The Function Of LPTS Gene And Its Associated Proteins

cDNA microarray permits a high throughput identification of changes ingene expression. Parallel analysis of the hybridized signals enabled us to get anexpression profile of genes in which about 256 genes were upregulated and 267were downregulated in the paired liver tumor tissues. We identified 4 genes, theexpression of three (Beclin 1, RbAp48 and Pir51) were increased and one(aldolase b) was decreased in liver tumor tissues. In addition, the expression ofthese genes in 6 hepatoma cell lines was also showed by RT-PCR analysis. Ourdata suggested that the genes encoding for Beclin 1, RbAp48, Pir51 and aldolaseb might be related with hepatocarcinoma. The expression of LPTS gene was also detected and showed to bedifferentially expressed in the paired liver tumors. LPTS, identified previouslyby allelic-loss mapping and positional candidate cloning, is a human putativetumor suppressor gene located at chromosome 8p23. LPTS plays an importantrole in both of inhibition of cell growth and telomerase activity. Wedemonstrated that the expression of LPTS gene was significantly reduced or notdetectable in liver tumor tissues at both of mRNA and protein levels. The genefor LPTS might be involved in liver carcinogenesis. For insight into the function of LPTS, we used an inducible-expressionyeast two-hybrid system with LPTS as the bait to identify LPTS-associatedproteins. After screening the human brain cDNA library, we obtained three 3 positive peptides, which could bind to LPTS protein in GST-pulldown assay. Wecloned these three novel genes for LPTS-binding protein and named these genesas MCRS2, hSMG5 and LRP1 respectively. MCRS2, an isoform of MCRS1/p78 is a cell-cycle dependent protein,accumulating in the very early S phase. MCRS2 interacts with LPTS in vitro, invivo and colocalizes with LPTS in nucleolus and on telomeres in cells. MCRS2or its N-terminus inhibits telomerase activity in vitro and long-termoverexpression of MCRS2 in SMMC-7721 cells results in a gradual andprogressive shorting of telomeres. Our findings suggest that MCRS2 might beinvolved in telomere shortening and cell-cycle regulation. Human SMG5 is a human homolog of C.elegans, which encodes a proteinof 1,016 amino acids and has a conserved PIN domain at the C-terminus. Wefound that hSMG5 interacted with LPTS and hTERT both in vitro and in vivoand inhibited telomerase activity via direct binding to the human telomerasereverse transcriptase (hTERT). Endogenous hSMG5 or GFP-hSMG5 wasdetected in the cytoplasm mainly around the nucleus. After cotransfection ofcells with GFP-hSMG5 and red fluorescence protein-tagged LPTS (RFP-LPTS)expression plasmids, GFP-hSMG5 and RFP-LPTS were colocalized ontelomeres and in nucleoli. It appears that LPTS recruits the hSMG5 from thecytoplasm to the telomeres and nucleolus. We found that hSMG5 also associatedwith hUpf1p and hUpf2p, the human orthologs of UPF in Saccharomycescerevisiae, which are required for the nonsense-mediated mRNA decay (NMD),and dephosphorylated for hUpf1p. Our results clearly demonstrate that thehSMG5 plays an important role in both the regulation of telomerase activity andthe nonsense-mediated mRNA decay. 4 LRP1 encodes a 756 aa protein (pI=4.5). LRP1 shares no homology withknown genes or proteins. It has few structural motifs, aside from one highlybasic region (550-756 aa, pI 10.4) and an adjacent acidic region (aa 500-549, pI4.0). LRP1 interacts with LPTS in cells and localizes on telomeres and nucleolus.We found that LRP1 could translate from second ATG to produce a short protein,LRP2, which contained 551 aa. Overexpression of LRP2 in cells could shortentelomeres and induce crisis. The basic regions of LRP1 bound hTERT directlyand inhibited telomerase activity in vitro. Our results demonstrate that LRP1 is anovel LPTS-binding protein and involved in telomere regulation. Telomerase activity ha...