Transcription gets to the checkpoint.

The rapid cell proliferation characteristic of early animal embryos is accomplished with an abbreviated cell cycle and no DNA replication checkpoint. Blythe and Wieschaus provide evidence that nascent zygotic transcription precedes­and may trigger­this checkpoint at the midblastula transition.

Regulation of the Drosophila transcriptome by Pumilio and the CCR4-NOT deadenylase complex.

The sequence-specific RNA-binding protein Pumilio (Pum) controls Drosophila development; however, the network of mRNAs that it regulates remains incompletely characterized. In this study, we use knockdown and knockout approaches coupled with RNA-seq to measure the impact of Pum on the transcriptome of Drosophila cells in culture. We also use an improved RNA coimmunoprecipitation method to identify Pum-bound mRNAs in Drosophila embryos. Integration of these data sets with the locations of Pum-binding motifs across the transcriptome reveals novel direct Pum target genes involved in neural, muscle, wing, and germ cell development and in cellular proliferation. These genes include components of Wnt, TGF-ß, MAPK/ERK, and Notch signaling pathways, DNA replication, and lipid metabolism. We identify the mRNAs regulated by the CCR4-NOT deadenylase complex, a key factor in Pum-mediated repression, and observe concordant regulation of Pum:CCR4-NOT target mRNAs. Computational modeling reveals that Pum binding, binding site number, clustering, and sequence context are important determinants of regulation. In contrast, we show that the responses of direct mRNA targets to Pum-mediated repression are not influenced by the content of optimal synonymous codons. Moreover, contrary to a prevailing model, we do not detect a role for CCR4-NOT in the degradation of mRNAs with low codon optimality. Together, the results of this work provide new insights into the Pum regulatory network and mechanisms and the parameters that influence the efficacy of Pum-mediated regulation.

A vertebrate Polycomb response element governs segmentation of the posterior hindbrain.

Chromatin remodeling by Polycomb group (PcG) and trithorax group (trxG) proteins regulates gene expression in all metazoans. Two major complexes, Polycomb repressive complexes 1 and 2 (PRC1 and PRC2), are thought to mediate PcG-dependent repression in flies and mammals. In Drosophila, PcG/trxG protein complexes are recruited by PcG/trxG response elements (PREs). However, it has been unclear how PcG/trxG are recruited in vertebrates. Here we have identified a vertebrate PRE, PRE-kr, that regulates expression of the mouse MafB/Kreisler gene. PRE-kr recruits PcG proteins in flies and mouse F9 cells and represses gene expression in a PcG/trxG-dependent manner. PRC1 and 2 bind to a minimal PRE-kr region, which can recruit stable PRC1 binding but only weak PRC2 binding when introduced ectopically, suggesting that PRC1 and 2 have different binding requirements. Thus, we provide evidence that similar to invertebrates, PREs act as entry sites for PcG/trxG chromatin remodeling in vertebrates.

Cutoff Suppresses RNA Polymerase II Termination to Ensure Expression of piRNA Precursors.

Small non-coding RNAs called piRNAs serve as guides for an adaptable immune system that represses transposable elements in germ cells of Metazoa. In Drosophila the RDC complex, composed of Rhino, Deadlock and Cutoff (Cuff) bind chromatin of dual-strand piRNA clusters, special genomic regions, which encode piRNA precursors. The RDC complex is required for transcription of piRNA precursors, though the mechanism by which it licenses transcription remained unknown. Here, we show that Cuff prevents premature termination of RNA polymerase II. Cuff prevents cleavage of nascent RNA at poly(A) sites by interfering with recruitment of the cleavage and polyadenylation specificity factor (CPSF) complex. Cuff also protects processed transcripts from degradation by the exonuclease Rat1. Our work reveals a conceptually different mechanism of transcriptional enhancement. In contrast to other factors that regulate termination by binding to specific signals on nascent RNA, the RDC complex inhibits termination in a chromatin-dependent and sequence-independent manner.

Opposing roles for Egalitarian and Staufen in transport, anchoring and localization of oskar mRNA in the Drosophila oocyte.

