Syncrip/hnRNP Q is required for activity-induced Msp300/Nesprin-1 expression and new synapse formation.

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

Drosophila gurken (TGFα) mRNA Localizes as Particles that Move within the Oocyte in Two Dynein-Dependent Steps

AbstractIn Drosophila oocytes, gurken mRNA localization orientates the TGF-α signal to establish the anteroposterior and dorsoventral axes. We have elucidated the path and mechanism of gurken mRNA localization by time-lapse cinematography of injected fluorescent transcripts in living oocytes. gurken RNA assembles into particles that move in two distinct steps, both requiring microtubules and cytoplasmic Dynein. gurken particles first move toward the anterior and then turn and move dorsally toward the oocyte nucleus. We present evidence suggesting that the two steps of gurken RNA transport occur on distinct arrays of microtubules. Such distinct microtubule networks could provide a general mechanism for one motor to transport different cargos to distinct subcellular destinations

Subcellular mRNA localisation at a glance.

mRNA localisation coupled to translational regulation provides an important means of dictating when and where proteins function in a variety of model systems. This mechanism is particularly relevant in polarised or migrating cells. Although many of the models for how this is achieved were first proposed over 20 years ago, some of the molecular details are still poorly understood. Nevertheless, advanced imaging, biochemical and computational approaches have started to shed light on the cis-acting localisation signals and trans-acting factors that dictate the final destination of localised transcripts. In this Cell Science at a Glance article and accompanying poster, we provide an overview of mRNA localisation, from transcription to degradation, focusing on the microtubule-dependent active transport and anchoring mechanism, which we will use to explain the general paradigm. However, it is clear that there are diverse ways in which mRNAs become localised and target protein expression, and we highlight some of the similarities and differences between these mechanisms.This work was supported by a Wellcome Trust Senior Research Fellowship to I.D. supporting R.M.P. [grant number: 096144], a studentship from the Wellcome Trust to A.D. [grant number: 097304], the University of Cambridge, ISSF to T.T.W. [grant number 097814].This is the final version of the article. It first appeared from the Company of Biologists via http://dx.doi.org/10.1242/jcs.11427

Imp/IGF2BP levels modulate individual neural stem cell growth and division through myc mRNA stability.

The numerous neurons and glia that form the brain originate from tightly controlled growth and division of neural stem cells, regulated systemically by important known stem cell-extrinsic signals. However, the cell-intrinsic mechanisms that control the distinctive proliferation rates of individual neural stem cells are unknown. Here, we show that the size and division rates of Drosophila neural stem cells (neuroblasts) are controlled by the highly conserved RNA binding protein Imp (IGF2BP), via one of its top binding targets in the brain, myc mRNA. We show that Imp stabilises myc mRNA leading to increased Myc protein levels, larger neuroblasts, and faster division rates. Declining Imp levels throughout development limit myc mRNA stability to restrain neuroblast growth and division, and heterogeneous Imp expression correlates with myc mRNA stability between individual neuroblasts in the brain. We propose that Imp-dependent regulation of myc mRNA stability fine-tunes individual neural stem cell proliferation rates

Localized Translation of gurken/TGF-α mRNA during Axis Specification Is Controlled by Access to Orb/CPEB on Processing Bodies.

In Drosophila oocytes, gurken/TGF-α mRNA is essential for establishing the future embryonic axes. gurken remains translationally silent during transport from its point of synthesis in nurse cells to its final destination in the oocyte, where it associates with the edge of processing bodies. Here we show that, in nurse cells, gurken is kept translationally silent by the lack of sufficient Orb/CPEB, its translational activator. Processing bodies in nurse cells have a similar protein complement and ultrastructure to those in the oocyte, but they markedly less Orb and do not associate with gurken mRNA. Ectopic expression of Orb in nurse cells at levels similar to the wild-type oocyte dorso-anterior corner at mid-oogenesis is sufficient to cause gurken mRNA to associate with processing bodies and translate prematurely. We propose that controlling the spatial distribution of translational activators is a fundamental mechanism for regulating localized translation.This work was supported by a studentship from the Wellcome Trust (grant 097304 to A.D.), a Wellcome Trust Senior Research Fellowship (grant 096144 to I.D and supporting R.M.P), the University of Cambridge, ISSF (grant 097814 to T.T.W), and Wellcome Trust Strategic Awards 091911 and 107457 supporting advanced microscopy at Micron Oxford (http://micronoxford.com).This is the author accepted manuscript. The final version is available from Cell Press via http://dx.doi.org/10.1016/j.celrep.2016.02.03

Expanding horizons: new roles for non-canonical RNA-binding proteins in cancer

Cancer development involves the stepwise accumulation of genetic lesions that overcome the normal regulatory pathways that prevent unconstrained cell division and tissue growth. Identification of the genetic changes that cause cancer has long been the subject of intensive study, leading to the identification of several RNA-binding proteins (RBPs) linked to cancer. Cross-reference of the complement of RBPs recently identified by RNA interactome capture with cancer-associated genes and biological processes led to the identification of a set of 411 proteins with potential implications in cancer biology. These involve a broad spectrum of cellular processes including response to stress, metabolism and cell adhesion. Future studies should aim to understand these proteins and their connection to cancer from an RNA-centred perspective, holding the promise of new mechanistic understanding of cancer formation and novel approaches to diagnosis and treatment

A single and rapid calcium wave at egg activation in Drosophila.

