Dynein-Dependent Transport ofnanosRNA inDrosophilaSensory Neurons Requires Rumpelstiltskin and the Germ Plasm Organizer Oskar

Abstract

Intracellular mRNA localization is a conserved mechanism for spatially regulating protein production in polarized cells, such as neurons. The mRNA encoding the translational repressor Nanos (Nos) forms ribonucleoprotein (RNP) particles that are dendritically localized in Drosophila larval class IV dendritic arborization (da) neurons. In nos mutants, class IV da neurons exhibit reduced dendritic branching complexity, which is rescued by transgenic expression of wild-type nos mRNA but not by a localization-compromised nos derivative. While localization is essential for nos function in dendrite morphogenesis, the mechanism underlying the transport of nos RNP particles was unknown. We investigated the mechanism of dendritic nos mRNA localization by analyzing requirements for nos RNP particle motility in class IV da neuron dendrites through live imaging of fluorescently labeled nos mRNA. We show that dynein motor machinery components mediate transport of nos mRNA in proximal dendrites. Two factors, the RNA-binding protein Rumpelstiltskin and the germ plasm protein Oskar, which are required for diffusion/entrapment-mediated localization of nos during oogenesis, also function in da neurons for formation and transport of nos RNP particles. Additionally, we show that nos regulates neuronal function, most likely independent of its dendritic localization and function in morphogenesis. Our results reveal adaptability of localization factors for regulation of a target transcript in different cellular contexts.

In the right place at the right time: visualizing and understanding mRNA localization

“…It is also intriguing that a single mRNA species may be subject to different mechanisms of transport in different cell types; for example, β-actin mRNA in glia cells is largely diffusive, whereas in neuronal dendrites β-actin and ARC mRNAs are static or motored 58, 62, 100, 101 (see Supplementary information S3 (movie)). Although it is tempting to assume that altered mRNA behaviour is the result of disparate RBP expression, enigmatically the same regulatory proteins that localize nanos mRNA to the posterior pole of D. melanogaster embryos through diffusion and entrapment (Box 2) have a role in mediating its dynein-dependant transport in larval neurons 102 .…”

Fixed and live visualization of RNAs in Drosophila oocytes and embryos

“…The hsp83 promoter produces higher levels of MCP than the nos promoter but is also active in the somatic follicle cells surrounding the egg chamber, which can interfere with visualization of mRNA in the oocyte from early to mid-oogenesis. For zygotic expression, the Gal4/UAS system affords the most versatility [50, 51] but ubiquitous promoters, including hsp83 , have also been used [51, 52]. …”

“…We have also had good results imaging mRNA transport in larval neurons by spinning disc confocal microscopy [44, 51]. The choice of microscope will depend on the specific specimen and the particular application.…”

The RNA-binding protein Rumpelstiltskin antagonizes gypsy chromatin insulator function in a tissue-specific manner

“…This result suggests that a certain threshold of Shep is sufficient to reduce insulator activity. By contrast, Rump is ubiquitously expressed and is also known to function in nos localization in sensory neurons (Xu et al, 2013); therefore, an alternative mechanism must exist to impart the specificity of its regulatory activity on insulator function.…”

“…Based on this function, a lack of maternal Rump expression results in defects in early posterior patterning of the embryo. Recent work shows that Rump is also required for nos transcript localization in sensory neuron dendrites (Xu et al, 2013). In fact, Rump is expressed throughout all stages of development and in all cell lines tested (Cherbas et al, 2011;Chintapalli et al, 2007), suggesting that it might have yet undiscovered roles.…”