Sequence, structure and function of the p27Kip1 5' untranslated region

The cyclin-dependent kinase inhibitor p27Kip1 is essential for proper control of cell cycle progression. p27Kip1 belongs to the Cip/Kip family of cyclin-dependent kinase inhibitors and inhibits G 1 cyclin dependent kinase activity. Cellular levels of p27Kip1 are tightly regulated by several mechanisms including transcriptional and translational controls. The 5' untranslated region (UTR) of the p27Kip1 mRNA has been reported to be important for translational control of p27Kip1 levels. p27 Kip1 translation is highest in quiescent cells when global, cap-dependent translation rates are down-regulated. The p27 Kip1 5' UTR has been shown to mediate cap-independent translation initiation via an internal ribosome entry site (IRES).; Messenger RNA 5' UTRs containing IRESs are known to fold into complex secondary structures and be regulated by trans-acting factors. In order to delineate the molecular details of the regulatory mechanism of internal ribosome entry, the first step was to map the p27Kip1 transcription initiation sites and thereby define the size of the p27Kip1 mRNA 5' UTR. This allowed studies of the structure and function of the 5' UTR to be done. The human p27 Kip1 major transcription initiation site, as determined by RNA ligase-mediated rapid amplification of cDNA ends and/or RNase protection assays, was found to be 472 base pairs upstream of the protein coding region. Minor transcription start sites were also identified at 403, 575, and 677 base pairs upstream of the ATG translation start codon. Transfections of bicistronic DNA and mRNA, along with in vitro translation assays, have shown that the 472 nucleotide p27Kip1 5' UTR also contains the highest IRES activity. Site-directed mutagenesis to insert upstream translation start codons was used to map the area where ribosome scanning begins to between 36 and 50 nucleotides upstream of the actual translation start codon. Also, enzymatic secondary structure probing was performed to obtain data to use in computer prediction analysis to derive a secondary structure model of the 472 nucleotide p27Kip1 5' UTR. These experiments identify the major human p27Kip1 5' UTR as a relatively long 472 nucleotide sequence that is predicted to form extensive secondary structures and mediate cap-independent translation initiation of the cell cycle inhibitor p27Kip1.