Tenfold lengthening of unstructured 5′ UTR moderately reduces translation of GFP reporter in yeast cells. (A) Design of GFP mRNAs containing unstructured 5′ UTRs (NUS1 series) of different lengths. (B) Mean GFP and RFP fluorescence measured in yeast cells, which were transformed with different RNA-ID constructs as indicated. (C) Mean GFP fluorescence in strains expressing RNA-ID mRNA with unstructured 5′ UTRs of different lengths normalized by GFP fluorescence produced in the GO RNA-ID control reporter with a 6 nt-long 5′ UTR. (D) Mean GFP/RFP fluorescence ratios. GFP/RFP ratio in the GO RNA-ID control was set to 1. Spearman rank correlation coefficient R (Virtanen et al. 2020) between length and GFP/RFP ratio was determined to be −0.841 (P < 0.0001). (E) Mean GFP/RFP fluorescence ratios normalized to the ratio of GFP and RFP mRNA levels, which were determined by RT-qPCR. NUS1 and NUS2 are independently designed, divergent, nonrepetitive, unstructured sequences. Error bars show standard deviations determined from three biological replicates.
