Periodic Reporting for period 2 - NeuroRibo (Specialized Ribosomes for Neuronal Protein Synthesis)
Reporting period: 2019-02-01 to 2020-07-31
In a second work package, we analyzed the nature and impact of dendritically localized ribosomal protein mRNAs. We found a dendritic enrichment of 16 different RP mRNAs in hippocampal slices and cultured neurons and quantified those via fluorescence in vitro hybridization (FISH). Using 3’UTR sequencing and bioinformatic approaches to describe and identify the ribosomal protein mRNAs, we discovered that 69 (out of 80) RP mRNAs were localized in the neuropil. Many of these exhibit novel and multiple 3’UTR isoforms, with some enriched in neurons and neuropil (Tushev et al., 2018). When asking the question whether ribosomal mRNAs can be translated into proteins in dendrites, we detected and quantified via the FUNCAT-PLA/Puro-PLA method 9 different RPs that were synthetized in hippocampal cultured neurons.
We have also started to examine the regulation of the local RP transcripts and translation by synaptic activity and plasticity, and asked if synaptic plasticity changes the quality or quantity globally and locally. So far we have tested for the ribosomal protein RPL26 with a protocol for pharmacologically induced plasticity, but we did not observe any change in RPL26 synthesis. In order to determine how the global RP proteome is modified by synaptic activity and plasticity, we are measuring the turnover of ribosomal proteins after action-potential blockade, e.g. with tetrodoxin (TTX). In this context, we could also recently show that ribosomal proteins exhibit different half-lives, suggesting that there is remodeling in ribosomes (Doerrbaum et al., 2018).