Off Target, but Sequence-Specific, shRNA-Associated Trans-Activation of Promoter Reporters in Transient Transfection Assays

Tomohiro Masuda,Cindy Berlinicke,Donald J Zack,Jun Wan,Alyssa Kallman,Jiang Qian,Anitha Yerrabelli

doi:10.1371/journal.pone.0167867

Tomohiro Masuda, Cindy Berlinicke + Show 5 more

Open Access

https://doi.org/10.1371/journal.pone.0167867

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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.

Highlights

Rhodopsin, the visual pigment of rod photoreceptors (PRs), is essential for normal retinal development and for visual function [1,2,3]
Through use of this approach, we initiated this study with the goal of exploring the potential role of PIAS2 in modulating rhodopsin gene expression
We found that some of the PIAS2 short hairpin RNAs (shRNAs) tested demonstrated strong stimulation of rho promoter activity, which at first suggested that PIAS2 could act as a negative regulator of rhodopsin gene expression

Summary

Introduction

The visual pigment of rod photoreceptors (PRs), is essential for normal retinal development and for visual function [1,2,3]. A number of the studies that have characterized the rhodopsin promoter and the factors that contribute to its expression were performed using transient transfection reporter assays. CRX enhances photoreceptor-specific gene expression in both rod and cone PRs [7,8,9,10]. NRL promotes PR precursor cells to the rod lineage by simultaneously activating rod-specific genes, such as rhodopsin, and suppressing cone-specific genes [11,12,13]. CRX and NRL physically interact with each other [14], and promoter reporter assays show that they work together to synergistically enhance rhodopsin promoter activity [7]

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