Studies on prediction of SPS1 gene function and microRNA transcriptional regulatory element by bioinformatical analysis

Abstract

Bioinformatics is an important area to analyze the massive biological data and predict the biological meanings using computational and statistical methods. Since the massive and highly qualified data have been accumulated by development of microarray technology, researches for finding biological meanings through predicting gene functions and transcriptional regulatory elements by using bioinformatical approaches are actively progressed. In these studies, we show the predicted and confirmed results for gene function and transcriptional regulatory element through two examples, selenophosphate synthetase 1 (SPS1) and microRNA genes. For example predicting gene function, we used SPS1 which functions are unknown yet. There are two selenophosphate synthetases (SPSs) in higher eukaryotes, SPS1 and SPS2. Of these two isotypes, only SPS2 catalyzes selenophosphate synthesis. Although SPS1 does not contain selenophosphate synthesis activity, it was found to be essential for cell growth and embryogenesis. The function of SPS1, however, has not been elucidated. Using microarray data from obtained SPS1 knockdown, differentially expressed genes were identified using two-way analysis of variance methods and clustered according to their temporal expression pattern. Gene ontology analysis was performed against differentially expressed genes and gene ontology terms related to vitamin B6 biosynthesis were found to be significantly affected at the early stage (day 3). Interestingly, genes related to defense and amino acid metabolism were affected at the later stage (day 5) following knockdown. Levels of pyridoxal phosphate, an active form of vitamin B6, were decreased by SPS1 knockdown. Treatment of SL2 cells with an inhibitor of pyridoxal phosphate synthesis resulted in both a similar pattern of expression as that found by SPS1 knockdown and the formation of megamitochondria, which is the major phenotypic change observed by SPS1 knockdown. These results indicate that SPS1 regulates vitamin B6 synthesis, which in turn impacts various cellular systems such as amino acid metabolism, defense and other important metabolic activities. For example for predicting transcriptional regulatory elements, we selected miRNA genes. miRNAs are important post-transcriptional regulators of various biological processes. Although our knowledge of miRNA expression and regulation has increased considerably in recent years, the regulatory elements for miRNA gene expression, especially for intergenic miRNAs, are not fully understood. We identified the differentially methylated regions (DMRs) occurring 1000 bp upstream from all miRNAs in human neuroglioma cells using microarrays and discovered a unique sequence motif C[N]6CT. This motif was preferentially located within 400 bp or from 800–1000 bp upstream of the intergenic miRNA start, corresponding to the highly methylated region. Interestingly, treatment of cells with a methyl transferase inhibitor (5-aza-2-deoxycytidine, DAC) significantly increased expression of miRNA genes with a high frequency of the C[N]6CT motif in DMRs. Statistical analysis showed that the frequency of the C[N]6CT motif in DMRs is highly correlated with intergenic miRNA gene expression, suggesting that C[N]6CT motifs associated with DNA methylation regions play a role as regulatory elements for intergenic miRNA gene expression.