Ji and Erin to present at Student Seminar on Thursday (18.November at 12:30 in 2-501 BSB
Posted by ppretorius
Ji and Erin will be presenting Student Seminar this week in 2-501 BSB at 12:30. I have asked the speakers to provide a brief overview for their talks. Hope to see you there!
Background for Ji’s talk
Transcriptome-wide identification of alternative polyadenylation events regulated by epithelial splicing factor ESRP1
The translation of messenger RNA (mRNA) is strongly influenced by regulatory elements in the 3’ untranslated region (3’ UTR). Different isoforms of 3’ UTR can be generated due to multiple polyadenylation (polyA) sites within a single mRNA. The differential usage of alternative polyA sites in different biological or physiological conditions is called APA switch. In this study, we aim to investigate the regulation of APA switch events by an epithelial-cell-specific RNA binding protein — Epithelial Splicing Regulatory Protein 1 (ESRP1) (OMIM). ESRP1 can regulate diverse types of alternative splicing events and control a genome-wide alternative splicing network during the epithelial-to-mesenchymal transition (EMT). However, whether ESRP1 can regulate APA switch events has not been investigated before.
To study the effects of ESRP1 on alternative splicing and APA switch events, we ectopically expressed a cDNA for mouse ESRP1 in the MDA-MB-231 mesenchymal human breast cancer cell line by viral transduction. Using high-throughput mRNA sequencing (RNA-Seq), we generated a total of 256 million 76bp RNA-Seq reads on cells with ESRP1 overexpression or empty vector control. In order to identify APA switch events upon ESRP1 overexpression, we developed a computational pipeline to quantify the usage of individual polyA sites using RNA-Seq data. Using this pipeline, we identified hundreds of genes with APA switch events after ESRP1 overexpression. We also found consistent APA switch patterns by comparing RNA-Seq data to Affymetrix Human Exon Array ST 1.0 data for the same samples. These results show that ESRP1 regulates polyadenylation of specific mRNAs and could contribute to the generation of 3’ UTR isoforms during EMT.
Background for Erin’s talk
A Hyperactive piggyBac DNA Transposon Achieves Persistent Gene Transfer
Nonviral vector systems are used increasingly in gene targeting technologies and as tools for gene transfer applications. The piggyBac (PB) transposon represents an alternative integrating vector for in vivo gene transfer. To date, there are no reports of gene transfer to mammals using the hyperactive piggyBac (PB7) transposon system. We hypothesized that this system could achieve persistent gene transfer to the liver when administered systemically. Here we report that the PB7 transposase generated higher transposition efficiency than a codon-optimized transposase in human liver cells. Following hydrodynamic tail-vein injection, we show that firefly luciferase expression driven by a liver-specific promoter persisted up to eight months in mice even following partial hepatectomy. This finding is consistent with expression from an integrated transgene. We extended these studies and delivered the human α1 anti-trypsin (hAAT) gene to murine liver along with PB7 and achieved stable hAAT expression in serum. To determine the integration pattern, we mapped 853 integration sites in human cells. We found an altered pattern of integration than previously reported for insect PB. Our results support the PB transposon combined with the hyperactive PB7 transposase is an efficient integrating vector system for in vitro and in vivo applications.
Posted on November 16, 2010, in Student Seminar and tagged alternative splicing, Erin B., ESRP1, gene transfer, Ji W., nonviral vector systems, piggyBAC, polyadenylation. Bookmark the permalink. Leave a comment.