| Nuclear proteins play a key role in transcription, mRNA processing, replication, chromosomal organization, cell proliferation and differentiation. Many nuclear proteins have intrinsic signals that mediate active transportation across nunclear pore complex (NPC). This is mediated by two essential elements: primary sequences for selective transportation factors-nuclear localization signals (NLS) and nuclear export signals (NES), and their cognate transportation factors. Transportation process can be divided into two phases: docking, or the binding of NLS-containing protein to the NPC and translocation, an energy-driving process of moving through the NPC. Although the precise mechanism for transportation across the NPC is not fully understood, the machinery is well conserved from yeast through mammals in terms of the structure of NPCs and homology of transportation factors.In order to enrich and identify cDNAs encoding nuclear targeted proteins systematically, we established a genetic screening method based on yeast. We employed an engineered NES-contaimng transcription factor, which is excluded from the nucleus, together with a reporter gene to provide a simple genetic selection in yeast. By fusing proteins of interest to the NES-containmg transcription factor, the system can identify NLS activity in the protein easily by its ability to counter the effects of theNES and enter the nucleus to activate the reporter gene expression.1. Construction ofNLS trapping system. We tested the system by construction a series oi defined fusion genes. The control vectors demonstrated that this system can specifically detect basic NLSs through positive selection in yeast. And the position of NLS in proteins does not affect the results of selection.2. Screening of mouse embryonic cDNA library. A mouse embryonic cDNA library was then inserted into the cloning site of the screening vector and 1-2% clones were shown to be positive after screening using the system. The cDNA inserts of these clones were sequenced and typical and untypical NLS were identified. We further confirmed the nuclear localizing activity of the cDNA inserts using the EGFP fusion protein. These results prove that the system will be useful in the screening of genes encoding NLS-bearing proteins from cDNA library. We selected two positive genes from screening, representing the known or unknown genes and with classical NLS or without classical NLS, for further analysis.3. Investigation ofTaxreblO? RpL6. We characterized the nuclear localization signal (NLS^ of the ribosomal protein L6 (RpL6, also called TaxreblO?) using the yeast-based NLS-trapping system as well as cultured mammalian cells. The result shows that while Taxrebl07/RpL6 is predicted to possess three putative NLS motifs, only the first two, located at amino acids 30-41 and 68-84, have nuclear localization activity in both yeast and mammalian cells. They also target the Taxrebl07/RpL6 protein to the nucleoli. The expression of Taxrebl07/RpL6 is much higher in embryonic tissues and undifferentiated cells than adult tissues and differentiated cells. Taxrebl07/RpL6 is a kind of HTLV-I Tax responsive element binding protein. To understand the molecular mechanisms of Taxrebl07/RpL6 gene regulation, we cloned the genomic gene by screening a phage library and analyzed the exon/intron structure as well as the 5'-flanking region by DNA sequencing. By reporter gene assay, we defined a 600-bpDNA fragment as a minimal promoter for expression and the proximal 350-bp for constitutive expression. In the followed 250-bp DNA fragment of the promoter, it contains recognition sites for many transcription factors including NF-KB. Transfection of Tax expression vector mildly up-regulated Taxrebl07/RpL6 expression and promoter activity. We further provide evidence that this up-regulation requires NF-icB site in the promoter of Taxrebl07/RpL6. Moreover, Taxrebl07/RpL6 could interact with Tax directly. Taken together, Tax may up-regulate Taxrebl07/RpL6 via NF-icB and this up-regulation may provid...
|
PDF Full Text Request