title: Off Target, but Sequence-Specific, shRNA-Associated Trans-Activation of Promoter Reporters in Transient Transfection Assays.
abstract: Transient transfection promoter reporter assays are commonly used in the study of transcriptional regulation, and can be used to define and characterize both cis-acting regulatory sequences and trans-acting factors. In the process of using a variety of reporter assays designed to study regulation of the rhodopsin (rho) promoter, we discovered that rhodopsin promoter-driven reporter expression could be activated by certain species of shRNA in a gene-target-independent but shRNA sequence-specific manner, suggesting involvement of a specific shRNA associated pathway. Interestingly, the shRNA-mediated increase of rhodopsin promoter activity was synergistically enhanced by the rhodopsin transcriptional regulators CRX and NRL. Additionally, the effect was cell line-dependent, suggesting that this pathway requires the expression of cell-type specific factors. Since microRNA (miRNA) and interferon response-mediated processes have been implicated in RNAi off-target phenomena, we performed miRNA and gene expression profiling on cells transfected with shRNAs that do target a specific gene but have varied effects on rho reporter expression in order to identify transcripts whose expression levels are associated with shRNA induced rhodopsin promoter reporter activity. We identified a total of 50 miRNA species, and by microarray analysis, 320 protein-coding genes, some of which were predicted targets of the identified differentially expressed miRNAs, whose expression was altered in the presence of shRNAs that stimulated rhodopsin-promoter activity in a non-gene-targeting manner. Consistent with earlier studies on shRNA off-target effects, a number of interferon response genes were among those identified to be upregulated. Taken together, our results confirm the importance of considering off-target effects when interpreting data from RNAi experiments and extend prior results by focusing on the importance of including multiple and carefully designed controls in the design and analysis of the effects of shRNA on transient transfection-based transcriptional assays.
authors.affiliation: Department of Ophthalmology, Wilmer Institute, Johns Hopkins University School of Medicine, Baltimore, Maryland, United States of America.
authors.forename: Tomohiro
authors.identifier: http://orcid.org/0000-0002-6668-1246
authors.initials: T
authors.lastname: Masuda
authors.affiliation: Department of Ophthalmology, Wilmer Institute, Johns Hopkins University School of Medicine, Baltimore, Maryland, United States of America.
authors.forename: Jun
authors.initials: J
authors.lastname: Wan
authors.affiliation: Department of Ophthalmology, Wilmer Institute, Johns Hopkins University School of Medicine, Baltimore, Maryland, United States of America.
authors.forename: Anitha
authors.initials: A
authors.lastname: Yerrabelli
authors.affiliation: Department of Ophthalmology, Wilmer Institute, Johns Hopkins University School of Medicine, Baltimore, Maryland, United States of America.
authors.forename: Cindy
authors.initials: C
authors.lastname: Berlinicke
authors.affiliation: Institute of Genetic Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland, United States of America.
authors.forename: Alyssa
authors.initials: A
authors.lastname: Kallman
authors.affiliation: Department of Ophthalmology, Wilmer Institute, Johns Hopkins University School of Medicine, Baltimore, Maryland, United States of America.
authors.forename: Jiang
authors.initials: J
authors.lastname: Qian
authors.affiliation: Department of Ophthalmology, Wilmer Institute, Johns Hopkins University School of Medicine, Baltimore, Maryland, United States of America.|Institute of Genetic Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland, United States of America.|Department of Molecular Biology and Genetics, Johns Hopkins University School of Medicine, Baltimore, Maryland, United States of America.|Department of Neuroscience, Johns Hopkins University School of Medicine, Baltimore, Maryland, United States of America.|Institut de la Vision, University Pierre and Marie Curie, Paris, France.
authors.forename: Donald J
authors.initials: DJ
authors.lastname: Zack
mesh_terms: D000818:Animals; D019556:COS Cells; D002522:Chlorocebus aethiops; D005434:Flow Cytometry; D057809:HEK293 Cells; D006801:Humans; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic / Q000235:genetics*; D034741:RNA, Small Interfering / Q000235:genetics*; D012243:Rhodopsin / Q000235:genetics; D015533:Transcriptional Activation / Q000235:genetics; D014162:Transfection
chemical_list: D034741:RNA, Small Interfering; D012243:Rhodopsin