Abstract
RNA regulatory elements (RREs) are an important yet relatively under-explored facet of gene regulation. Deciphering the prevalence and functional impact of this post-transcriptional control layer requires technologies for disrupting RREs without perturbing cellular homeostasis. Here we describe genome-engineering based evaluation of RNA regulatory element activity (GenERA), a clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 platform for in situ high-content functional analysis of RREs. We use GenERA to survey the entire regulatory landscape of a 3′UTR, and apply it in a multiplex fashion to analyse combinatorial interactions between sets of miRNA response elements (MREs), providing strong evidence for cooperative activity. We also employ this technology to probe the functionality of an entire MRE network under cellular homeostasis, and show that high-resolution analysis of the GenERA dataset can be used to extract functional features of MREs. This study provides a genome editing-based multiplex strategy for direct functional interrogation of RNA cis-regulatory elements in a native cellular environment.
Highlights
RNA regulatory elements (RREs) are an important yet relatively under-explored facet of gene regulation
We reasoned that coupling efficient induction of genomic deletions by error prone NHEJ with generation sequencing (NGS) as readout of activity, could alleviate this limitation enabling rapid and multiplexed analysis of RRE functionality under normal cellular homeostasis
These findings suggest that the impact of post-transcriptional control imparted by intragenic RNA cis-regulatory elements has significantly evolved with organismal complexity[53]
Summary
RNA regulatory elements (RREs) are an important yet relatively under-explored facet of gene regulation. RREs are defined by characteristic sequence motifs that serve as docking sites for trans-acting factors, such as short non-coding regulatory RNAs (e.g. microRNAs) and RNA binding proteins (RBPs)[2] This knowledge enabled the development of in silico tools for genome-wide identification of putative RREs, studying the regulatory impact of these elements in an endogenous cellular context has been challenging[3]. Studies aiming to decipher the functional role of RREs have relied primarily on fusing RRE-containing UTRs (or a region flanking the predicted element) to reporter genes, which provide an indirect readout of their activity[4,5,6] Such constructs are often overexpressed and inherently remove the RREs from their native RNA context. Assessing the regulatory significance of MRE networks under endogenous conditions remains one of the most important yet unmet technical challenges in this field
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