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

Abstract

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 Me31B's 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.

FMRP and Ataxin-2 function together in long-term olfactory habituation and neuronal translational control

“…tains, binding sites for several miRNAs and translational regulators in its 3′ UTR (48,55,56). We tested whether expression of this reporter mRNA is subject to common repression by dFmr1, and Atx2 in dendrites of PNs, as well as whether the reporter is subject to miRNA regulation in vivo, an issue that has not been clearly established by previous work (48,49). For these experiments, we used RNAi to k/d target proteins in PNs expressing the CaMKII translational reporter.…”

“…In addition, Atx2 function seems to be essential for the formation of Me31B-containing mRNP assemblies both in yeast as well as in mammalian cultured cells (39). Given the potential significance of these assemblies for translational control, we examined how loss of dFmr1 or Atx2 affected Me31B foci that can be visualized and quantified in both cell bodies and synapses of single genetically marked projection neurons in vivo (49) (Fig. 6 A and F).…”

“…Somatic spots were analyzed by "Spotnik," a MATLAB plug-in developed in collaboration with Kangyu Pan and Anil Kokaram (49). Dendritic spots were analyzed in Photoshop.…”

“…This potentially allows us to ask whether dFmr1 and Atx2 function in the same cells for LTH formation. In addition, the translation of the Ca2+/calmodulin-dependent protein kinase II (CaMKII) mRNA in PN dendrites, previously shown to be activity-regulated in vivo, is conveniently quantified using GFPbased translational reporters under control of the CaMKII 3′ UTR (48,49). Finally, mRNP assemblies containing the translational repressor Me31B/RCK may be visualized and quantified in cell bodies and synapses in vivo (49).…”

Long-term memory consolidation: The role of RNA-binding proteins with prion-like domains

“…Consistently several neuronal RNA-granule components such as Ataxin2, Staufen and FMRP have been shown to be required for long-term memory formation as well. 85,86,[183][184][185] Prion-like domains may regulate RNA granule assembly…”