EXPRESSION, INTRACELLULAR LOCALIZATION AND PTMS IN DIFFERENT CELL LINES OF THE MRNA DECAY REGULATORS OF THE ZFP36 FAMILY

CRISTIAN JORGE ALEJANDRO ASENSIO

BUENOS AIRES

Congreso; LXII REUNIÓN ANUAL DE LA SOCIEDAD ARGENTINA DE INVESTIGACIÓN CLÍNICA (SAIC); 2017

SAIC

Abstract: 3 nucleo-cytoplasmic shuttling, mRNA binding, CCCHzinc finger, PPPP containing, low-abundant phosphoproteins (zfp36,zfp36L1, zfp36L2) compose the zfp36 family. In the cytosol trough2 central tandem zinc fingers, they bind to AU-rich elements (ARE)in the mRNA´s 3?UTR resulting in mRNA instability/ degradation byother proteins. Several ARE-containing mRNAs, especially of inflammatorymediators, are stabilised by the p38 pathway through inactivatingphosphosites in these proteins. To understand the elusiveparticular role(s) of the L1 member, different cell lines were studiedassessing their expression by WB and qPCR. Several antibodieswere tested. Zfp36 protein was detected for the first time in HeLa byan improved WB and IP. L1 and L2 were KD in HeLa by RNAi. Theeffects on p38-regulated inflammatory mediator expression were examined.L1 function was investigated in L1 -/- cell lines (n=17). L1 orL2 did not contribute to post-transcriptional regulation of inflammatorymediators by the p38 pathway in HeLa or L1 null cells. L1 nullcells over-expressed IL-6 protein/mRNA but L1 was not regulatingIL-6 mRNA stability. Instead, IL-6 underwent fastest transcription inthe L1´s absence suggesting that the IL-6 transcript is an indirecttarget through some factor being itself a L1 target. On the otherhand, L1 and Zfp36 showed reproducible (p≤0.01, n=19) inversebasal protein expression levels in macrophages, with L1 increasingand Zfp36 decreasing during differentiation, suggesting thatL1 has actually a role in sustaining a Mc-MF differentiation switch.Concerning PTMs, the phosphorylation extent was regulating theintra-cytosolic localization with hyperphosphorylated forms of Zfp36and L1 becoming insoluble in the cytosol and putatively associatedwith the cytoskeleton. Other results suggest roles for Pro isomerisationand for electrostatic interactions in the fine-tuning, rheostaticregulation of the activity, level and localization of these proteins.Keywords: mRNA