ADAR3 modulates neuronal differentiation and regulates mRNA stability and translation.

ADAR3 is a catalytically inactive member of the family of adenosine deaminases acting on RNA (ADARs). Here we have investigated its function in the context of the developing mouse brain. The expression of ADAR3 gradually increases throughout embryogenesis and drops after birth. Using primary cortical neurons, we show that ADAR3 is only expressed in a subpopulation of in vitro differentiated neurons, which suggests specific functions rather than being a general regulator of ADAR editing in the brain. The analysis of the ADAR3 interactome suggested a role in mRNA stability and translation, and we show that expression of ADAR3 in a neuronal cell line that is otherwise ADAR3-negative changes the expression and stability of a large number of mRNAs. Notably, we show that ADAR3 associates with polysomes and inhibits translation. We propose that ADAR3 binds to target mRNAs and stabilizes them in non-productive polysome complexes. Interestingly, the expression of ADAR3 downregulates genes related to neuronal differentiation and inhibits neurofilament outgrowth in vitro. In summary, we propose that ADAR3 negatively regulates neuronal differentiation, and that it does so by regulating mRNA stability and translation in an editing-independent manner.

Isolation, Expansion, and Nucleofection of Neural Stem Cells from Adult Murine Subventricular Zone.

Isolation and expansion of neural stem cells (NSCs) from the subventricular zone (SVZ) of the adult mouse brain can be achieved in a medium supplemented with basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF) as mitogens, producing clonal aggregates known as neurospheres. This in vitro system is a valuable tool for studying NSC potential. Transfection of siRNAs or genes carried in plasmids can be used to induce perturbations to gene expression and study NSC biology. However, the exogenous nucleic acid delivery to NSC cultures is challenging due to the low efficiency of central nervous system (CNS) cells transfection. Here, we present an improved nucleofection system that achieves high efficiency of gene delivery in expanded NSCs from adult murine SVZ. We demonstrate that this relatively simple method enhances gene perturbation in adult NSCs, surpassing traditional transfection protocols with survival rates exceeding 80%. Moreover, this method can also be applied in primary isolated NSCs, providing a crucial advancement in gene function studies through gene expression manipulation via knockdown or overexpression in neurosphere cultures.

Genetic inactivation of essential HSF1 reveals an isolated transcriptional stress response selectively induced by protein misfolding.

Heat Shock Factor 1 (Hsf1) in yeast drives the basal transcription of key proteostasis factors and its activity is induced as part of the core heat shock response. Exploring Hsf1 specific functions has been challenging due to the essential nature of the HSF1 gene and the extensive overlap of target promoters with environmental stress response (ESR) transcription factors Msn2 and Msn4 (Msn2/4). In this study, we constructed a viable hsf1∆ strain by replacing the HSF1 open reading frame with genes that constitutively express Hsp40, Hsp70, and Hsp90 from Hsf1-independent promoters. Phenotypic analysis showed that the hsf1∆ strain grows slowly, is sensitive to heat as well as protein misfolding and accumulates protein aggregates. Transcriptome analysis revealed that the transcriptional response to protein misfolding induced by azetidine-2-carboxylic acid is fully dependent on Hsf1. In contrast, the hsf1∆ strain responded to heat shock through the ESR. Following HS, Hsf1 and Msn2/4 showed functional compensatory induction with stronger activation of the remaining stress pathway when the other branch was inactivated. Thus, we provide a long-overdue genetic test of the function of Hsf1 in yeast using the novel hsf1∆ construct. Our data highlight that the accumulation of misfolded proteins is uniquely sensed by Hsf1-Hsp70 chaperone titration inducing a highly selective transcriptional stress response.

Modulation of protein synthesis and degradation maintains proteostasis during yeast growth at different temperatures.

To understand how cells regulate each step in the flow of gene expression is one of the most fundamental goals in molecular biology. In this work, we have investigated several protein turnover-related steps in the context of gene expression regulation in response to changes in external temperature in model yeast Saccharomyces cerevisiae. We have found that the regulation of protein homeostasis is stricter than mRNA homeostasis. Although global translation and protein degradation rates are found to increase with temperature, the increase of the catalytic activity of ribosomes is higher than the global translation rate suggesting that yeast cells adapt the amount of translational machinery to the constraints imposed by kinetics in order to minimize energy costs. Even though the transcriptional machinery is subjected to the same constraints, we observed interesting differences between transcription and translation, which may be related to the different energy costs of the two processes as well as the differential functions of mRNAs and proteins.