Stress granules and Processing bodies oscillate in a circadian manner

MALCOLM, MELISA; PENAZZI, LAURA GABRIELA; GARBARINO-PICO, EDUARDO

Congreso; Reunión conjunta SAIB-SAMIGE 2020 -Edición on-line; 2020

STRESS GRANULES AND PROCESSING BODIES OSCILLATE IN A CIRCADIAN MANNERMalcolm M, Pennazi L. G, Garbarino Pico E.1.CIQUIBIC (CONICET) ? Dpto. de Química Biológica Ranwel Caputto, Facultad de Ciencias Químicas, Universidad Nacional de Córdoba. Haya de la Torre y Medina Allende - Ciudad Universitaria - X5000HUA CórdobaE-mail: mmalcolm@fcq.unc.edu.ar.Stress granules (SG) and processing bodies (PB) are membraneless organelles, responsible for storage and cytoplasmic processing of mRNA, which share some components such as mRNA and RNA binding proteins. SGs, which are also constituted by translation initiation factors, the minor subunit of the ribosome 40s, and translation stalled mRNAs, are formed in response to different stress stimuli, typically through phosphorylation of the eIF2 factor. PBs, on the other hand, are enriched in factors involved in mRNA degradation, translational repression, and RNA-mediated silencing, and they are constitutive membranless organelles, although they increase in number with stress. In previous studies we found that SG formation presents temporary changes in number and size in mouse fibroblast synchronized cultures (NIH/3T3). We wonder whether the circadian clock controls these temporal changes. The cells were synchronized with dexamethasone and harvested every 4 h for 68 h. We induced the formation of SGs with sodium arsenite (oxidative stress). On the other hand, we analyzed PBs in not stressed N2A cells (neuroblastome cell line). We also studied the temporal expression profile of p-eIF2α (factor involved in SG aggregation) and eIF3, the marker we used for detecting SGs, by Western blot. Cell cultures were arrested in order to prevent cell cycle progression, and we confirm their quiescence state by flow cytometry. We performed a double immunolabeling for SGs (eIF3 and G3BP1) and PB (GE-1/HEDLS and DDX6) by immunocytochemistry. We observed that NIH/3T3 and N2A cells show daily rhythms in SGs and PBs, respectively, for three variables: number, area, and signal intensity with the estimated parameters showed periods of approximately 24h, for two cycles. The subtle differences that we found between both translation initiation factors eIF3 and eIF2α, would not be responsible for generating the changes observed in SGs. These findings strongly suggest that the molecular circadian clock rhythmically controls SGs and PBs. The results presented here reveal new ways in which circadian clocks modulated mRNA translation, stability and storage in the cytoplasm.