Celia Alecki

Cells rapidly adapt to survive harsh environmental conditions through the potent upregulation of molecular chaperones or heat shock proteins (HSPs). The inducible members of the HSP70 family are the fastest and most transcriptionally induced chaperone upon stress. The HSP70 mRNA life cycle regulation in the cytoplasm is unique because it is translated during stress when general translation is repressed and rapidly degraded once conditions are optimal for growth. Contrary to the role of the HSP70 mRNA 5’ untranslated region in maximizing the synthesis of HSP70, we discovered that the coding sequence (CDS) represses its translation through the ribosome quality control (RQC) mechanism. The CDS of the most inducible HSP70 in Saccharomyces cerevisiae, SSA4 , is uniquely biased with low-frequency codons that promote ribosome stalling during heat stress. The stalled ribosomes are recognized by RQC components Asc1p and Hel2p and two ribosome proteins, Rps28A and Rps19B, that we identified as new RQC components. Surprisingly, RQC does not signal the degradation of the SSA4 mRNA by no-go-decay (NGD). Instead, Asc1p destabilizes the SSA4 mRNA during recovery from heat stress by a mechanism independent of its ribosome binding and SSA4 CDS codon optimality. Therefore, Asc1p operates two synergistic mechanisms that converge to regulate the life cycle of HSP70 mRNA during stress and recovery. Our research identifies Asc1p as a critical regulator of the stress response and RQC as the system tuning HSP70 synthesis.

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.