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Characterization of processing bodies in T and B lymphocytes Tebaykina, Zinaida

Abstract

Processing bodies (P-bodies) are cytoplasmic aggregates that contain translationally-repressed mRNAs in complex with repressor proteins (GW182, RCK/p54, and DCP1a), facilitate mRNA storage or degradation, and can be identified by the αGW-body (GWB) serum that detects several P-body proteins. The partitioning of mRNAs between a translationally-competent cytoplasmic pool and a translationally-repressed P-body pool could be an important mechanism for dynamically controlling the synthesis of key proteins. Memory CD8⁺ T lymphocytes contain translationally-repressed RANTES and IFN-γ mRNAs, enabling the secretion of these cytokines within 30 minutes of T cell receptor (TCR) engagement. Although P-bodies have not been characterized in lymphocytes, I hypothesized that storage of RANTES and IFN-γ mRNAs in P-bodies could contribute to the ability of memory CD8⁺ T cells to mount rapid recall responses. Using immunoblotting, flow cytometry, and confocal microscopy, I established that T and B lymphocytes contain GWBs and express GW182, RCK/p54, and DCP1a, which are concentrated in cytoplasmic granules. Co-localization analysis identified multiple subsets of P-bodies, raising the possibility that P-bodies with different protein compositions have distinct functional properties. Moreover, I found that P-bodies partially dissociate and move towards the model immune synapse in both T and B lymphocytes. To explore the role of P-bodies in the recall response, I utilized the OT-I model to generate effector and memory CD8⁺ T lymphocytes in vitro. Compared to naïve CD8⁺ T cells from OT-I mice, effector T cells had elevated levels of P-body proteins and a greater number of P-bodies. In contrast, memory T cells had similar numbers of P-bodies as naïve T cells, but contained larger GWBs and RCK/p54 granules. Remarkably, RANTES mRNA did not co-localize with P-bodies in memory T cells, but was distributed diffusely in the cytoplasm. Conversely, IFN-γ mRNA co-localized with GWBs and RCK/p54 granules in memory T cells. The abundance of P-body-targeting AU-rich elements (AREs) in IFN-γ mRNA and the absence of AREs in RANTES mRNA suggests that IFN-γ mRNA transcribed following activation of naïve T cells, is directed for storage into GWB⁺ RCK/p54⁺ P-bodies to be reused during the recall response, whereas RANTES mRNA is stored by an undefined P-body-independent mechanism.

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