Design and function of the RNA-ID reporter for RNA cis-regulatory elements. (A) Diagram of the dual GFP and RFP vector. Expression of superfolder GFP (Pedelacq et al. 2006) and yeast-optimized mCherry RFP (Keppler-Ross et al. 2008) are under the control of the bidirectional, galactose-inducible promoter GAL1,10. The MET15 gene is used for selection in yeast, and integration is directed to the ADE2 locus. (B) Schematic illustrating LIC cloning into the sites upstream of GFP. Single-stranded sequences on the 5′ and 3′ ends of the vector pEKD1024 (green lines) are created by digestion of the vector with restriction endonucleases PacI and BbrPI, followed by treatment with T4 DNA polymerase in the presence of dGTP to generate 17 and 12 base single-stranded ends. Two overlapping oligonucleotides with homology with the single-stranded vector ends suffice for cloning, and allow use of a single oligonucleotide containing a randomized sequence at a defined position without requiring a full-length base-paired complement. The top oligonucleotide (blue line) contains a sequence complementary to the 5′ LIC site, the sequence of interest, including the ATG, and a 12-base sequence complementary to the bottom oligonucleotide. The bottom oligonucleotide (brown line) minimally contains a sequence that is complementary to the 3′ LIC site sequence, followed by a sequence that base pairs with the indicated complementary sequence of the top oligonucleotide. Sequences can also be inserted near the 5′ end of the RFP gene after digestion with the restriction endonuclease SwaI and resection with T4 DNA polymerase to create different single-stranded ends (see Materials and Methods). (C,D) Comparison of fluorescence outputs from a reporter on a multicopy plasmid versus an integrated reporter. To express GFP, an in-frame ATG is inserted upstream of GFP, as described above. (C) Histogram of GFP fluorescence profile versus cell number from yeast cells bearing the GFP and RFP genes on a 2μ plasmid (orange), the identical GFP/RFP construct integrated at the ade2 locus (blue), and an integrated plasmid lacking the GFP and RFP genes (gray). (D) Scatter plot of cells expressing GFP and RFP. Cells and the colors are identical to those in C. (E) Comparison of the signal and noise, with or without the RFP cutoff, from multicopy versus integrated GFP constructs.