Activation is an essential process that accompanies fertilisation in all animals and heralds major cellular changes, most notably, resumption of the cell cycle. While activation involves wave-like oscillations in intracellular Ca(2+) concentration in mammals, ascidians and polychaete worms and a single Ca(2+) peak in fish and frogs, in insects, such as Drosophila, to date, it has not been shown what changes in intracellular Ca(2+) levels occur. Here, we utilise ratiometric imaging of Ca(2+) indicator dyes and genetically encoded Ca(2+) indicator proteins to identify and characterise a single, rapid, transient wave of Ca(2+) in the Drosophila egg at activation. Using genetic tools, physical manipulation and pharmacological treatments we demonstrate that the propagation of the Ca(2+) wave requires an intact actin cytoskeleton and an increase in intracellular Ca(2+) can be uncoupled from egg swelling, but not from progression of the cell cycle. We further show that mechanical pressure alone is not sufficient to initiate a Ca(2+) wave. We also find that processing bodies, sites of mRNA decay and translational regulation, become dispersed following the Ca(2+) transient. Based on this data we propose the following model for egg activation in Drosophila: exposure to lateral oviduct fluid initiates an increase in intracellular Ca(2+) at the egg posterior via osmotic swelling, possibly through mechano-sensitive Ca(2+) channels; a single Ca(2+) wave then propagates in an actin dependent manner; this Ca(2+) wave co-ordinates key developmental events including resumption of the cell cycle and initiation of translation of mRNAs such as bicoid.This work was supported by the University of Cambridge, ISSF to T.T.W. [grant number 097814]; and Wellcome Trust Senior Research Fellowship to I.D. [grant number 096144].This is the final version of the article. It first appeared from the Company of Biologists via http://dx.doi.org/10.1242/bio.20141129

ParticleStats: open source software for the analysis of particle motility and cytoskeletal polarity

The study of dynamic cellular processes in living cells is central to biology and is particularly powerful when the motility characteristics of individual objects within cells can be determined and analysed statistically. However, commercial programs only offer a limited range of inflexible analysis modules and there are currently no open source programs for extensive analysis of particle motility. Here, we describe ParticleStats (http://www.ParticleStats.com), a web server and open source programs, which input the X,Y coordinate positions of objects in time, and output novel analyses, graphical plots and statistics for motile objects. ParticleStats comprises three separate analysis programs. First, ParticleStats:Directionality for the global analysis of polarity, for example microtubule plus end growth in Drosophila oocytes. Second, ParticleStats:Compare for the analysis of saltatory movement in terms of runs and pauses. This can be applied to chromosome segregation and molecular motor-based movements. Thirdly ParticleStats:Kymographs for the analysis of kymograph images, for example as applied to separation of chromosomes in mitosis. These analyses have provided key insights into molecular mechanisms that are not possible from qualitative analysis alone and are widely applicable to many other cell biology problems

A PAR-1–dependent orientation gradient of dynamic microtubules directs posterior cargo transport in the Drosophila oocyte

A PAR-1–mediated bias in microtubule organization in the Drosophila oocyte underlies posterior-directed mRNA transport

RNA:RNA Interaction Can Enhance RNA Localisation in Drosophila Oocytes

RNA localization is a key mechanism for targeting proteins to particular subcellular domains. Sequences necessary and sufficient for localization have been identified, but little is known about factors that affect its kinetics. Transcripts of gurken and the I factor, a non-LTR retrotransposon, colocalize at the nucleus in the dorso-antero corner of the Drosophila oocyte directed by localization signals, the GLS and ILS. I factor RNA localizes faster than gurken after injection into oocytes, due to a difference in the intrinsic localization ability of the GLS and ILS. The kinetics of localization of RNA containing the ILS are enhanced by the presence of a stem-loop, the A loop. This acts as an RNA:RNA interaction element in vivo and in vitro, and stimulates localization of RNA containing other localization signals. RNA:RNA interaction may be a general mechanism for modulating RNA localization and could allow an mRNA that lacks a localization signal to hitchhike on another RNA that has one