tailieunhanh - Báo cáo khoa học: The 3¢-UTR of the mRNA coding for the major protein kinase C substrate MARCKS contains a novel CU-rich element interacting with the mRNA stabilizing factors HuD and HuR

The expression of the major protein kinase C substrate MARCKS (myristoylatedalanine-richCkinase substrate) is controlled by the stability of its mRNA. While the MARCKS mRNA is long living in quiescent fibroblasts (t1/2¼14 h), its half-life time is drastically reduced (t1/2¼2 h) in cells treated with phorbol esters to activate protein kinase C (PKC) or treatedwith growth factors. In a first step to study the underlying mechanism we identified both acis-element on the MARCKS mRNA and the cor-responding trans-acting factors. . | Eur. J. Biochem. 270 350-365 2003 FEBS 2003 doi The 3 -UTR of the mRNA coding for the major protein kinase C substrate MARCKS contains a novel CU-rich element interacting with the mRNA stabilizing factors HuD and HuR Georg Wein1 Marek Rossler1 Roland Klug1 and Thomas Herget1 2 1 Laboratory of Molecular Neurobiology Institute of Physiological Chemistry and Pathobiochemistry Johannes Gutenberg-University Mainz Germany 2Axxima Pharmaceuticals AG Martinsried Germany The expression of the major protein kinase C substrate MARCKS myristoylated alanine-rich C kinase substrate is controlled by the stability of its mRNA. While the MARCKS mRNA is long living in quiescent fibroblasts t1 2 14 h its half-life time is drastically reduced t1 2 2 h in cells treated with phorbol esters to activate protein kinase C PKC or treated with growth factors. In a first step to study the underlying mechanism we identified both a cis-element on the MARCKS mRNA and the corresponding trans-acting factors. Fusing the complete 3 -UTR or specific regions of the 3 -UTR of the MARCKS gene to a luciferase reporter gene caused a drastic decrease in luciferase expression to as low as 5-10 of controls. This down-regulation was a result of destabilization of the chimeric transcript as shown by NNA nm-off and Northern blot-assays. By RNase EMSA and UV-cross-linking experiments we identified a stretch of 52 nucleotides CuUU 11 U 8 in the 3 -UTR of the MARCKS mRNA specifically recognized by two RNA-binding proteins HuD and HuR. These trans-acting factors are members of the ELAV gene family and bind the MARCKS CU-rich sequence with high affinity. Overexpression of HuD and HuR in murine fibroblasts caused a striking stabilization of the endogenous MARCKS mRNA even under conditions when the MARCKS mRNA is normally actively degraded . after treating cells with phorbol ester. These data imply that the identified CU-rich cis-element of the MARCKS 3 -UTR is involved in conferring

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