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The neuronal transcriptome changes dynamically to adapt to stimuli from the extracellular and intracellular environment. In this study, we adapted for the first time a click chemistry technique to label the newly synthesized RNA in cultured hippocampal neurons and intact larval zebrafish brain. Ethynyl uridine (EU) was incorporated into neuronal RNA in a time- and concentration-dependent manner. Newly synthesized RNA granules observed throughout the dendrites were colocalized with mRNA and rRNA markers. In zebrafish larvae, the application of EU to the swim water resulted in uptake and labeling throughout the brain. Using a GABA receptor antagonist, PTZ (pentylenetetrazol), to elevate neuronal activity, we demonstrate that newly transcribed RNA signal increased in specific regions involved in neurogenesis.
Local protein synthesis has re-defined our ideas on the basic cellular mechanisms that underlie synaptic plasticity and memory formation. The population of messenger RNAs that are localised to dendrites, however, remains sparsely identified. Furthermore, neuronal morphological complexity and spatial compartmentalisation require efficient mechanisms for messenger RNA localisation and control over translational efficiency or transcript stability. 3’ untranslated regions, downstream from stop codons, are recognised for providing binding platforms for many regulatory units, thus encoding the processing of the above processes. The hippocampus, a part of the brain involved in the formation, organisation and storage of memories, provides a natural platform to investigate patterns of RNA localisation. The hippocampus comprises tissue layers, which naturally separate the principle neuronal cell bodies from their processes (axons and dendrites). Identifying the full-complement of localised transcripts and associated 3’UTR isoforms is of great importance to understand both basic neuronal functions and principles of synaptic plasticity. These findings can be used to study the properties of neuronal networks as well as to understand how these networks malfunction in neuronal diseases.
Here, deep sequencing is used to identify the mRNAs resident in the synaptic neuropil in the hippocampus. Analysis of a neuropil data set yields a list of 8,379 transcripts of which 2,550 are localised in dendrites and/or axons. Using a fluorescent barcode strategy to label individual mRNAs shows that the relative abundance of different mRNAs in the neuropil varies over 5 orders of magnitude. High-resolution in situ hybridisation validated the presence of mRNAs in both cultured neurons and hippocampal slices. Among the many mRNAs identified, a large fraction of known synaptic proteins including signaling molecules, scaffolds and receptors is discovered. These results reveal a previously unappreciated enormous potential for the local protein synthesis machinery to supply, maintain and modify the dendritic and synaptic proteome.
Using advances in library preparation for next generation sequencing experiments, the diversity of 3’UTR isoforms present in localised transcripts from the rat hippocampus is examined. The obtained results indicate that there is an increase in 3’UTR heterogeneity and 3’UTR length in neuronal tissue. The evolutionary importance of the 3’UTR diversity and correlation with changes in species,tissue and cell complexity is investigated. The conducted analysis reveals the population of 3’UTR isoforms required for transcript localisation in overall neuronal transcriptome as well as the regulatory elements and binding sites specific for neuronal compartments. The configuration of poly(A) signals is correlated with gene function and can be further exploit to determine similar mechanisms for alternative polyadenylation.
Usage of custom specified methods for next-generation sequencing as well as novel approaches for RNA quantification and visualisation necessitate the development and implementation of new downstream analytic methods. Library methods for data-mining transcripts annotation, expression and ontology relations is provided. Usage of a specialised search engine targeting key features of previous experiments is proposed. A processing pipeline for NanoString technology, defining experimental quality and exploiting methods for data normalisation is developed. High-resolution in situ images are analysed by custom application, showing a correlation between RNA quantity and spatial distribution. The vast variety of bioinformatic methods included in this work indicates the importance of downstream analysis to reach biological conclusions. Maintaining the integrability and modularity of our implementations is of great priority, as the dynamic nature of many experimental techniques requires constant improvement in computational analysis.