Scott A. Barbee

A novel function for the Sm proteins in germ granule localization during C. elegans embryogenesis.

General mRNA processing factors are traditionally thought to function only in the control of global gene expression. Here we show that the Sm proteins, core components of the splicesome, also regulate germ granules during early C. elegans development. Germ granules are large cytoplasmic particles that localize to germ cells and their precursors during embryogenesis of diverse organisms. In C. elegans, germ granules, called P granules, are segregated to the germline precursor cells during embryogenesis by asymmetric cell division, and they remain in germ cells at all stages of development. We found that at least some Sm proteins are components of P granules. Moreover, disruption of Sm activity caused defects in P granule localization to the germ cell precursors during early embryogenesis. In contrast, loss of other splicing factor activities had no effect on germ granule control in the embryo. These observations suggest that the Sm proteins control germ granule integrity and localization in the early C. elegans embryo and that this role is independent of pre-mRNA splicing. Thus, a highly conserved splicing factor may have been adapted to control both snRNP biogenesis and the localization of components important for germ cell function.

GLD2 poly(A) polymerase is required for long-term memory

The formation of long-term memory is believed to require translational control of localized mRNAs. In mammals, dendritic mRNAs are maintained in a repressed state and are activated upon repetitive stimulation. Several regulatory proteins required for translational control in early development are thought to be required for memory formation, suggesting similar molecular mechanisms. Here, using Drosophila, we identify the enzyme responsible for poly(A) elongation in the brain and demonstrate that its activity is required specifically for long-term memory. These findings provide strong evidence that cytoplasmic polyadenylation is critical for memory formation, and that GLD2 is the enzyme responsible.

Genetic Modifiers of dFMR1 Encode RNA Granule Components in Drosophila

Mechanisms of neuronal mRNA localization and translation are of considerable biological interest. Spatially regulated mRNA translation contributes to cell-fate decisions and axon guidance during development, as well as to long-term synaptic plasticity in adulthood. The Fragile-X Mental Retardation protein (FMRP/dFMR1) is one of the best-studied neuronal translational control molecules and here we describe the identification and early characterization of proteins likely to function in the dFMR1 pathway. Induction of the dFMR1 in sevenless-expressing cells of the Drosophila eye causes a disorganized (rough) eye through a mechanism that requires residues necessary for dFMR1/FMRPs translational repressor function. Several mutations in dco, orb2, pAbp, rm62, and smD3 genes dominantly suppress the sev-dfmr1 rough-eye phenotype, suggesting that they are required for dFMR1-mediated processes. The encoded proteins localize to dFMR1-containing neuronal mRNPs in neurites of cultured neurons, and/or have an effect on dendritic branching predicted for bona fide neuronal translational repressors. Genetic mosaic analyses indicate that dco, orb2, rm62, smD3, and dfmr1 are dispensable for translational repression of hid, a microRNA target gene, known to be repressed in wing discs by the bantam miRNA. Thus, the encoded proteins may function as miRNA- and/or mRNA-specific translational regulators in vivo.

P-Body Components, microRNA Regulation, and Synaptic Plasticity

What is the protein apparatus required for microRNA (miRNA) function and translational repression in neurons? This article reviews our recent work on Me31B, a conserved P-body protein present on Staufen-containing neuronal and maternal ribonucleoprotein (RNP) particles, which is required for dendrite morphogenesis and miRNA function in vivo. In addition, it provides new data to show that Me31B is present on and regulates formation of P-bodies in the Drosophila wing disc, where it has a general role in the regulation of miRNA function. While illuminating the function of this important RNA regulatory molecule, it also brings into focus a hypothesis of potentially broad significance. Namely, that P-body proteins may play important roles in regulation of dendrite-localized mRNAs and, thereby, in synaptic plasticity. A wide range of protein localization and early functional data support this hypothesis. We also discuss current knowledge of RNP particles that mediate translational repression and the implications of these findings for understanding translational control in neurons.

The Me31B DEAD-Box Helicase Localizes to Postsynaptic Foci and Regulates Expression of a CaMKII Reporter mRNA in Dendrites of Drosophila Olfactory Projection Neurons.

mRNP granules at adult central synapses are postulated to regulate local mRNA translation and synapse plasticity. However, they are very poorly characterized in vivo. Here, in Drosophila olfactory synapses, we present early observations and characterization of candidate synaptic mRNP particles, one of which contains a widely conserved, DEAD-box helicase, Me31B. In Drosophila, Me31B is required for translational repression of maternal and miRNA-target mRNAs. A role in neuronal translational control is primarily suggested by Me31Bs localization, in cultured primary neurons, to neuritic mRNP granules that contain: (i) various translational regulators; (ii) CaMKII mRNA; and (iii) several P-body markers including the mRNA hydrolases, Dcp1, and Pcm/Xrn-1. In adult neurons, Me31B localizes to P-body like cytoplasmic foci/particles in neuronal soma. In addition it is present to synaptic foci that may lack RNA degradative enzymes and localize predominantly to dendritic elements of olfactory sensory and projection neurons (PNs). MARCM clones of PNs mutant for Me31B show loss of both Me31B and Dcp1-positive dendritic puncta, suggesting potential interactions between these granule types. In PNs, expression of validated hairpin-RNAi constructs against Me31B causes visible knockdown of endogenous protein, as assessed by the brightness and number of Me31B puncta. Knockdown of Me31B also causes a substantial elevation in observed levels of a translational reporter of CaMKII, a postsynaptic protein whose mRNA has been shown to be localized to PN dendrites and to be translationally regulated, at least in part through the miRNA pathway. Thus, neuronal Me31B is present in dendritic particles in vivo and is required for repression of a translationally regulated synaptic mRNA.

