Global transcriptional repression in C. elegans germline precursors by regulated sequestration of TAF-4.

In C. elegans, four asymmetric divisions, beginning with the zygote (P0), generate transcriptionally repressed germline blastomeres (P1-P4) and somatic sisters that become transcriptionally active. The protein PIE-1 represses transcription in the later germline blastomeres but not in the earlier germline blastomeres P0 and P1. We show here that OMA-1 and OMA-2, previously shown to regulate oocyte maturation, repress transcription in P0 and P1 by binding to and sequestering in the cytoplasm TAF-4, a component critical for assembly of TFIID and the pol II preinitiation complex. OMA-1/2 binding to TAF-4 is developmentally regulated, requiring phosphorylation by the DYRK kinase MBK-2, which is activated at meiosis II after fertilization. OMA-1/2 are normally degraded after the first mitosis, but ectopic expression of wild-type OMA-1 is sufficient to repress transcription in both somatic and later germline blastomeres. We propose that phosphorylation by MBK-2 serves as a developmental switch, converting OMA-1/2 from oocyte to embryo regulators.

Developmental inhibition of miR-iab8-3p disrupts mushroom body neuron structure and adult learning ability.

MicroRNAs are small non-coding RNAs that inhibit protein expression post-transcriptionally. They have been implicated in many different physiological processes, but little is known about their individual involvement in learning and memory. We recently identified several miRNAs that either increased or decreased intermediate-term memory when inhibited in the central nervous system, including miR-iab8-3p. We report here a new developmental role for this miRNA. Blocking the expression of miR-iab8-3p during the development of the organism leads to hypertrophy of individual mushroom body neuron soma, a reduction in the field size occupied by axonal projections, and adult intellectual disability. We further identified four potential mRNA targets of miR-iab8-3p whose inhibition modulates intermediate-term memory including ceramide phosphoethanolamine synthase, which may account for the behavioral effects produced by miR-iab8-3p inhibition. Our results offer important new information on a microRNA required for normal neurodevelopment and the capacity to learn and remember normally.

Regulation of maternal Wnt mRNA translation in C. elegans embryos.

The restricted spatiotemporal translation of maternal mRNAs, which is crucial for correct cell fate specification in early C. elegans embryos, is regulated primarily through the 3'UTR. Although genetic screens have identified many maternally expressed cell fate-controlling RNA-binding proteins (RBPs), their in vivo targets and the mechanism(s) by which they regulate these targets are less clear. These RBPs are translated in oocytes and localize to one or a few blastomeres in a spatially and temporally dynamic fashion unique for each protein and each blastomere. Here, we characterize the translational regulation of maternally supplied mom-2 mRNA, which encodes a Wnt ligand essential for two separate cell-cell interactions in early embryos. A GFP reporter that includes only the mom-2 3'UTR is translationally repressed properly in oocytes and early embryos, and then correctly translated only in the known Wnt signaling cells. We show that the spatiotemporal translation pattern of this reporter is regulated combinatorially by a set of nine maternally supplied RBPs. These nine proteins all directly bind the mom-2 3'UTR in vitro and function as positive or negative regulators of mom-2 translation in vivo. The net translational readout for the mom-2 3'UTR reporter is determined by competitive binding between positive- and negative-acting RBPs for the 3'UTR, along with the distinct spatiotemporal localization patterns of these regulators. We propose that the 3'UTR of maternal mRNAs contains a combinatorial code that determines the topography of associated RBPs, integrating positive and negative translational inputs.

Functional neuroanatomy of Drosophila olfactory memory formation.

New approaches, techniques and tools invented over the last decade and a half have revolutionized the functional dissection of neural circuitry underlying Drosophila learning. The new methodologies have been used aggressively by researchers attempting to answer three critical questions about olfactory memories formed with appetitive and aversive reinforcers: (1) Which neurons within the olfactory nervous system mediate the acquisition of memory? (2) What is the complete neural circuitry extending from the site(s) of acquisition to the site(s) controlling memory expression? (3) How is information processed across this circuit to consolidate early-forming, disruptable memories to stable, late memories? Much progress has been made and a few strong conclusions have emerged: (1) Acquisition occurs at multiple sites within the olfactory nervous system but is mediated predominantly by the γ mushroom body neurons. (2) The expression of long-term memory is completely dependent on the synaptic output of α/ß mushroom body neurons. (3) Consolidation occurs, in part, through circuit interactions between mushroom body and dorsal paired medial neurons. Despite this progress, a complete and unified model that details the pathway from acquisition to memory expression remains elusive.

Multiple RNA-binding proteins function combinatorially to control the soma-restricted expression pattern of the E3 ligase subunit ZIF-1.

