A widely conserved mechanism of polarity establishment is the localization of mRNA to specific cellular regions. While it is clear that many mRNAs are transported to their destinations along microtubule tracks, much less is known regarding the mechanism by which these mRNAs are linked to microtubule motors. The RNA binding protein Egalitarian (Egl) is necessary for localization of several mRNAs in Drosophila oocytes and embryos. In addition to binding RNA, Egl also interacts with Dynein light chain (Dlc) and Bicaudal-D (BicD). The role of Dlc and BicD in mRNA localization has remained elusive. Like Egl, both proteins are required for oocyte specification. Null alleles in these genes result in an oogenesis block. In this report, we used an innovative approach to overcome the oogenesis block. Our findings reveal that the primary function of Dlc is to promote Egl dimerization. Loss of dimerization compromises the ability of Egl to bind RNA. Consequently, Egl is not bound to cargo, and is not able to efficiently associate with BicD and the Dynein motor. Our results therefore identify the key molecular steps required for assembling a localization competent mRNP.

A conserved mechanism of polarity establishment is the localization of mRNA to specific cellular regions. Although it is clear that many mRNAs are transported along microtubules, much less is known about the mechanism by which these mRNAs are linked to microtubule motors. The RNA binding protein Egalitarian (Egl) is necessary for localization of several mRNAs in Drosophila oocytes and embryos. Egl also interacts with Dynein light chain (Dlc) and Bicaudal-D (BicD). The role of Dlc and BicD in mRNA localization has remained elusive. Both proteins are required for oocyte specification, as is Egl. Null alleles in these genes result in an oogenesis block. In this report, we used an shRNA-depletion strategy to overcome the oogenesis block. Our findings reveal that the primary function of Dlc is to promote Egl dimerization. Loss of dimerization compromises the ability of Egl to bind RNA. Consequently, Egl is not bound to cargo, and is not able to efficiently associate with BicD and the Dynein motor. Our results therefore identify the key molecular steps required for assembling a localization-competent mRNP.

Messenger RNA (mRNA) localization is a powerful and prevalent mechanism of post-transcriptional gene regulation, enabling the cell to produce protein at the exact location at which it is needed. The phenomenon of mRNA localization has been observed in many types of cells in organisms ranging from yeast to man. Thus, the process appears to be widespread and highly conserved. Several model systems have been used to understand the mechanism by which mRNAs are localized. One such model, and the focus of this chapter, is the egg chamber of the female Drosophila melanogaster. The polarity of the developing Drosophila oocyte and resulting embryo relies on the specific localization of three critical mRNAs: gurken, bicoid, and oskar. If these mRNAs are not localized during oogenesis, the resulting progeny will not survive. The study of these mRNAs has served as a model for understanding the general mechanisms by which mRNAs are sorted. In this chapter, we will discuss how the localization of these mRNAs enables polarity establishment. We will also discuss the role of motor proteins in the localization pathway. Finally, we will consider potential mechanisms by which mRNAs can be anchored at their site of localization. It is likely that the lessons learned using the Drosophila oocyte model system will be applicable to mRNAs that are localized in other organisms as well.

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Egalitarian (Egl) is an RNA adaptor for the Dynein motor and is thought to link numerous, perhaps hundreds, of mRNAs with Dynein. Dynein, in turn, is responsible for the transport and localization of these mRNAs. Studies have shown that efficient mRNA binding by Egl requires the protein to dimerize. We recently demonstrated that Dynein light chain (Dlc) is responsible for facilitating the dimerization of Egl. Mutations in Egl that fail to interact with Dlc do not dimerize, and as such, are defective for mRNA binding. Consequently, this mutant does not efficiently associate with BicaudalD (BicD), the factor responsible for linking the Egl/mRNA complex with Dynein. In this report, we tested whether artificially dimerizing this Dlc-binding mutant using a leucine zipper would restore mRNA binding and rescue mutant phenotypes in vivo. Interestingly, we found that although artificial dimerization of Egl restored BicD binding, it only partially restored mRNA binding. As a result, Egl-dependent phenotypes, such as oocyte specification and mRNA localization, were only partially rescued. We hypothesize that Dlc-mediated dimerization of Egl results in a three-dimensional conformation of the Egl dimer that is best suited for mRNA binding. Although the leucine zipper restores Egl dimerization, it likely does not enable Egl to assemble into the conformation required for maximal mRNA binding activity.