Drosophila Doublefault protein coordinates multiple events during male meiosis by controlling mRNA translation.
ABSTRACT: During the extended prophase of Drosophila gametogenesis, spermatocytes undergo robust gene transcription and store many transcripts in the cytoplasm in a repressed state, until translational activation of select mRNAs in later steps of spermatogenesis. Here, we characterize the Drosophila Doublefault (Dbf) protein as a C2H2 zinc-finger protein, primarily expressed in testes, that is required for normal meiotic division and spermiogenesis. Loss of Dbf causes premature centriole disengagement and affects spindle structure, chromosome segregation and cytokinesis. We show that Dbf interacts with the RNA-binding protein Syncrip/hnRNPQ, a key regulator of localized translation in Drosophila We propose that the pleiotropic effects of dbf loss-of-function mutants are associated with the requirement of dbf function for translation of specific transcripts in spermatocytes. In agreement with this hypothesis, Dbf protein binds cyclin B mRNA and is essential for translation of cyclin B in mature spermatocytes.
Project description:In the Drosophila oocyte, mRNA transport and localised translation play a fundamental role in axis determination and germline formation of the future embryo. gurken mRNA encodes a secreted TGF-? signal that specifies dorsal structures, and is localised to the dorso-anterior corner of the oocyte via a cis-acting 64 nucleotide gurken localisation signal. Using GRNA chromatography, we characterised the biochemical composition of the ribonucleoprotein complexes that form around the gurken mRNA localisation signal in the oocyte. We identified a number of the factors already known to be involved in gurken localisation and translational regulation, such as Squid and Imp, in addition to a number of factors with known links to mRNA localisation, such as Me31B and Exu. We also identified previously uncharacterised Drosophila proteins, including the fly homologue of mammalian SYNCRIP/hnRNPQ, a component of RNA transport granules in the dendrites of mammalian hippocampal neurons. We show that Drosophila Syncrip binds specifically to gurken and oskar, but not bicoid transcripts. The loss-of-function and overexpression phenotypes of syncrip in Drosophila egg chambers show that the protein is required for correct grk and osk mRNA localisation and translational regulation. We conclude that Drosophila Syncrip is a new factor required for localisation and translational regulation of oskar and gurken mRNA in the oocyte. We propose that Syncrip/SYNCRIP is part of a conserved complex associated with localised transcripts and required for their correct translational regulation in flies and mammals.
Project description:Localized mRNA translation is thought to play a key role in synaptic plasticity, but the identity of the transcripts and the molecular mechanism underlying their function are still poorly understood. Here, we show that Syncrip, a regulator of localized translation in the Drosophila oocyte and a component of mammalian neuronal mRNA granules, is also expressed in the Drosophila larval neuromuscular junction, where it regulates synaptic growth. We use RNA-immunoprecipitation followed by high-throughput sequencing and qRT-PCR to show that Syncrip associates with a number of mRNAs encoding proteins with key synaptic functions, including msp-300, syd-1, neurexin-1, futsch, highwire, discs large, and ?-spectrin. The protein levels of MSP-300, Discs large, and a number of others are significantly affected in syncrip null mutants. Furthermore, syncrip mutants show a reduction in MSP-300 protein levels and defects in muscle nuclear distribution characteristic of msp-300 mutants. Our results highlight a number of potential new players in localized translation during synaptic plasticity in the neuromuscular junction. We propose that Syncrip acts as a modulator of synaptic plasticity by regulating the translation of these key mRNAs encoding synaptic scaffolding proteins and other important components involved in synaptic growth and function.
Project description:Exosomal miRNA transfer is a mechanism for cell-cell communication that is important in the immune response, in the functioning of the nervous system and in cancer. Syncrip/hnRNPQ is a highly conserved RNA-binding protein that mediates the exosomal partition of a set of miRNAs. Here, we report that Syncrip's amino-terminal domain, which was previously thought to mediate protein-protein interactions, is a cryptic, conserved and sequence-specific RNA-binding domain, designated NURR (N-terminal unit for RNA recognition). The NURR domain mediates the specific recognition of a short hEXO sequence defining Syncrip exosomal miRNA targets, and is coupled by a non-canonical structural element to Syncrip's RRM domains to achieve high-affinity miRNA binding. As a consequence, Syncrip-mediated selection of the target miRNAs implies both recognition of the hEXO sequence by the NURR domain and binding of the RRM domains 5' to this sequence. This structural arrangement enables Syncrip-mediated selection of miRNAs with different seed sequences.