In C. elegans embryos, transcriptional repression in germline blastomeres requires PIE-1 protein. Germline blastomere-specific localization of PIE-1 depends, in part, upon regulated degradation of PIE-1 in somatic cells. We and others have shown that the temporal and spatial regulation of PIE-1 degradation is controlled by translation of the substrate-binding subunit, ZIF-1, of an E3 ligase. We now show that ZIF-1 expression in embryos is regulated by five maternally-supplied RNA-binding proteins. POS-1, MEX-3, and SPN-4 function as repressors of ZIF-1 expression, whereas MEX-5 and MEX-6 antagonize this repression. All five proteins bind directly to the zif-1 3' UTR in vitro. We show that, in vivo, POS-1 and MEX-5/6 have antagonistic roles in ZIF-1 expression. In vitro, they bind to a common region of the zif-1 3' UTR, with MEX-5 binding impeding that by POS-1. The region of the zif-1 3' UTR bound by MEX-5/6 also partially overlaps with that bound by MEX-3, consistent with their antagonistic functions on ZIF-1 expression in vivo. Whereas both MEX-3 and SPN-4 repress ZIF-1 expression, neither protein alone appears to be sufficient, suggesting that they function together in ZIF-1 repression. We propose that MEX-3 and SPN-4 repress ZIF-1 expression exclusively in 1- and 2-cell embryos, the only period during embryogenesis when these two proteins co-localize. As the embryo divides, ZIF-1 continues to be repressed in germline blastomeres by POS-1, a germline blastomere-specific protein. MEX-5/6 antagonize repression by POS-1 and MEX-3, enabling ZIF-1 expression in somatic blastomeres. We propose that ZIF-1 expression results from a net summation of complex positive and negative translational regulation by 3' UTR-binding proteins, with expression in a specific blastomere dependent upon the precise combination of these proteins in that cell.

zif-1 translational repression defines a second, mutually exclusive OMA function in germline transcriptional repression.

Specification of primordial germ cells requires global repression of transcription. In C. elegans, primordial germ cells are generated through four rounds of asymmetric divisions, starting from the zygote P0, each producing a transcriptionally repressed germline blastomere (P1-P4). Repression in P2-P4 requires PIE-1, which is provided maternally in oocytes and segregated to all germline blastomeres. We have shown previously that OMA-1 and OMA-2 repress global transcription in P0 and P1 by sequestering TAF-4, an essential component of TFIID. Soon after the first mitotic cycle, OMA proteins undergo developmentally regulated degradation. Here, we show that OMA proteins also repress transcription in P2-P4 indirectly, through a completely different mechanism that operates in oocytes. OMA proteins bind to both the 3' UTR of the zif-1 transcript and the eIF4E-binding protein, SPN-2, repressing translation of zif-1 mRNA in oocytes. zif-1 encodes the substrate-binding subunit of the E3 ligase for PIE-1 degradation. Inhibition of zif-1 translation in oocytes ensures high PIE-1 levels in oocytes and germline blastomeres. The two OMA protein functions are strictly regulated in both space and time by MBK-2, a kinase activated following fertilization. Phosphorylation by MBK-2 facilitates the binding of OMA proteins to TAF-4 and simultaneously inactivates their function in repressing zif-1 translation. Phosphorylation of OMA proteins displaces SPN-2 from the zif-1 3' UTR, releasing translational repression. We propose that MBK-2 phosphorylation serves as a developmental switch, converting OMA proteins from specific translational repressors in oocytes to global transcriptional repressors in embryos, together effectively repressing transcription in all germline blastomeres.

miR-92a Suppresses Mushroom Body-Dependent Memory Consolidation in Drosophila.

MicroRNAs (miRNAs) fine tune gene expression to regulate many aspects of nervous system physiology. Here, we show that miR-92a suppresses memory consolidation that occurs in the αß and γ mushroom body neurons (MBns) of Drosophila, making miR-92a a memory suppressor miRNA. Bioinformatics analyses suggested that mRNAs encoding kinesin heavy chain 73 (KHC73), a protein that belongs to Kinesin-3 family of anterograde motor proteins, may be a functional target of miR-92a. Behavioral studies that employed expression of khc73 with and without its 3' untranslated region (UTR) containing miR-92a target sites, luciferase assays in HEK cells with reporters containing wild-type and mutant target sequences in the khc73 3'UTR, and immunohistochemistry experiments involving KHC73 expression with and without the wild-type khc73 3'UTR, all point to the conclusion that khc73 is a major target of miR-92a in its functional role as a miRNA memory suppressor gene.

MicroRNA function in Drosophila memory formation.

MicroRNAs (miRs) are small non-coding RNAs that regulate protein expression through post-transcriptional mechanisms. They participate in broad aspects of biology from the control of developmental processes to tumorigenesis. Recent studies in Drosophila show that they also regulate activity-dependent and sensory-specific protein expression and support olfactory memory formation. Among the hundreds of miRs described, several have been demonstrated to be required for normal learning, memory, or for the development of neuronal circuits that support memory formation. Fly models of human diseases offer promise of identifying miRs whose expression becomes dysregulated and part of the pathological state, providing models for understanding brain disorders and drug discovery.

