driving various aspects of germ cell formation and function. Many of these mRNA binding proteins, including Pumilio, Nanos, Vasa and Dazl have been conserved through evolution, specifically mark germ cells, and carry out similar functions across species. These proteins typically influence mRNA translation by binding to specific elements within the 3' untranslated region (UTR) of target messages. Emerging evidence indicates that the global regulation of mRNA translation also plays an important role in germ cell development. For example, ribosome biogenesis is often regulated in a stage specific manner during gametogenesis. Moreover, oocytes need to produce and store a sufficient number of ribosomes to support the development of the early embryo until the initiation of zygotic transcription. Accumulating evidence indicates that disruption of mRNA translation regulatory mechanisms likely contributes to infertility and reproductive aging in humans. These findings highlight the importance of gaining further insights into the mechanisms that control mRNA translation within germ cells. Future work in this area will likely have important impacts beyond germ cell biology.
Specialized microenvironments called niches keep stem cells in an undifferentiated and self-renewing state. Dedicated stromal cells form niches by producing a variety of factors that act directly on ...stem cells. The size and signaling output of niches must be finely tuned to ensure proper tissue homeostasis. Although advances have been made in identifying factors that promote niche cell fate, the mechanisms that restrict niche cell formation during development and limit niche signaling output in adults remain poorly understood. Here, we show that the histone lysine-specific demethylase 1 (Lsd1) regulates the size of the germline stem cell (GSC) niche in Drosophila ovaries. GSC maintenance depends on bone morphogenetic protein (BMP) signals produced by a small cluster of cap cells located at the anterior tip of the germarium. Lsd1 null mutant ovaries carry small germline tumors containing an expanded number of GSC-like cells with round fusomes that display ectopic BMP signal responsiveness away from the normal niche. Clonal analysis and cell type-specific rescue experiments demonstrate that Lsd1 functions within the escort cells (ECs) that reside immediately adjacent to cap cells and prevents them from ectopically producing niche-specific signals. Temporally restricted gene knockdown experiments suggest that Lsd1 functions both during development, to specify EC fate, and in adulthood, to prevent ECs from forming ectopic niches independent of changes in cell fate. Further analysis shows that Lsd1 functions to repress decapentaplegic (dpp) expression in adult germaria. The role of Lsd1 in regulating niche-specific signals may have important implications for understanding how disruption of its mammalian homolog contributes to cancer and metastasis.
Glioblastoma is the most common and aggressive type of cancer in the brain; its poor prognosis is often marked by reoccurrence due to resistance to the chemotherapeutic agent temozolomide, which is ...triggered by an increase in the expression of DNA repair enzymes such as MGMT. The poor prognosis and limited therapeutic options led to studies targeted at understanding specific vulnerabilities of glioblastoma cells. Metabolic adaptations leading to increased synthesis of nucleotides by de novo biosynthesis pathways are emerging as key alterations driving glioblastoma growth. In this study, we show that enzymes necessary for the de novo biosynthesis of pyrimidines, DHODH and UMPS, are elevated in high grade gliomas and in glioblastoma cell lines. We demonstrate that DHODH's activity is necessary to maintain ribosomal DNA transcription (rDNA). Pharmacological inhibition of DHODH with the specific inhibitors brequinar or ML390 effectively depleted the pool of pyrimidines in glioblastoma cells grown in vitro and in vivo and impaired rDNA transcription, leading to nucleolar stress. Nucleolar stress was visualized by the aberrant redistribution of the transcription factor UBF and the nucleolar organizer nucleophosmin 1 (NPM1), as well as the stabilization of the transcription factor p53. Moreover, DHODH inhibition decreased the proliferation of glioblastoma cells, including temozolomide-resistant cells. Importantly, the addition of exogenous uridine, which reconstitutes the cellular pool of pyrimidine by the salvage pathway, to the culture media recovered the impaired rDNA transcription, nucleolar morphology, p53 levels, and proliferation of glioblastoma cells caused by the DHODH inhibitors. Our in vivo data indicate that while inhibition of DHODH caused a dramatic reduction in pyrimidines in tumor cells, it did not affect the overall pyrimidine levels in normal brain and liver tissues, suggesting that pyrimidine production by the salvage pathway may play an important role in maintaining these nucleotides in normal cells. Our study demonstrates that glioblastoma cells heavily rely on the de novo pyrimidine biosynthesis pathway to generate ribosomal RNA (rRNA) and thus, we identified an approach to inhibit ribosome production and consequently the proliferation of glioblastoma cells through the specific inhibition of the de novo pyrimidine biosynthesis pathway.
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