Project description:Spermatogenesis is a differentiation process during which diploid spermatogonial stem cells (SSCs) produce haploid spermatozoa. This highly specialized process is precisely controlled at the transcriptional, posttranscriptional, and translational levels. Here we report that N6-methyladenosine (m6A), an epitranscriptomic mark regulating gene expression, plays essential roles during spermatogenesis. We present comprehensive m6A mRNA methylomes of mouse spermatogenic cells from five developmental stages: undifferentiated spermatogonia, type A1 spermatogonia, preleptotene spermatocytes, pachytene/diplotene spermatocytes, and round spermatids. Germ cell-specific inactivation of the m6A RNA methyltransferase Mettl3 or Mettl14 with Vasa-Cre causes loss of m6A and depletion of SSCs. m6A depletion dysregulates translation of transcripts that are required for SSC proliferation/differentiation. Combined deletion of Mettl3 and Mettl14 in advanced germ cells with Stra8-GFPCre disrupts spermiogenesis, whereas mice with single deletion of either Mettl3 or Mettl14 in advanced germ cells show normal spermatogenesis. The spermatids from double-mutant mice exhibit impaired translation of haploid-specific genes that are essential for spermiogenesis. This study highlights crucial roles of mRNA m6A modification in germline development, potentially ensuring coordinated translation at different stages of spermatogenesis.
Project description:During Drosophila and vertebrate brain development, the conserved transcription factor Prospero/Prox1 is an important regulator of the transition between proliferation and differentiation. Prospero level is low in neural stem cells and their immediate progeny, but is upregulated in larval neurons and it is unknown how this process is controlled. Here, we use single molecule fluorescent in situ hybridisation to show that larval neurons selectively transcribe a long prospero mRNA isoform containing a 15 kb 3’ untranslated region, which is bound in the brain by the conserved RNA-binding protein Syncrip/hnRNPQ. Syncrip binding increases the mRNA stability of the long prosperoisoform, which allows an upregulation of Prospero protein production. Our findings highlight a regulatory strategy involving alternative polyadenylation followed by differential post-transcriptional regulation. Overall design: RNA-seq in Drosophila L3 larval brain.
Project description:Memory and learning involve activity-driven expression of proteins and cytoskeletal reorganization at new synapses, requiring posttranscriptional regulation of localized mRNA a long distance from corresponding nuclei. A key factor expressed early in synapse formation is Msp300/Nesprin-1, which organizes actin filaments around the new synapse. How Msp300 expression is regulated during synaptic plasticity is poorly understood. Here, we show that activity-dependent accumulation of Msp300 in the postsynaptic compartment of the Drosophila larval neuromuscular junction is regulated by the conserved RNA binding protein Syncrip/hnRNP Q. Syncrip (Syp) binds to msp300 transcripts and is essential for plasticity. Single-molecule imaging shows that msp300 is associated with Syp in vivo and forms ribosome-rich granules that contain the translation factor eIF4E. Elevated neural activity alters the dynamics of Syp and the number of msp300:Syp:eIF4E RNP granules at the synapse, suggesting that these particles facilitate translation. These results introduce Syp as an important early acting activity-dependent regulator of a plasticity gene that is strongly associated with human ataxias.
Project description:Differentiation from a haploid round spermatid to a highly streamlined, motile sperm requires temporal and spatial regulation of the expression of numerous proteins. One form of regulation is the storage of translationally repressed mRNAs. In Drosophila spermatocytes, the transcription of many of these translationally delayed mRNAs during spermiogenesis is in turn directly or indirectly regulated by testis-specific homologs of TATA-box-binding-protein-associated factors (tTAFs). Here we present evidence that expression of Mst77F, which is a specialized linker histone-like component of sperm chromatin, and of protamine B (ProtB), which contributes to formation of condensed sperm chromatin, is regulated at three levels. Transcription of Mst77F is guided by a short, promoter-proximal region, while expression of the Mst77F protein is regulated at two levels, early by translational repression via sequences mainly in the 5' part of the ORF and later by either protein stabilization or translational activation, dependent on sequences in the ORF. The protB gene is a direct target of tTAFs, with very short upstream regulatory regions of protB (-105 to +94 bp) sufficient for both cell-type-specific transcription and repression of translation in spermatocytes. In addition, efficient accumulation of the ProtB protein in late elongating spermatids depends on sequences in the ORF. We present evidence that spermatocytes provide the transacting mechanisms for translational repression of these mRNAs, while spermatids contain the machinery to activate or stabilize protamine accumulation for sperm chromatin components. Thus, the proper spatiotemporal expression pattern of major sperm chromatin components depends on cell-type-specific mechanisms of transcriptional and translational control.