MiR-980 Is a Memory Suppressor MicroRNA that Regulates the Autism-Susceptibility Gene A2bp1.

MicroRNAs have been associated with many different biological functions, but little is known about their roles in conditioned behavior. We demonstrate that Drosophila miR-980 is a memory suppressor gene functioning in multiple regions of the adult brain. Memory acquisition and stability were both increased by miR-980 inhibition. Whole cell recordings and functional imaging experiments indicated that miR-980 regulates neuronal excitability. We identified the autism susceptibility gene, A2bp1, as an mRNA target for miR-980. A2bp1 levels varied inversely with miR-980 expression; memory performance was directly related to A2bp1 levels. In addition, A2bp1 knockdown reversed the memory gains produced by miR-980 inhibition, consistent with A2bp1 being a downstream target of miR-980 responsible for the memory phenotypes. Our results indicate that miR-980 represses A2bp1 expression to tune the excitable state of neurons, and the overall state of excitability translates to memory impairment or improvement.

microRNAs That Promote or Inhibit Memory Formation in Drosophila melanogaster.

microRNAs (miRNAs) are small noncoding RNAs that regulate gene expression post-transcriptionally. Prior studies have shown that they regulate numerous physiological processes critical for normal development, cellular growth control, and organismal behavior. Here, we systematically surveyed 134 different miRNAs for roles in olfactory learning and memory formation using "sponge" technology to titrate their activity broadly in the Drosophila melanogaster central nervous system. We identified at least five different miRNAs involved in memory formation or retention from this large screen, including miR-9c, miR-31a, miR-305, miR-974, and miR-980. Surprisingly, the titration of some miRNAs increased memory, while the titration of others decreased memory. We performed more detailed experiments on two miRNAs, miR-974 and miR-31a, by mapping their roles to subpopulations of brain neurons and testing the functional involvement in memory of potential mRNA targets through bioinformatics and a RNA interference knockdown approach. This screen offers an important first step toward the comprehensive identification of all miRNAs and their potential targets that serve in gene regulatory networks important for normal learning and memory.

Translational control of the oogenic program by components of OMA ribonucleoprotein particles in Caenorhabditis elegans.

The oocytes of most sexually reproducing animals arrest in meiotic prophase I. Oocyte growth, which occurs during this period of arrest, enables oocytes to acquire the cytoplasmic components needed to produce healthy progeny and to gain competence to complete meiosis. In the nematode Caenorhabditis elegans, the major sperm protein hormone promotes meiotic resumption (also called meiotic maturation) and the cytoplasmic flows that drive oocyte growth. Prior work established that two related TIS11 zinc-finger RNA-binding proteins, OMA-1 and OMA-2, are redundantly required for normal oocyte growth and meiotic maturation. We affinity purified OMA-1 and identified associated mRNAs and proteins using genome-wide expression data and mass spectrometry, respectively. As a class, mRNAs enriched in OMA-1 ribonucleoprotein particles (OMA RNPs) have reproductive functions. Several of these mRNAs were tested and found to be targets of OMA-1/2-mediated translational repression, dependent on sequences in their 3'-untranslated regions (3'-UTRs). Consistent with a major role for OMA-1 and OMA-2 in regulating translation, OMA-1-associated proteins include translational repressors and activators, and some of these proteins bind directly to OMA-1 in yeast two-hybrid assays, including OMA-2. We show that the highly conserved TRIM-NHL protein LIN-41 is an OMA-1-associated protein, which also represses the translation of several OMA-1/2 target mRNAs. In the accompanying article in this issue, we show that LIN-41 prevents meiotic maturation and promotes oocyte growth in opposition to OMA-1/2. Taken together, these data support a model in which the conserved regulators of mRNA translation LIN-41 and OMA-1/2 coordinately control oocyte growth and the proper spatial and temporal execution of the meiotic maturation decision.

An eIF4E-binding protein regulates katanin protein levels in C. elegans embryos.

In Caenorhabditis elegans, the MEI-1-katanin microtubule-severing complex is required for meiosis, but must be down-regulated during the transition to embryogenesis to prevent defects in mitosis. A cullin-dependent degradation pathway for MEI-1 protein has been well documented. In this paper, we report that translational repression may also play a role in MEI-1 down-regulation. Reduction of spn-2 function results in spindle orientation defects due to ectopic MEI-1 expression during embryonic mitosis. MEL-26, which is both required for MEI-1 degradation and is itself a target of the cullin degradation pathway, is present at normal levels in spn-2 mutant embryos, suggesting that the degradation pathway is functional. Cloning of spn-2 reveals that it encodes an eIF4E-binding protein that localizes to the cytoplasm and to ribonucleoprotein particles called P granules. SPN-2 binds to the RNA-binding protein OMA-1, which in turn binds to the mei-1 3' untranslated region. Thus, our results suggest that SPN-2 functions as an eIF4E-binding protein to negatively regulate translation of mei-1.