Localization of oskar mRNA includes two distinct phases: transport from nurse cells to the oocyte, a process typically accompanied by cortical anchoring in the oocyte, followed by posterior localization within the oocyte. Signals within the oskar 3' UTR directing transport are individually weak, a feature previously hypothesized to facilitate exchange between the different localization machineries. We show that alteration of the SL2a stem-loop structure containing the oskar transport and anchoring signal (TAS) removes an inhibitory effect such that in vitro binding by the RNA transport factor, Egalitarian, is elevated as is in vivo transport from the nurse cells into the oocyte. Cortical anchoring within the oocyte is also enhanced, interfering with posterior localization. We also show that mutation of Staufen recognized structures (SRSs), predicted binding sites for Staufen, disrupts posterior localization of oskar mRNA just as in staufen mutants. Two SRSs in SL2a, one overlapping the Egalitarian binding site, are inferred to mediate Staufen-dependent inhibition of TAS anchoring activity, thereby promoting posterior localization. The other three SRSs in the oskar 3' UTR are also required for posterior localization, including two located distant from any known transport signal. Staufen, thus, plays multiple roles in localization of oskar mRNA.

The Canadian Rare Diseases Models and Mechanisms (RDMM) Network: Connecting Understudied Genes to Model Organisms.

Advances in genomics have transformed our ability to identify the genetic causes of rare diseases (RDs), yet we have a limited understanding of the mechanistic roles of most genes in health and disease. When a novel RD gene is first discovered, there is minimal insight into its biological function, the pathogenic mechanisms of disease-causing variants, and how therapy might be approached. To address this gap, the Canadian Rare Diseases Models and Mechanisms (RDMM) Network was established to connect clinicians discovering new disease genes with Canadian scientists able to study equivalent genes and pathways in model organisms (MOs). The Network is built around a registry of more than 500 Canadian MO scientists, representing expertise for over 7,500 human genes. RDMM uses a committee process to identify and evaluate clinician-MO scientist collaborations and approve 25,000 Canadian dollars in catalyst funding. To date, we have made 85 clinician-MO scientist connections and funded 105 projects. These collaborations help confirm variant pathogenicity and unravel the molecular mechanisms of RD, and also test novel therapies and lead to long-term collaborations. To expand the impact and reach of this model, we made the RDMM Registry open-source, portable, and customizable, and we freely share our committee structures and processes. We are currently working with emerging networks in Europe, Australia, and Japan to link international RDMM networks and registries and enable matches across borders. We will continue to create meaningful collaborations, generate knowledge, and advance RD research locally and globally for the benefit of patients and families living with RD.

The maternal-to-zygotic transition revisited.

The development of animal embryos is initially directed by maternal gene products. Then, during the maternal-to-zygotic transition (MZT), developmental control is handed to the zygotic genome. Extensive research in both vertebrate and invertebrate model organisms has revealed that the MZT can be subdivided into two phases, during which very different modes of gene regulation are implemented: initially, regulation is exclusively post-transcriptional and post-translational, following which gradual activation of the zygotic genome leads to predominance of transcriptional regulation. These changes in the gene expression program of embryos are precisely controlled and highly interconnected. Here, we review current understanding of the mechanisms that underlie handover of developmental control during the MZT.

Regulation of the RNA-binding protein Smaug by the GPCR Smoothened via the kinase Fused.

From fly to mammals, the Smaug/Samd4 family of prion-like RNA-binding proteins control gene expression by destabilizing and/or repressing the translation of numerous target transcripts. However, the regulation of its activity remains poorly understood. We show that Smaug's protein levels and mRNA repressive activity are downregulated by Hedgehog signaling in tissue culture cells. These effects rely on the interaction of Smaug with the G-protein coupled receptor Smoothened, which promotes the phosphorylation of Smaug by recruiting the kinase Fused. The activation of Fused and its binding to Smaug are sufficient to suppress its ability to form cytosolic bodies and to antagonize its negative effects on endogenous targets. Importantly, we demonstrate in vivo that HH reduces the levels of smaug mRNA and increases the level of several mRNAs downregulated by Smaug. Finally, we show that Smaug acts as a positive regulator of Hedgehog signaling during wing morphogenesis. These data constitute the first evidence for a post-translational regulation of Smaug and reveal that the fate of several mRNAs bound to Smaug is modulated by a major signaling pathway.

