Characterization of the microRNA pathway in neurons.

DE LA MATA, M., NGUYEN, T.-M.-P., FILIPOWICZ, W.

Kovalam

Encuentro; 10th Annual HFSP Awardees Meeting.; 2010

Human Frontier Science Program

Increasing evidence indicates that the development of the nervous system is dependent on the spatiotemporal expression of a wide repertoire of small RNAs and longer non-coding RNAs. At the stage of mature neurons, local post-synaptic translation seems to have a particularly important role in the most persistent forms of synaptic plasticity and memory. Several mRNAs are transported along the dendrites as repressed mRNPs and become translated at dendritic spines upon synaptic activation. A few reports demonstrate that miRNAs regulate local protein translation in neurons dendrites, however, little is known about the localization of the proteins involved and molecular mechanisms of this process. We have tagged several components of the miRNA pathway (e.g. Ago2, Tnrc6b, Dicer) including a neuron specific alternative splicing isoform of Tnrc6b (a member of the GW182 family of translational repressors) that we recently discovered. This neuron specific isoform of Trnc6b includes and exon (171nt) which does not code for any of the well characterized domains of the protein but it is located just upstream of the Q-rich domain previously shown to be involved in P-body localization. This suggests that this exon might be important for specific localization in neurons. We will express these constructs in neurons which will be then subjected to different culture conditions. We are particularly interested in following the localization and movement of these proteins upon different culture conditions such as those that mimic an activated neuron state (e.g. using neurotransmitter agonists) or by inhibiting neuron activity (e.g. with drugs such as tetrodotoxin). We also plan to do FRET analysis and EM imaging to get additional insight into the localization, structure and possible function of these proteins, which may be important for fine tuning synaptic tagging and plasticity in memory formation.