Molecular mechanisms underlying dendritic transport and processing of neuronal microRNAs
Prof. Dr. Gerhard Schratt
Institut für Physiologische Chemie
Karl-von-Frisch Str. 1
microRNAs (miRNAs) are an extensive class of small, non-coding RNAs that constitute a major new layer of post-transcriptional control of gene expression throughout the animal and plant kingdom. In vertebrates, miRNAs mostly bind to the 3’ untranslated region (UTR) of mRNAs, thereby interfering with productive translation and/or inducing mRNA degradation. Hence, miRNAs are emerging as crucial regulators of many cellular processes, including differentiation, metabolic function, homeostasis and transformation.
In neurons, the local translation of mRNAs that are transported into the synapto-dendritic compartment plays a crucial role in synapse development and plasticity. Over the past four years, work from our laboratory has identified specific neuronal miRNAs that dynamically regulate local mRNA translation at the synapse. In particular, we found that miR-134 and miR-138, two dendritically localized miRNAs, negatively regulate the size of dendritic spines, specialized postsynaptic contact sites. However, the molecular mechanisms underlying directed transport of miRNAs to the synapto-dendritic compartment are completely unknown.
To tackle this question, we have recently embarked on genome-wide expression profiling of miRNAs and miRNA precursors (pre-miRs) using microarray technology. Surprisingly, we found that several pre-miRs, including pre-miR-134, are highly enriched in synaptosomes, a biochemical preparation of purified synaptic terminals. Dendritic localization of pre-miR-134 in hippocampal neurons could be validated by in situ hybridization. This raises the intriguing possibility that at least part of the synaptic pool of miR-134 (and possibly other miRNAs) is derived from active transport of pre-miR-134 to the synapto-dendritic compartment followed by local processing at the synapse.
Based on these preliminary observations, we plan to pursue two major lines of investigation. First, we will attempt to identify the protein(s) mediating dendritic transport of pre-miR-134 using affinity-tag purification from synaptosome protein extracts followed by mass spectrometry. We will then be in a position to interrogate the functional importance of the identified candidates for dendritic targeting using RNA interference and genetic tools. Furthermore, we will address how neural activity modulates pre-miR transport using high-resolution time-lapse imaging. Second, we will assess how stimulation of individual synapses affects pre-miR-134 processing. Given the presence of the processing enzyme Dicer at synapses, this could represent an additional layer of regulation for miR-134 activity. Towards this aim, we will develop novel fluorescent miR-134 sensor constructs that allow the spatial and temporal visualization of miR-134 activity at synapses. This will be complemented by the measurement of structural and functional determinants of miR-134 activity, including spine morphogenesis and synapse physiology.
In conclusion, the proposed project promises to unravel a completely novel layer of miRNA regulation with implications for synaptic plasticity and other processes that are dependent on compartmentalized gene expression.
Working model for molecular mechanism underlying pre-miR-134 nuclear export, transport to and local processing within the synapto-dendritic compartment. Pre-miR-134 is bound by an unknown factor („X“) in the nucleear that is involved in nuclear export. Alternatively, „X“ assembles with the pre-miR after nuclear export in the cell body, facilitating dendritic transport while blocking premature processing of pre-miR-134. In the postsynaptic compartment, stimulation of specific synapses triggers Dicer-dependent pre-miR-134 processing, thereby resulting in enhanced silencing of the local translation of miR-134 target mRNAs.
Bicker, Silvia (PhD student), firstname.lastname@example.org, phone: +49-6221-566214
Publications since 2007
Siegel, G., Saba, R. and Schratt G. (2011) microRNAs in neurons: manifold regulatory roles at the synapse. Curr. Op. Genet. Dev., in press.
Bicker, S. and Schratt, G. (2010) Not miR-ly aging: SIRT1 boosts memory via a microRNA-dependent mechanism. Cell Res. 20 (11):1175-7.
Krol J, Busskamp V, Markiewicz I, Stadler MB, Ribi S, Richter J, Duebel J, Bicker S, Fehling HJ, Schübeler D, Oertner TG, Schratt G, Bibel M, Roska B and Filipowicz W. (2010) Characterizing light-regulated retinal microRNAs reveals rapid turnover as a common property of neuronal microRNAs. Cell, 14;141(4):618-31.
Saba, R. and Schratt G. (2010) MicroRNAs in neuronal development, function and dysfunction. Brain Res. 1338:3-13.
Christensen, M., Larsen, L.A., Kauppinen, S. & Schratt G. (2010) Recombinant adeno-associated virus-mediated microRNA delivery into the postnatal mouse brain reveals a role for miR-134 in dendritogenesis in vivo. Frontiers in Neural Circuits,3:16.
Khudayberdiev S., Fiore R. and Schratt G. (2009) microRNAs as modulators of neuronal responses. Commun Integr Biol. 2(5): 411-3.
Schratt, G. (2009) microRNAs at the synapse. Nat Rev Neurosci. 10(12): 842-9.
Schratt G. (2009) Fine-tuning neural gene expression with microRNAs. Curr. Op. Neurobiol., 19(2):213-9.
Siegel G.*, Obernosterer G.*, Fiore R., Oehmen, M., Bicker, S., Christensen M., Khudayberdiev, S., Leuschner, P., Busch, C., Kane, C., Hübel K., Dekker, F., Hedberg, C., Rengarajan, B., Drepper, C., Waldmann H., Kauppinen S., Greenberg M.E., Draguhn, A., Rehmsmeier M., Martinez J. and Schratt G. (2009) A functional microRNA screen implicates miR-138-dependent regulation of the depalmitoylation enzyme APT1 in dendritic spine morphogenesis. Nature Cell Biology, 11(6):705-716.
Christensen, M. and Schratt, G. (2009) microRNA involvement in developmental and functional aspects of the nervous system and in neurological diseases. Neurosci. Lett. 4;466(2):55-62.
Fiore R.*, Khudayberdiev S.*, Christensen M., Siegel G., Flavell S., Kim T.K., Greenberg M.E. and Schratt G. (2009) Mef2-dependent activation of the miR-379-410 cluster promotes dendritic outgrowth by fine-tuning protein levels of the translational repressor Pumilio2. EMBO J.18;28(6):697-710.
Bicker S. and Schratt G. (2008) microRNAs: Tiny regulators of synapse function in development and disease. J Cell Mol Med., 12(5A):1466-76.
Fiore R., Siegel G. and Schratt G. (2008) microRNA function in neuronal development, plasticity and disease. Biochim Biophys Acta 1779(8): 471-8.
Fiore R. and Schratt G. (2007) microRNAs in vertebrate synapse development. The Scientific World Journal 7: 167-77.
Fiore R. and Schratt G. (2007) microRNAs in synapse development: Tiny molecules to remember? Expert Opinion in Biological Therapies 7(12): 1823-31.
Satterlee, J., Barbee, S., Jin, P., Krichevsky, A., Salama, S., Schratt, G. and Wu, D.Y. (2007) Non-coding RNAs in the Brain. J. Neurosci 27(44): 11856-9.