RNA Localization in flies and mammals: the contribution of translational silencing and mRNA Degradation factors / LSD

Translational control of localized mRNAs is a common mechanism for regulating protein expression in specific cellular subdomains and plays an important role in a number of processes, such as axes formation, asymmetric cell division, cell motility, and neuronal synaptic plasticity (1-3). Localized mRNAs are usually transported in ribonucleoprotein particles (RNPs) and must be translationally repressed until the RNA reaches its final destination. This is achieved by translational repressor molecules, e.g. Bruno, CPEB,eIF4AIII, FMRP, Staufen and ZBP1 that are present in these transport RNPs. It has recently emerged that mRNA degradation factors also play an essential role in mRNA localization. For example, the transport of oskar (osk) mRNA to the posterior of the Drosophila oocyte requires the DEAD-box RNA helicase Me31b/DDX-6, a decapping activator, and the Dcp-1 subunit of the decapping enzyme that removes the 5'-cap from mRNAs to trigger their degradation. Both proteins colocalize with osk mRNA at the oocyte posterior. Furthermore, osk mRNA localization also depends on the exon junction complex (EJC) and Staufen, both of which have been implicated in mRNA decay in mammals. The EJC must be bound to an mRNA downstream of a stop codon to trigger nonsense mediated decay (NMD), while Staufen 1 recruits the NMD factor Upf1 to specific mRNA 3'-UTRs and thereby induces a novel form of mRNA decay. In mammals, Staufen 1 is a component of dendritic mRNA transport complexes. Moreover, the DEAD-box protein DDX-6, the mammalian homolog of Me31b, is found in kinesin-associated RNA granules isolated from rat brain. osk mRNA localization is also disrupted by mutations in armitage (armi), spindle-E (spn-E) and maelstrom, all RISC components mediating siRNA-dependent RNA degradation and miRNA-dependent translational silencing. In contrast, the Argonaute proteins, Aubergine (Aub) and Piwi, are required for efficient osk mRNA translation once it is localized, and both proteins accumulate at the posterior with the mRNA. Both proteins have been shown to associate with a new class of small RNAs called repeat-associated small interfering RNAs (rasiRNAs) to repress the activity of transposable elements in the germline. However, it is unclear whether Aub and Piwi that localize with osk mRNA are bound to rasiRNAs, nor whether the latter play any role in osk mRNA translation or degradation.Finally, there is recent evidence that not only RNA degradation but also translational silencing is coupled to RNA transport in mammals, since non-coding RNAs, such as microRNAs (miRNAs) and longer regulatory RNAs, e.g. BC1, can repress translation of mRNAs during transport (22-24). It has been postulated that this miRNA-guided silencing occurs in another class of RNPs, called processing bodies (Pbodies),which are the major sites of mRNA degradation in both invertebrate and vertebrate cells , and that repressed mRNAs can even be released from P-bodies upon specific signals into the cytoplasm for further translation. This raises the question of whether P-bodies provide a platform for the transport of translationally repressed RNAs, and function as centers that co-ordinate degradation, translation and localization. We would therefore like to investigate whether P-body components are involved in mRNA transport by addressing the following specific questions: Do protein components of the RNA degradation pathway and the RISC complex play a direct role in localizing RNAs in Drosophila oocytes or mammalian neurons? Do small non-coding RNAs silence mRNA during their transport in both systems? Do localized mRNAs associate with P-bodies, before, during or after their transport to their destination? What is the molecular function of the Argonaute family members Aub and Piwi in osk mRNA localization and translation in Drosophila oocytes? Are individual members of the mammalian Ago family, e.g. Ago1-5, associated with specific miRNAs in dendritic RNPs?

Although degradation factors and RISC components are required for osk mRNA localization , it remains to be proven that they are components of osk transport RNPs. We plan to take several approaches to investigate their function in osk mRNA localization. 1) Using wide-field deconvolution microscopy, and the MS2-GFP technique to label osk mRNA , we have recently succeeded in visualizing the kinesin-dependent movement of osk mRNPs to the oocyte posterior. We plan to investigate whether GFP-tagged Dcp-1, Me31b, and RISC proteins are components of these osk mRNA transport complexes by performing two-color timelapse videomicroscopy with MS2-RFP labeled osk mRNA. This will reveal whether these proteins associate with the mRNA during its transport, or before or after its localization to the posterior. The latter results would suggest that they play a role in a different process, such as the assembly of transport RNPs, the translational regulation of localized mRNAs or the degradation of unlocalized mRNA. 2) Since the EJC is thought to be removed from the mRNA by translation, the osk localization defects of mutants in degradation and RISC components could be caused by premature translation of osk mRNA. To test this hypothesis, we will determine whether their localization defects are rescued by expressing non-translatable osk mRNA. 3) We have now shown that different components of the osk transport complex (Hrp48, EJC and Staufen) disrupt its motility in distinct ways. We will therefore examine how mutants in P-body (Me31b, Dcp-1) or in RNAi pathway proteins (aub, armi, spn-E) affect the motility of osk transport RNPs to determine at which step in the pathway they act. 4) We will purify RNPs containing tandem affinity-tagged Aub and Piwi from Drosophila ovaries andcompare the composition of these complexes with that of osk RNPs that we are purifying in parallel using TAP-tagged MS2-GFP/osk RNA and Staufen.