Elav Like HuR-Dependent MRNA Stability Regulates Post-Transcriptional Expression Of Cyclin Dependent Kinase Inhibitor P27^Kip1

The progression of mammalian cells through the various phases of the cell cycle requires orderly activation of cyclin-dependent protein kinases(CDKs). Gene product of the cyclin dependent kinase inhibitor p27Kip1 controls entry into and exit from the cell cycle in mammalian cells by regulating the activity of CDK-cyclin complexes. p27Kipl integrates extracellular signals with the cellular machinery, thus playing a critical role in the regulation of the cell growth, differentiation, apoptosis and tumorigenesis. And reduced expression of p27Kipl has been reported in various human malignancies, such as breast cancer, lung cancer and epithelial ovarian cancer, and it's related with shorter survival time and disease-free intervals. So post-transcriptional regulation of p27Kipl expression is of significant interest. ELAV like RNA binding protein HuR is thought be important for the translation of p27Kipl, however different reports attributed diametrically opposite roles to HuR. Millard has reported that basal and cell cycle-dependent translation of p27Kipl is stimulated by the embryonic lethal abnormal visual system (ELAV) like RNA binding protein HuR binding to the 5'UTR of p27Kipl mRNA, while Kullmann reported that repression of internal ribosome entry site (IRES)-dependent translation by HuR in proliferating but not quiescent cells is responsible for cell cycle-dependent expression of p27Kip1. More recently, a p27K'pl mRNA isoform with an upstream open reading frame has been proposed to be responsible for the cell cycle-dependent regulation of p27Kipl expression. And an endonuclease binding site in the 5'UTR of p27Kipl has been reported, suggesting that mRNA stability may also play a role in regulating the post-transcriptional expression of p27Kipl.Given the critical role of p27Kipl in the regulation of cell proliferation and cancer patients'outcome, understanding the regulation of its expression is of great interest. We therefore undertook a comprehensive study to investigate cis-and trans-regulatory elements that govern the posttranscriptional expression of p27Kipl:(1) Based on the full-length p27Kip1 which has been cloned by screening a mouse embryonic cDNA library, we performed a modified RACE procedure for mouse and human cDNA which aimed to obtain all of the mRNA isoforms.(2) To find cis-regulatory elements of mouse p27Kip1 mRNA we developed a dual Luciferase reporter assay in which the firefly luciferase has been fused with either individual fragments or different combinations of 5'and/or 3'UTR of mouse p27Kipl mRNA. The amount of firefly and renilla luciferase was tested in proliferating and quiescent cells by Dual Luciferase reporter assay. And the corresponding amount of mRNA was measured by performing real-time PCR.(3) For fragments with putative cis-regulatory elements of mouse and human p27Kip1 mRNA, radioactive labeled mRNA was produced and crosslinked with nuclear cell extracts from either mouse fibroblast or human brain nuclear cell extracts. And crosslinked proteins were immunoprecipitated with different antibodies specific to various RNA binding protein suspected of interacting with p27Kipl mRNAs.(4) To better define the biological role of HuR in regulating stability of endogenous p27Kip1 mRNA and expression of endogenous p27Kipl protein p27Kipl expression, we studied effect of HuR knockdown on the stability the of p27Kipl mRNA and crosslinked proteins and then immunoprecipitated in vivo, and based on sucrose density gradient centrifugation followed by real time PCR quantification of reporter mRNA in translated and untranslated fractions we confirmed that deletion of HuR binding sites had very little effect on translatability of the reporters but fusion with UTRs of p27Kipl mRNA reduces the translation efficiency.We report here an alternative mechanism wherein HuR regulates stability of the p27Kip1 mRNA thereby modulate posttranscriptional expression this gene. Specifically, human and mouse p27Kipl mRNAs interact with HuR protein through multiple U-rich elements in both 5'and 3'UTRs. These interactions which, occurs in vitro and in vivo, stabilize p27Kipl mRNA and play a critical role in its accumulation. Deleting HuR binding sites or knocking down HuR expression destabilizes p27Kip1 mRNA and reduce its accumulation. Our studies further identified a CT repeat in the 5'UTR of full-length p27Kip1 mRNA isoforms which interacts with a-41-kd protein and represses p27Kip1 expression. This CT-rich element and diffuse elements in the 3'UTR regulate post-transcriptional expression of p27Kip1 at the level of translation. This is the first demonstration that HuR dependent mRNA stability and HuR independent mRNA translation plays a critical role in the regulation of posttranscriptional p27Kip1 expression.