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 (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 RNA run-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, i.e. 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 instability to mRNAs and that members of the ELAV gene family oppose this effect. Based on its structural and functional properties, the (CUUU)11(U)8 sequence described here can be grouped into class III of AU-rich elements.
Wein
Georg
G
Institute of Physiological Chemistry and Pathobiochemistry, Johannes Gutenberg-University, Mainz, Germany.
Rössler
Marek
M
Klug
Roland
R
Herget
Thomas
T
D020413:3' Untranslated Regions; D016475:3T3 Cells; D000818:Animals; D000954:Antigens, Surface; D015536:Down-Regulation; D051959:ELAV Proteins; D000067780:ELAV-Like Protein 1; D000067779:ELAV-Like Protein 4; D005347:Fibroblasts; D017930:Genes, Reporter; D047908:Intracellular Signaling Peptides and Proteins; D008156:Luciferases / Q000235:genetics / Q000378:metabolism; D008565:Membrane Proteins; D051379:Mice; D000076250:Myristoylated Alanine-Rich C Kinase Substrate; D009419:Nerve Tissue Proteins / Q000378:metabolism*; D011493:Protein Kinase C / Q000502:physiology; D011506:Proteins / Q000235:genetics*; D012333:RNA, Messenger / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism
D020413:3' Untranslated Regions; D000954:Antigens, Surface; D051959:ELAV Proteins; D000067780:ELAV-Like Protein 1; D000067779:ELAV-Like Protein 4; C098770:ELAVL1 protein, human; C498011:Elavl4 protein, mouse; D047908:Intracellular Signaling Peptides and Proteins; C000619710:MARCKS protein, human; C000619711:Marcks protein, mouse; D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; D011506:Proteins; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; D011993:Recombinant Fusion Proteins; D000076250:Myristoylated Alanine-Rich C Kinase Substrate; D008156:Luciferases; D011493:Protein Kinase C