Identification of C17ORF45 protein and the effects of its snornas on cell cycle

Abstract

The Wnt / β-catenin signaling has many important roles in cellular responses and dysregulation of this pathway are linked to tumor formation. Priorly, SAGE and microarray analyses were performed in our laboratory in order to detect differentially expressed genes in Wnt / β-catenin pathway and c17orf45 gene was found to be upregulated in the presence of constitutively active β-catenin. It was characterized as a novel Wnt / β-catenin target. A bioinformatics tool designed by our former lab member was used to detect differentially expressed genes in different tumor samples from online data. This indicated that the expression level of c17orf45 gene was altered in distinct tumors. Besides, the upregulation of c17orf45 was experimentally observed in meningi-oma samples. Moreover, c17orf45 gene is accepted as the host gene for 3 snoRNAs in its introns. SnoRNAs are mainly involved in post-transcriptional modification of ribo-somal RNAs; however the roles of snoRNAs in cancer are currently identified. In this study, the putative protein product of c17orf45 gene and the effects of 2 snoRNAs resid-ing in introns of c17orf45 on different cancer cells were analyzed. Bioinformatics and PAML analyses for c17orf45 ORF revealed that there might be a positive selection for putative protein of the gene. Western Blot analysis done to identify the putative protein resulted in that the antibody did not target the protein of c17orf45. Besides, to examine the possible effects of snoRNAs, snoRNAs were cloned into intronic region of HBB gene encompassed by exonic parts in order to splice intron out. This would lead to ob-tain functional, processed snoRNAs. Splicing were verified with RT-PCR, Real Time PCR and YFP fused HBB construct which depended on joining of exons and splicing out of intron. Then, snoRNA constructs were introduced to different cancer cell lines to determine the effects of snoRNAs on cell cycle. However, none of the cells with 2 dif-ferent time points indicated an alteration on cell cycle.