Abstract
Posttranscriptional regulatory programs governing diverse aspects of RNA biology remain largely uncharacterized. Understanding the functional roles of RNA cis-regulatory elements is essential for decoding complex programs that underlie the dynamic regulation of transcript stability, splicing, localization, and translation. Here, we describe a combined experimental/computational technology to reveal a catalog of functional regulatory elements embedded in 3' UTRs of human transcripts. We used a bidirectional reporter system coupled with flow cytometry and high-throughput sequencing to measure the effect of short, noncoding, vertebrate-conserved RNA sequences on transcript stability and translation. Information-theoretic motif analysis of the resulting sequence-to-gene-expression mapping revealed linear and structural RNA cis-regulatory elements that positively and negatively modulate the posttranscriptional fates of human transcripts. This combined experimental/computational strategy can be used to systematically characterize the vast landscape of posttranscriptional regulatory elements controlling physiological and pathological cellular state transitions.
Highlights
Gene expression is highly regulated in order to achieve the requisite repertoire of RNA and protein products across the vast space of possible cellular phenotypes encompassing physiological and developmental contingencies
Gene expression can be heavily influenced by the fate of mRNAs post-transcriptionally
To construct the post-transcriptional reporter library, the generated pool of DNA was cloned downstream of a fluorescent reporter system stably integrated into a single chromosomal locus in the Flp-In 293 cell line, a human embryonic kidney cell line with a single Flp Recombination Target (FRT) site (Life Technologies)
Summary
Gene expression is highly regulated in order to achieve the requisite repertoire of RNA and protein products across the vast space of possible cellular phenotypes encompassing physiological and developmental contingencies. Messenger RNAs pass through several steps of regulation in the nucleus and cytoplasm that control their processing, surveillance, localization, translation and stability (Martin and Ephrussi, 2009; Moore, 2005). Such post-transcriptional processing allows the cell to fine-tune gene expression in a fast, precise and cost effective manner (Keene, 2007). Trans-factors including microRNAs (Bartel, 2004; He and Hannon, 2004) and RNA-binding proteins (Glisovic et al, 2008) can exert significant influence on the stability and the translation of their target mRNAs. many species rely on post-transcriptional regulation to maintain mRNAs in a translationally silent state in different cell-types and developmental stages (Carrington and Ambros, 2003; Farh et al, 2005). Perturbations to these regulatory programs can lead to disease, including neurodegenerative disorders and cancer (Cooper et al, 2009; Lukong et al, 2008)
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