Identification of Process-Localized mRNAs from Cultured Rodent Hippocampal Neurons

Abstract

The regulated translation of localized mRNAs in neurons provides a mechanism for spatially restricting gene expression in a synapsespecific manner. To identify the population of mRNAs present in distal neuronal processes of rodent hippocampal neurons, we grew neurons on polycarbonate filters etched with 3 m pores. Although the neuronal cell bodies remained on the top surface of the filters, dendrites, axons, and glial processes penetrated through the pores to grow along the bottom surface of the membrane where they could be mechanically separated from cell bodies. Quantitative PCR and immunochemical analyses of the process preparation revealed that it was remarkably free of somatic contamination. Microarray analysis of RNA isolated from the processes identified over 100 potentially localized mRNAs. In situ hybridization studies of 19 of these transcripts confirmed that all 19 were present in dendrites, validating the utility of this approach for identifying dendritically localized transcripts. Many of the identified mRNAs encoded components of the translational machinery and several were associated with the RNA-binding protein Staufen. These findings indicate that there is a rich repertoire of mRNAs whose translation can be locally regulated and support the emerging idea that local protein synthesis serves to boost the translational capacity of synapses.

Ubiquitin ligase TRIM3 controls hippocampal plasticity and learning by regulating synaptic γ-actin levels

“…We also established that Actg1 mRNA is present in mRNP granules that are immunoprecipitated with antibodies against either TRIM3 or PURA. Actg1 mRNA lacks the zip code sequence that is known to target Actb mRNA to dendrites (Eom et al, 2003;Poon et al, 2006). However, a recent deep sequencing study showed that both Actg1 and Actb transcript are identified with high confidence as dendritic/axonal transcripts in the hippocampal neuropil (Cajigas et al, 2012).…”

Regulation of global and specific mRNA translation by the mTOR signaling pathway

“…88,89 In fact, TOP mRNAs represent some of the most abundant transcripts localized within these structures. 90,91 While it seems unlikely that new ribosomes are assembled in this compartment, as this process is thought to strictly occur in the nucleolus, 92 the translation of TOP mRNAs may serve to selectively replace or replete translation factors and ribosomal proteins during times of increased demand in protein synthesis. 78 Another possibility is that certain elongation factors encoded by TOP mRNAs may be present in dendrites and axons in limiting amounts, such that increasing their expression by promoting TOP mRNA translation would significantly increase overall translation rates.…”

Identification of a cis-acting element that localizes mRNA to synapses

“…In the brain, local protein synthesis is critical to the development and experience-driven refinement of neural circuits, playing roles in axon guidance, synaptogenesis, and synaptic plasticity (1,2). A large but select population of transcripts localizes to axons and dendrites (3)(4)(5)(6)(7)(8), indicating that local translation subserves diverse cell biological functions. Where studied, the localization of mRNAs to axons or dendrites has been shown to depend on cisacting localization elements (LEs) usually found in the 3′ UTR, although occasionally present in the coding sequence or 5′ UTR (1,2,9).…”