RNA-ID, a highly sensitive and robust method to identify cis-regulatory sequences using superfolder GFP and a fluorescence-based assay

Abstract

We have developed a robust and sensitive method, called RNA-ID, to screen for cis-regulatory sequences in RNA using fluorescence-activated cell sorting (FACS) of yeast cells bearing a reporter in which expression of both superfolder green fluorescent protein (GFP) and yeast codon-optimized mCherry red fluorescent protein (RFP) is driven by the bidirectional GAL1,10 promoter. This method recapitulates previously reported progressive inhibition of translation mediated by increasing numbers of CGA codon pairs, and restoration of expression by introduction of a tRNA with an anticodon that base pairs exactly with the CGA codon. This method also reproduces effects of paromomycin and context on stop codon read-through. Five key features of this method contribute to its effectiveness as a selection for regulatory sequences: The system exhibits greater than a 250-fold dynamic range, a quantitative and dose-dependent response to known inhibitory sequences, exquisite resolution that allows nearly complete physical separation of distinct populations, and a reproducible signal between different cells transformed with the identical reporter, all of which are coupled with simple methods involving ligation-independent cloning, to create large libraries. Moreover, we provide evidence that there are sequences within a 9-nt library that cause reduced GFP fluorescence, suggesting that there are novel cis-regulatory sequences to be found even in this short sequence space. This method is widely applicable to the study of both RNA-mediated and codon-mediated effects on expression.

Translation of CGA codon repeats in yeast involves quality control components and ribosomal protein L1

“…3B). As we observed previously (Dean and Grayhack 2012), expression of the TAA-GFP construct increased from background expression levels (approximately 0.8) to nearly half the (CGA) 3 -GFP/RFP expression levels (6.9 GFP/RFP), and there was no change in GFP/RFP from a construct lacking the ATG initiation codon for GFP (data not shown). To determine if the effects of paromomycin on (CGA) 3 -GFP are specific or due to a general inhibition of translation, we examined the effects of cycloheximide on read-through of CGA codons.…”

“…2C). In this system, which is derived from the RNA-ID reporter (Dean and Grayhack 2012), a single bidirectional promoter (GAL1,10 promoter) drives expression of the Renilla luciferase-GFP fusion in one direction and red fluorescent protein (RFP) in the other direction. Standardizing measurements based on RFP expression reduces noise (Dean and Grayhack 2012) and facilitates comparisons between strains.…”

“…To test this idea, we grew strains with the integrated GFP and RFP reporters in media containing increasing concentrations of translational inhibitors, paromomycin or cycloheximide. We used a range of concentrations for the inhibitors, in each case reaching a maximal concentration at which growth inhibition was evident, but in which >89% of the cells expressed RFP at ≥5 × 10 3 units, an arbitrary cutoff for robust induction of the GAL1,10 promoter, which was established with the nondrug treated sample (Dean and Grayhack 2012). For paromomycin, we have previously demonstrated that improved read-through of the TAA stop codon at amino acid 7 of GFP in a poor context is detectable with this GFP-RFP reporter (Dean and Grayhack 2012), even at the lowest concentration of paromomycin used here.…”

“…For GFP and RFP fluorescence measurements, strains were grown and analytical flow cytometry was conducted as described previously (Dean and Grayhack 2012).…”

“…Inhibitory effects of CGA codons are observed with CGA codons inserted at either amino acid 4 or 314 upstream of firefly luciferase or at amino acid 4 upstream of Renilla luciferase (Letzring et al 2010); even two adjacent CGA codons at amino acid 6 upstream of green fluorescent protein (GFP) cause a substantial reduction in GFP expression (Dean and Grayhack 2012). The defect in efficient translation is due to I•A wobble decoding of CGA, rather than to tRNA abundance, since a single integrated copy of an anticodon-mutated tRNA Arg(UCG) that exactly base pairs with CGA strongly suppresses the expression defect caused by CGA codons (Letzring et al 2010).…”