Analysis of the Expression and Subcellular Distribution of eEF1A1 and eEF1A2 mRNAs during Neurodevelopment

Abstract

Neurodevelopment is accompanied by a precise change in the expression of the translation elongation factor 1A variants from eEF1A1 to eEF1A2. These are paralogue genes that encode 92% identical proteins in mammals. The switch in the expression of eEF1A variants has been well studied in mouse motor neurons, which solely express eEF1A2 by four weeks of postnatal development. However, changes in the subcellular localization of eEF1A variants during neurodevelopment have not been studied in detail in other neuronal types because antibodies lack perfect specificity, and immunofluorescence has a low sensitivity. In hippocampal neurons, eEF1A is related to synaptic plasticity and memory consolidation, and decreased eEF1A expression is observed in the hippocampus of Alzheimer’s patients. However, the specific variant involved in these functions is unknown. To distinguish eEF1A1 from eEF1A2 expression, we have designed single-molecule fluorescence in-situ hybridization probes to detect either eEF1A1 or eEF1A2 mRNAs in cultured primary hippocampal neurons and brain tissues. We have developed a computational framework, ARLIN (analysis of RNA localization in neurons), to analyze and compare the subcellular distribution of eEF1A1 and eEF1A2 mRNAs at specific developmental stages and in mature neurons. We found that eEF1A1 and eEF1A2 mRNAs differ in expression and subcellular localization over neurodevelopment, and eEF1A1 mRNAs localize in dendrites and synapses during dendritogenesis and synaptogenesis. Interestingly, mature hippocampal neurons coexpress both variant mRNAs, and eEF1A1 remains the predominant variant in dendrites.

Localized molecular chaperone synthesis maintains neuronal dendrite proteostasis

“…3G ). To differentiate regulated from a random coexistence of two mRNAs, we randomly simulated the position of Hsp90aa , Hsp90ab , or Hsp110 mRNAs over the dendritic shaft using a new module in ARLIN ( 88 ). ARLIN binned the dendritic shaft into 25 μm segments and organized them depending on their distance from the soma to consider changes in the number of HSP mRNAs ( Fig.…”

“…Because dendritic spines have high levels of localized translation and protein concentration, we proposed they would require more HSPs to prevent aberrant interactions among unfolded proteins. Thus, we used ARLIN to analyzed stress-induced changes in the contiguity between HSP mRNAs, detected by smFISH, and the postsynaptic density, labeled by IF with PSD95 antibody ( 88 ) ( Fig. 3H ).…”

“…Single mRNAs, peptides, and postsynaptic densities were identified with the Matlab version of the software FISH-quant v3 ( 86 ). The post detection analysis for RNA subcellular distribution in neurons and simulations was done with the second version of ARLIN ( 88 ). The code for ARLIN v1.0 and ARLIN v2.0 can be found here: https://github.com/LR-MVU/neuron.…”

Endogenous epitope tagging of eEF1A2 in mice reveals early embryonic expression of eEF1A2 and subcellular compartmentalisation of neuronal eEF1A1 and eEF1A2

“…Although proteins can be directly targeted to specific subcellular locations by virtue of specific motifs, protein localisation in neurons is frequently controlled by targeting of the cognate mRNA, which is then translated locally, and this seems the most likely mechanism here. Indeed, a number of studies of both the mRNA content and translatome of axons have identified eEF1A1 but not eEF1A2 in axons of mice and rats (Taylor et al, 2009;Gumy et al, 2011;Shigeoka et al, 2016;Nijssen et al, 2018) and in dendrites, albeit in cultured cells (Wefers et al, 2022); other studies using TRAP and Riboseq methodology have shown enrichment of eEF1A1 in axons relative to cell bodies (Ostroff et al, 2019;Glock et al, 2021;Jung et al, 2023). It seems likely that there are one or more RNA binding proteins controlling the developmental switch resulting in the transport and subsequent translation of eEF1A1 in axons; the UTRs of eEF1A1 and eEF1A2, unlike the coding regions, are largely dissimilar (41% identity in 3'UTRs compared with 80% in the coding regions).…”

Impact of histone deacetylase inhibition and arimoclomol on heat shock protein expression and disease biomarkers in primary culture models of familial ALS

“…See Wefers et al . for detailed methods [28]. Separate analysis of somal cytoplasm and dendritic projections was carried out using the FISH-Quant polygon tool to outline regions of interest.…”