Lengthening unstructured 5′ UTR enriched with UCC and ACCAC sequences progressively reduces translation of GFP reporter in yeast cells. (A) Schematic depiction of RNA-ID reporter that includes ADE2 loci for integration into the yeast genome, URA3 or MET15 selection marker gene (details in Materials and Methods), Gal 1,10 bidirectional promoter, RFP, and sfGFP ORFs. (B) Design of GFP mRNAs containing unstructured 5′ UTRs (NUS′) of different lengths. The constructs share the same NUS sequence but differ in how much of the NUS sequence is included from the 5′ end. (C) Mean GFP/RFP fluorescence ratios measured in yeast cells, which were transformed with different RNA-ID 5′ UTR NUS′ constructs as indicated. GFP/RFP ratio in GO RNA-ID with 6 nt-long 5′ UTR was set to 1. Spearman rank correlation coefficient R (Virtanen et al. 2020) between length and GFP/RFP ratio was determined to be −0.915 (P < 0.0001). (D) Mean GFP/RFP fluorescence ratios normalized to the ratio of GFP and RFP mRNA levels, which were determined by RT-qPCR. Error bars show standard deviations determined from three biological replicates.
