Comparison of PPIG knockdown in HEK293T cells compared to scrambled shRNA control

HEK293T cells transduced with shRNA from MISSION library TRCN0000049326 using lentiviral delivery system. HEK293T cells transduced with scrambled shRNA, gifted from Dr. Mauricio Reginato. Tara L Davis, S. RaElle Jackson, Beth Adams, Anh Trinh performed primary experimental contributions to cell lines, RNA/cDNA preparation, and validations, all Drexel University College of Medicine, Philadelphia, PA. Hetty Rodriguez and John Tobias performed Bioanalyzer and microarray expreriments, and initial data processing. Affiliation: Molecular Profiling Facility and Genomic Analysis Core Bioinformatics Group, University of Pennsylvania, Philadelphia, PA. Human PPIG (alias: SR-Cyp, CARS) is a cyclophilin, an enzyme that interconverts cis and trans isomers of proline. The PPIG gene, in addition to the cyclophilin domain, encodes for a multiple C-terminal SR motifs. PPIG associates with the human spliceosome, the complex and dynamic machinery that removes intronic sequence from pre-messenger RNA (pre-mRNA). Nothing is known about the function of PPIG in regulation of alternative splicing. To understand the function of PPIG, we knocked down PPIG in human cells. We characterized a set of alternative splicing and transcriptional events that are PPIG-responsive. We used these splicing and transcriptional bioassays to show that PPIG-responsive events are largely specific, even within the cyclophilin family. 3 replicates of PPIG knockdown cell lines (HEK293T cells, stably integrated, selected using puro, MISSION shRNA (TRCN0000049326, 5'-CCGGGCAGTCTGATTCTAAAGGAAACTCGAGTTTCCTTTAGAATCAGACTGCTTTTTG-3'). 3 replicates of SCR control cells lines (HEK293T cells, stably integrated, selected using puro, sequence 5′-CCTAAGGTTAAGTCGCCCTCGCTCTAGCGAGGGCGACTTAACCTT-3′). total RNA purified from ~1 million cells, cDNA converted using random hexamer primers, quantity measured using NanoDrop, quality quantified using BioAnaylzer). Affymatrix HTA2.0 array