Identification Of Alternative Polyadenylation Involved In Glioblastoma Multiforme And Its Effect On Protein Binding

Most eukaryotic cells produce mRNAs with polyA tail, which is required for mRNA stability. Alternative polyadenylation (APA) is widely present in human genome, resulting in one gene with various3’ends. Here we conducted a comprehensive analysis of the APA products for glioblastoma and normal brain tissues using a data set from massively parallel signature sequencing of mRNAs. As a result, we found24%of the expressed genes having more than one polyadenylation site. Among them,182APA isoforms from148genes showed significantly differential expression between normal and GBM brain tissues, suggesting that APA plays a role in glioma development. Moreover, we found that APA isoforms of a same gene could show contradict expression levels between normal and GBM brain tissues. In this study, three genes including MEF2D (myocyte enhancer factor2D), HSBP1(Heat shock factor binding protein1) and PHC1(Polyhomeotic homolog1(Drosophila)) fit the criteria. All the polyadenylation sites of the3genes were validated by3’RACE PCR in both GBM cell lines and tissues. The novel sites were further determined by sequencing.3’UTR is found to be binding sites for microRNAs or RNA binding proteins (RBP) that might affect RNA stability or translation efficiency. We thus wondered whether the differential3’UTR regions of APA isoforms contained RBP binding sites. In order to identify binding motifs for RNA binding proteins in the APA regions, we first developed a computational pipeline named RNAelements using covariance model from known RBP-RNA interaction datasets acquired by RIP methods, and tested the tool with segments from CLIP methods. Here the RBPs were now limited to AUF1, HNRNPA1, HuR, MSI1, NCL, PUM2and SSB. RNAelements can be accessed at:http://sysbio.zju.edu.cn/RNAelements/.Not surprisingly, we found that RBPs were enriched in the alternative regions between the upstream and the downstream polyadenylation sites using RNAelements. For MEF2D, HSBP1and PHC1, we found that most RBPs specifically bound to the regions between the two alternative polyadenylation sites, which would result in the shorter APA isoform to escape regulation by these RBPs. Moreover, doRiNA database was searched to find the experimental RBP binding segments, and AREsite database was applied to find the conserved ARE sites in the mRNA sequence of of MEF2D, HSBP1and PHC1. Both results showed that the shorter one would lose some regulation sites.In the end, we extracted all the3’ UTR SNPs from dbSNP137, and checked whether the SNPs were located in polyadenation signals or altered the RBP bining motifs. In conclusion, our data provided a novel RBP mediated mechanism controlling RNA stability and translation for APA isoforms.