Regulation of cellular function by localized synthesis of proteins

Many signaling molecules activate intracellular pathways that regulate gene transcription.  For example, growth factors and other signaling molecules act on cell receptors, which transduce a signal to the nucleus that results in increased transcription of specific genes, and a consequent increase in the level of the corresponding proteins.

However, it is becoming clear that protein expression in cells is also regulated by a variety of post-transcriptional mechanisms.  One mechanism involves the trafficking and "local" translation of specific mRNAs.  For example, in migrating cells, the leading edge is enriched in specific mRNAs that are subsequently translated, resulting in an enrichment of specific proteins at the leading edge.  The phenomenon of RNA trafficking and selective RNA localizations is especially prominent in neurons, in which specific mRNAs are selectively targeted to dendrites, synapses, axons, and growth cones.  Although the biological significance of these localized mRNAs remains largely unknown, local RNA translation appears to have critical roles in numerous fundamental processes in neurons.  Although local mRNA translation appears to have roles in numerous cell types, the extreme morphological polarization of neurons makes this system an excellent model system from which to gain fundamental insights into this widespread biological process. 

The Jaffrey laboratory is interested in several critical questions regarding this poorly understood signaling mechanism:

• Why are some mRNAs localized to specific sites in cells? 
• Which cellular processes require localized mRNAs and why is mRNA localization important for these processes? 
• Which mRNAs exhibit specific intracellular localizations?  How are mRNAs localized to specific subcellular sites?
• What are the signaling pathways that regulate the translation of these localized mRNAs?   

We have developed several novel tools to explore these questions.  We have developed novel culturing strategies that allow distal axons and growth cones to be selectively purified, which has allowed us to generate cDNA libraries and analyze the diversity of axonal mRNAs.  We have also developed new viral techniques using RNA-based viruses that allow us to selectively express proteins in axons, allowing us to ascertain the role of specific signaling proteins without affecting cellular processes in the cell body.  We have also utilized classical Campenot culturing chambers as well as newer microfluidic culturing devices to develop a siRNA-based technique to selectively abolish specific mRNAs from axons.  These techniques have allowed us to unambiguously define the role of the axonal pool of specific RNAs.  We have coupled these state of the art approaches with classical axonal guidance and synaptogenesis assays to begin to unravel the biological roles and regulatory pathways that control mRNA translation in axons.