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

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

RNA localization in neurite morphogenesis and synaptic regulation: current evidence and novel approaches

It is now generally accepted that RNA localization in the central nervous system conveys important roles both during development and in the adult brain. Of special interest is protein synthesis located at the synapse, as this potentially confers selective synaptic modification and has been implicated in the establishment of memories. However, the underlying molecular events are largely unknown. In this review, we will first discuss novel findings that highlight the role of RNA localization in neurons. We will focus on the role of RNA localization in neurotrophin signaling, axon outgrowth, dendrite and dendritic spine morphogenesis as well as in synaptic plasticity. Second, we will briefly present recent work on the role of microRNAs in translational control in dendrites and its implications for learning and memory. Finally, we discuss recent approaches to visualize RNAs in living cells and their employment for studying RNA trafficking in neurons

Dynactin suppresses the retrograde movement of apically localized mRNA in Drosophila blastoderm embryos

Motor dependent transport of mRNA is a key mechanism in axis specification during development. Apical transport and anchoring of wingless and pair-rule transcripts in the Drosophila syncytial blastoderm embryo is mediated by cytoplasmic Dynein, the major minus end directed microtubule dependent molecular motor. Here, we show that, despite apical transport of mRNA being highly directional, mRNA particles often pause and move backward toward the plus ends of microtubules. We suggest that this retrograde movement helps overcome cellular obstructions. We show that the plus end movement of apical mRNA is independent of the major plus end microtubule motors Kinesin-1 and Kinesin-2. In contrast, Dynactin, a Dynein processivity factor, is required to suppress retrograde mRNA movements, as well as for efficient minus end motility. We propose that Dynein itself, rather than the activity of a plus end motor, is responsible for the plus end movements of the mRNA and that Dynactin is involved in preventing short reverse movements of the Dynein motor, known to occur in vitro

A bioinformatics search pipeline, RNA2DSearch, identifies RNA localization elements in Drosophila retrotransposons

mRNA localization is a widespread mode of delivering proteins to their site of function. The embryonic axes in Drosophila are determined in the oocyte, through Dynein-dependent transport of gurken/TGF-α mRNA, containing a small localization signal that assigns its destination. A signal with a similar secondary structure, but lacking significant sequence similarity, is present in the I factor retrotransposon mRNA, also transported by Dynein. It is currently unclear whether other mRNAs exist that are localized to the same site using similar signals. Moreover, searches for other genes containing similar elements have not been possible due to a lack of suitable bioinformatics methods for searches of secondary structure elements and the difficulty of experimentally testing all the possible candidates. We have developed a bioinformatics approach for searching across the genome for small RNA elements that are similar to the secondary structures of particular localization signals. We have uncovered 48 candidates, of which we were able to test 22 for their localization potential using injection assays for Dynein mediated RNA localization. We found that G2 and Jockey transposons each contain a gurken/I factor-like RNA stem–loop required for Dynein-dependent localization to the anterior and dorso–anterior corner of the oocyte. We conclude that I factor, G2, and Jockey are members of a “family” of transposable elements sharing a gurken-like mRNA localization signal and Dynein-dependent mechanism of transport. The bioinformatics pipeline we have developed will have broader utility in fields where small RNA signals play important roles

A loss of function allele for murine Staufen1 leads to impairment of dendritic Staufen1-RNP delivery and dendritic spine morphogenesis

The dsRNA-binding protein Staufen was the first RNA-binding protein proven to play a role in RNA localization in Drosophila. A mammalian homolog, Staufen1 (Stau1), has been implicated in dendritic RNA localization in neurons, translational control, and mRNA decay. However, the precise mechanisms by which it fulfills these specific roles are only partially understood. To determine its physiological functions, the murine Stau1 gene was disrupted by homologous recombination. Homozygous stau1tm1Apa mutant mice express a truncated Stau1 protein lacking the functional RNA-binding domain 3. The level of the truncated protein is significantly reduced. Cultured hippocampal neurons derived from stau1tm1Apa homozygous mice display deficits in dendritic delivery of Stau1-EYFP and β-actin mRNA-containing ribonucleoprotein particles (RNPs). Furthermore, these neurons have a significantly reduced dendritic tree and develop fewer synapses. Homozygous stau1tm1Apa mutant mice are viable and show no obvious deficits in development, fertility, health, overall brain morphology, and a variety of behavioral assays, e.g., hippocampus-dependent learning. However, we did detect deficits in locomotor activity. Our data suggest that Stau1 is crucial for synapse development in vitro but not critical for normal behavioral function