Follow the mRNA: a new model for Bicoid gradient formation.

Morphogens are molecules that specify cell fate in a concentration-dependent manner. A classic example is the Bicoid (BCD) protein, for which the prevailing model is that translation of bcd mRNA occurs from a point source at the anterior pole of the Drosophila melanogaster embryo followed by diffusion to produce a protein gradient. This model has been challenged by experiments showing that the diffusion rate of BCD is too slow to establish the protein gradient. The work described in a recent paper has solved this conundrum by demonstrating that a bcd mRNA gradient prefigures the BCD protein gradient.

A compendium of RNA-binding motifs for decoding gene regulation.

RNA-binding proteins are key regulators of gene expression, yet only a small fraction have been functionally characterized. Here we report a systematic analysis of the RNA motifs recognized by RNA-binding proteins, encompassing 205 distinct genes from 24 diverse eukaryotes. The sequence specificities of RNA-binding proteins display deep evolutionary conservation, and the recognition preferences for a large fraction of metazoan RNA-binding proteins can thus be inferred from their RNA-binding domain sequence. The motifs that we identify in vitro correlate well with in vivo RNA-binding data. Moreover, we can associate them with distinct functional roles in diverse types of post-transcriptional regulation, enabling new insights into the functions of RNA-binding proteins both in normal physiology and in human disease. These data provide an unprecedented overview of RNA-binding proteins and their targets, and constitute an invaluable resource for determining post-transcriptional regulatory mechanisms in eukaryotes.

A high-throughput pipeline for the production of synthetic antibodies for analysis of ribonucleoprotein complexes.

Post-transcriptional regulation of mRNAs plays an essential role in the control of gene expression. mRNAs are regulated in ribonucleoprotein (RNP) complexes by RNA-binding proteins (RBPs) along with associated protein and noncoding RNA (ncRNA) cofactors. A global understanding of post-transcriptional control in any cell type requires identification of the components of all of its RNP complexes. We have previously shown that these complexes can be purified by immunoprecipitation using anti-RBP synthetic antibodies produced by phage display. To develop the large number of synthetic antibodies required for a global analysis of RNP complex composition, we have established a pipeline that combines (i) a computationally aided strategy for design of antigens located outside of annotated domains, (ii) high-throughput antigen expression and purification in Escherichia coli, and (iii) high-throughput antibody selection and screening. Using this pipeline, we have produced 279 antibodies against 61 different protein components of Drosophila melanogaster RNPs. Together with those produced in our low-throughput efforts, we have a panel of 311 antibodies for 67 RNP complex proteins. Tests of a subset of our antibodies demonstrated that 89% immunoprecipitate their endogenous target from embryo lysate. This panel of antibodies will serve as a resource for global studies of RNP complexes in Drosophila. Furthermore, our high-throughput pipeline permits efficient production of synthetic antibodies against any large set of proteins.

A Smaug2-Based Translational Repression Complex Determines the Balance between Precursor Maintenance versus Differentiation during Mammalian Neurogenesis.

Here, we have asked about post-transcriptional mechanisms regulating murine developmental neurogenesis, focusing upon the RNA-binding proteins Smaug2 and Nanos1. We identify, in embryonic neural precursors of the murine cortex, a Smaug2 protein/nanos1 mRNA complex that is present in cytoplasmic granules with the translational repression proteins Dcp1 and 4E-T. We show that Smaug2 inhibits and Nanos1 promotes neurogenesis, with Smaug2 knockdown enhancing neurogenesis and depleting precursors, and Nanos1 knockdown inhibiting neurogenesis and maintaining precursors. Moreover, we show that Smaug2 likely regulates neurogenesis by silencing nanos1 mRNA. Specifically, Smaug2 knockdown inappropriately increases Nanos1 protein, and the Smaug2 knockdown-mediated neurogenesis is rescued by preventing this increase. Thus, Smaug2 and Nanos1 function as a bimodal translational repression switch to control neurogenesis, with Smaug2 acting in transcriptionally primed precursors to silence mRNAs important for neurogenesis, including nanos1 mRNA, and Nanos1 acting during the transition to neurons to repress the precursor state. SIGNIFICANCE STATEMENT: The mechanisms instructing neural stem cells to generate the appropriate progeny are still poorly understood. Here, we show that the RNA-binding proteins Smaug2 and Nanos1 are critical regulators of this balance and provide evidence supporting the idea that neural precursors are transcriptionally primed to generate neurons but translational regulation maintains these precursors in a stem cell state until the appropriate developmental time.