Project description:BACKGROUND:Post-transcriptional regulation of gene expression can be achieved through the control of mRNA stability, cytoplasmic compartmentalization, 3' UTR length and translational efficacy. Spermiogenesis, a process through which haploid male germ cells differentiate into spermatozoa, represents an ideal model for studying post-transcriptional regulation in vivo because it involves a large number of transcripts that are physically sequestered in ribonucleoprotein particles (RNPs) and thus subjected to delayed translation. To explore how small RNAs regulate mRNA fate, we conducted RNA-Seq analyses to determine not only the levels of both mRNAs and small noncoding RNAs, but also their cytoplasmic compartmentalization during spermiogenesis. RESULT:Among all small noncoding RNAs studied, miRNAs displayed the most dynamic changes in both abundance and subcytoplasmic localization. mRNAs with shorter 3' UTRs became increasingly enriched in RNPs from pachytene spermatocytes to round spermatids, and the enrichment of shorter 3' UTR mRNAs in RNPs coincided with newly synthesized miRNAs that target these mRNAs at sites closer to the stop codon. In contrast, the translocation of longer 3' UTR mRNAs from RNPs to polysomes correlated with the production of new miRNAs that target these mRNAs at sites distal to the stop codon. CONCLUSIONS:miRNAs appear to control cytoplasmic compartmentalization of mRNAs based on 3' UTR length. Our data suggest that transcripts with longer 3' UTRs tend to contain distal miRNA binding sites and are thus targeted to polysomes for translation followed by degradation. In contrast, those with shorter 3' UTRs only possess proximal miRNA binding sites, which, therefore, are targeted into RNPs for enrichment and delayed translation.
Project description:Male germ cell differentiation proceeds to a large extent in the absence of active gene transcription. In Drosophila, hundreds of genes whose proteins are required during post-meiotic spermatid differentiation (spermiogenesis) are transcribed in primary spermatocytes. Transcription of these genes depends on the sequential action of the testis meiotic arrest complex (tMAC), Mediator complex, and testis-specific TFIID (tTFIID) complex. How the action of these protein complexes is coordinated and which other factors are involved in the regulation of transcription in spermatocytes is not well understood. Here, we show that the bromodomain proteins tBRD-1 and tBRD-2 regulate gene expression in primary spermatocytes and share a subset of target genes. The function of tBRD-1 was essential for the sub-cellular localization of endogenous tBRD-2 but dispensable for its protein stability. Our comparison of different microarray data sets showed that in primary spermatocytes, the expression of a defined number of genes depends on the function of the bromodomain proteins tBRD-1 and tBRD-2, the tMAC component Aly, the Mediator component Med22, and the tTAF Sa.
Project description:Gemcitabine is the first-line treatment for locally advanced and metastatic gallbladder cancer (GBC), but poor gemcitabine response is universal. Here, we utilize a genome-wide CRISPR screen to identify that loss of ELP5 reduces the gemcitabine-induced apoptosis in GBC cells in a P53-dependent manner through the Elongator complex and other uridine 34 (U34) tRNA-modifying enzymes. Mechanistically, loss of ELP5 impairs the integrity and stability of the Elongator complex to abrogate wobble U34 tRNA modification, and directly impedes the wobble U34 modification-dependent translation of hnRNPQ mRNA, a validated P53 internal ribosomal entry site (IRES) trans-acting factor. Downregulated hnRNPQ is unable to drive P53 IRES-dependent translation, but rescuing a U34 modification-independent hnRNPQ mutant could restore P53 translation and gemcitabine sensitivity in ELP5-depleted GBC cells. GBC patients with lower ELP5, hnRNPQ, or P53 expression have poor survival outcomes after gemcitabine chemotherapy. These results indicate that the Elongator/hnRNPQ/P53 axis controls gemcitabine sensitivity in GBC cells.