Regulation of local translation in glia

ABSTRACT To enable efficient specialization and dynamic regulation of subcellular regions, many cells have evolved local translation of mRNA - yet the fundamental principles of such translation regulation in astrocytes are unknown. Long studied in neurons, local translation of a variety of proteins is thought to be essential for the synapse- specific changes that underlay learning and memory. In the prior cycle of this award we provided evidence astrocytes also have a regulated local translation by using a variety of approaches. Here, we propose to continue this work, focusing on the following question regarding this fascinating new basic biological phenomenon: what is the regulatory grammar that determines when and which transcripts are locally translated in astrocytes? Our central model is that elements in the untranslated regions (UTRs) of transcripts are responsible for their enrichment or depletion from ribosomes in peripheral astrocyte processes (PAPs), via UTR interactions with RNA binding proteins (RBPs) and microRNAs (miRNAs). However, with hundreds of potential elements to screen, new, scalable methods are needed to systematically characterize how RNA localization and translation is regulated in astrocytes, both at baseline and in response to signaling cues. Furthermore, glial morphology only reaches full maturity in vivo, requiring in vivo functional studies. Therefore, we have developed a new method, a synaptoneurosome–massively parallel reporter assay (SN-MPRA) which allows us to assess functional effects of thousands of candidate UTR elements in vivo simultaneously. We will apply this to define the sequences that modulate RNA localization in astrocytes. Furthermore, to better understand how a subset of these elements function, we will define the role of a specific RBP, ‘quaking’ (QKI), in modulating local translation in astrocytes. Finally, to understand how sets of transcripts might be regulated in a coordinated fashion for local translation, we will examine the role of miRNA effector proteins (Ago2) along with specific miRNAs in regulating local translation in astrocytes.