HuR is required for IL-17-induced Act1-mediated CXCL1 and CXCL5 mRNA stabilization.

IL-17, a major inflammatory cytokine plays a critical role in the pathogenesis of many autoimmune inflammatory diseases. In this study, we report a new function of RNA-binding protein HuR in IL-17-induced Act1-mediated chemokine mRNA stabilization. HuR deficiency markedly reduced IL-17-induced chemokine expression due to increased mRNA decay. Act1-mediated HuR polyubiquitination was required for the binding of HuR to CXCL1 mRNA, leading to mRNA stabilization. Although IL-17 induced the coshift of Act1 and HuR to the polysomal fractions in a sucrose gradient, HuR deficiency reduced the ratio of translation-active/translation-inactive IL-17-induced chemokine mRNAs. Furthermore, HuR deletion in distal lung epithelium attenuated IL-17-induced neutrophilia. In summary, HuR functions to couple receptor-proximal signaling to posttranscriptional machinery, contributing to IL-17-induced inflammation.

Genome-wide analysis of Staufen-associated mRNAs identifies secondary structures that confer target specificity.

Despite studies that have investigated the interactions of double-stranded RNA-binding proteins like Staufen with RNA in vitro, how they achieve target specificity in vivo remains uncertain. We performed RNA co-immunoprecipitations followed by microarray analysis to identify Staufen-associated mRNAs in early Drosophila embryos. Analysis of the localization and functions of these transcripts revealed a number of potentially novel roles for Staufen. Using computational methods, we identified two sequence features that distinguish Staufen's target transcripts from non-targets. First, these Drosophila transcripts, as well as those human transcripts bound by human Staufen1 and 2, have 3' untranslated regions (UTRs) that are 3-4-fold longer than unbound transcripts. Second, the 3'UTRs of Staufen-bound transcripts are highly enriched for three types of secondary structures. These structures map with high precision to previously identified Staufen-binding regions in Drosophila bicoid and human ARF1 3'UTRs. Our results provide the first systematic genome-wide analysis showing how a double-stranded RNA-binding protein achieves target specificity.

Drosophila Hindsight and mammalian RREB-1 are evolutionarily conserved DNA-binding transcriptional attenuators.

The Drosophila Hindsight (hnt) gene encodes a C2H2-type Zinc-finger protein, HNT, that plays multiple developmental roles including control of embryonic germ band retraction and regulation of retinal cell fate and morphogenesis. While the developmental functions of the human HNT homolog, RREB-1, are unknown, it has been shown to function as a transcriptional modulator of several tumor suppressor genes. Here we investigate HNT's functional motifs, target genes and its regulatory abilities. We show that the C-terminal region of HNT, containing the last five of its 14 Zinc fingers, binds in vitro to DNA elements very similar to those identified for RREB-1. We map HNT's in vivo binding sites on salivary gland polytene chromosomes and define, at high resolution, where HNT is bound to two target genes, hnt itself and nervy (nvy). Data from both loss-of-function and over-expression experiments show that HNT attenuates the transcription of these two targets in a tissue-specific manner. RREB-1, when expressed in Drosophila, binds to the same polytene chromosome sites as HNT, attenuates expression of the hnt and nvy genes, and rescues the germ band retraction phenotype. HNT's ninth Zinc finger has degenerated or been lost in the vertebrate lineage. We show that a HNT protein mutant for this finger can also attenuate target gene expression and rescue germ band retraction. Thus HNT and RREB-1 are functional homologs at the level of DNA binding, transcriptional regulation and developmental control.

Post-transcriptional regulatory pre-complex assembly drives timely cell-state transitions during differentiation.