The miRNA Pathway Controls Rapid Changes in Activity-Dependent Synaptic Structure at the Drosophila melanogaster Neuromuscular Junction

It is widely accepted that long-term changes in synapse structure and function are mediated by rapid activity-dependent gene transcription and new protein synthesis. A growing amount of evidence suggests that the microRNA (miRNA) pathway plays an important role in coordinating these processes. Despite recent advances in this field, there remains a critical need to identify specific activity-regulated miRNAs as well as their key messenger RNA (mRNA) targets. To address these questions, we used the larval Drosophila melanogaster neuromuscular junction (NMJ) as a model synapse in which to identify novel miRNA-mediated mechanisms that control activity-dependent synaptic growth. First, we developed a screen to identify miRNAs differentially regulated in the larval CNS following spaced synaptic stimulation. Surprisingly, we identified five miRNAs (miRs-1, -8, -289, -314, and -958) that were significantly downregulated by activity. Neuronal misexpression of three miRNAs (miRs-8, -289, and -958) suppressed activity-dependent synaptic growth suggesting that these miRNAs control the translation of biologically relevant target mRNAs. Functional annotation cluster analysis revealed that putative targets of miRs-8 and -289 are significantly enriched in clusters involved in the control of neuronal processes including axon development, pathfinding, and growth. In support of this, miR-8 regulated the expression of a wingless 3′UTR (wg 3′ untranslated region) reporter in vitro. Wg is an important presynaptic regulatory protein required for activity-dependent axon terminal growth at the fly NMJ. In conclusion, our results are consistent with a model where key activity-regulated miRNAs are required to coordinate the expression of genes involved in activity-dependent synaptogenesis.

The conserved P body component HPat/Pat1 negatively regulates synaptic terminal growth at the larval Drosophila neuromuscular junction.

Summary The temporal and spatial regulation of protein synthesis plays an important role in the control of neural physiology. In axons and dendrites, translationally repressed mRNAs are actively transported to their destinations in a variety of ribonucleoprotein particles (RNPs). A subset of these neuronal RNPs has been shown to contain proteins associated with mRNA processing bodies (P bodies). P bodies are a class of highly conserved cytoplasmic granules that have been linked to both mRNA decay and translational repression via general and miRNA-mediated pathways. Here, we characterize functions for HPat/Pat1 (also known as Patr-1), a core component of P bodies, at the glutamatergic larval Drosophila neuromuscular junction (NMJ). We show that hpat mutants exhibit a strong synaptic hyperplasia at the NMJ. The synaptic defects observed in hpat mutants are associated with rearrangement of the axonal microtubule cytoskeleton suggesting that HPat negatively regulates presynaptic microtubule-based growth during NMJ development. Consistent with this, overexpression of HPat also blocks the rapid growth of presynaptic boutons induced by spaced depolarization. Finally, we demonstrate that HPat interacts genetically with the catalytic subunit of the deadenylase complex (twin/CCR4) and the miRNA pathway (Argonaute 1) to control bouton formation. We propose that HPat is required to target mRNAs involved in the control of microtubule architecture and synaptic terminal growth for repression, presumably in P bodies, via both general and miRNA-mediated mechanisms.

GW-Bodies and P-Bodies Constitute Two Separate Pools of Sequestered Non-Translating RNAs.

Non-translating RNAs that have undergone active translational repression are culled from the cytoplasm into P-bodies for decapping-dependent decay or for sequestration. Organisms that use microRNA-mediated RNA silencing have an additional pathway to remove RNAs from active translation. Consequently, proteins that govern microRNA-mediated silencing, such as GW182/Gw and AGO1, are often associated with the P-bodies of higher eukaryotic organisms. Due to the presence of Gw, these structures have been referred to as GW-bodies. However, several reports have indicated that GW-bodies have different dynamics to P-bodies. Here, we use live imaging to examine GW-body and P-body dynamics in the early Drosophila melanogaster embryo. While P-bodies are present throughout early embryonic development, cytoplasmic GW-bodies only form in significant numbers at the midblastula transition. Unlike P-bodies, which are predominantly cytoplasmic, GW-bodies are present in both nuclei and the cytoplasm. RNA decapping factors such as DCP1, Me31B, and Hpat are not associated with GW-bodies, indicating that P-bodies and GW-bodies are distinct structures. Furthermore, known Gw interactors such as AGO1 and the CCR4-NOT deadenylation complex, which have been shown to be important for Gw function, are also not present in GW-bodies. Use of translational inhibitors puromycin and cycloheximide, which respectively increase or decrease cellular pools of non-translating RNAs, alter GW-body size, underscoring that GW-bodies are composed of non-translating RNAs. Taken together, these data indicate that active translational silencing most likely does not occur in GW-bodies. Instead GW-bodies most likely function as repositories for translationally silenced RNAs. Finally, inhibition of zygotic gene transcription is unable to block the formation of either P-bodies or GW-bodies in the early embryo, suggesting that these structures are composed of maternal RNAs.