Complexes that control mRNA stability and translation promote timely cell-state transitions during differentiation by ensuring appropriate expression patterns of key developmental regulators. The Drosophila RNA-binding protein Brain tumor (Brat) promotes degradation of target transcripts during the maternal-to-zygotic transition in syncytial embryos and in uncommitted intermediate neural progenitors (immature INPs). We identified Ubiquitin-specific protease 5 (Usp5) as a Brat interactor essential for the degradation of Brat target mRNAs in both cell types. Usp5 promotes Brat-dedadenylase pre-complex assembly in mitotic neural stem cells (neuroblasts) by bridging Brat and the scaffolding components of deadenylase complexes lacking their catalytic subunits. The adaptor protein Miranda binds the RNA-binding domain of Brat, limiting its ability to bind target mRNAs in mitotic neuroblasts. Cortical displacement of Miranda activates Brat-mediated mRNA decay in immature INPs. We propose that the assembly of an enzymatically inactive and RNA-binding-deficient pre-complex poises mRNA degradation machineries for rapid activation driving timely developmental transitions.

Smaug regulates germ plasm assembly and primordial germ cell number in Drosophila embryos.

During Drosophila oogenesis, the Oskar (OSK) RNA binding protein (RBP) determines the amount of germ plasm that assembles at the posterior pole of the oocyte. Here, we identify mechanisms that subsequently regulate germ plasm assembly in the early embryo. We show that the Smaug (SMG) RBP is transported into the germ plasm of the early embryo where it accumulates in the germ granules. SMG binds to and represses translation of the osk messenger RNA (mRNA) as well as the bruno 1 (bru1) mRNA, which encodes an RBP that we show promotes germ plasm production. Loss of SMG or mutation of SMG's binding sites in the osk or bru1 mRNA results in excess translation of these transcripts in the germ plasm, accumulation of excess germ plasm, and budding of excess primordial germ cells (PGCs). Therefore, SMG triggers a posttranscriptional regulatory pathway that attenuates the amount of germ plasm in embryos to modulate the number of PGCs.

Smaug regulates germ plasm synthesis and primordial germ cell number in Drosophila embryos by repressing the oskar and bruno 1 mRNAs.

During Drosophila oogenesis, the Oskar (OSK) RNA-binding protein (RBP) determines the amount of germ plasm that assembles at the posterior pole of the oocyte. Here we identify the mechanisms that regulate the osk mRNA in the early embryo. We show that the Smaug (SMG) RBP is transported into the germ plasm of the early embryo where it accumulates in the germ granules. SMG binds to and represses translation of the osk mRNA itself as well as the bruno 1 (bru1) mRNA, which encodes an RBP that we show promotes germ plasm production. Loss of SMG or mutation of SMG's binding sites in the osk or bru1 mRNAs results in ectopic translation of these transcripts in the germ plasm and excess PGCs. SMG therefore triggers a post-transcriptional regulatory pathway that attenuates germ plasm synthesis in embryos, thus modulating the number of PGCs.

Regulation of the Drosophila transcriptome by Pumilio and CCR4-NOT deadenylase.

The sequence-specific RNA-binding protein Pumilio controls development of Drosophila; however, the network of mRNAs that it regulates remains incompletely characterized. In this study, we utilize knockdown and knockout approaches coupled with RNA-Seq to measure the impact of Pumilio on the transcriptome of Drosophila cells. We also used an improved RNA co-immunoprecipitation method to identify Pumilio bound mRNAs in Drosophila embryos. Integration of these datasets with the content of Pumilio binding motifs across the transcriptome revealed novel direct Pumilio target genes involved in neural, muscle, wing, and germ cell development, and cellular proliferation. These genes include components of Wnt, TGF-beta, MAPK/ERK, and Notch signaling pathways, DNA replication, and lipid metabolism. Additionally, we identified the mRNAs regulated by the CCR4-NOT deadenylase complex, a key factor in Pumilio-mediated repression, and observed concordant regulation of Pumilio:CCR4-NOT target mRNAs. Computational modeling revealed that Pumilio binding, binding site number, density, and sequence context are important determinants of regulation. Moreover, the content of optimal synonymous codons in target mRNAs exhibits a striking functional relationship to Pumilio and CCR4-NOT regulation, indicating that the inherent translation efficiency and stability of the mRNA modulates their response to these trans-acting regulatory factors. Together, the results of this work provide new insights into the Pumilio regulatory network and mechanisms, and the parameters that influence the efficacy of Pumilio-mediated regulation.