Fig. 1
From: mRNAs and lncRNAs intrinsically form secondary structures with short end-to-end distances
RNA ends are brought within FRET distance via the formation of intramolecular basepairing interactions. a An exemplary secondary structure from the ensemble of structures of rabbit β-globin mRNA lacking poly(A) tail predicted by free energy minimization. Base pair probabilities, predicted with a partition function, are color-coded. In order to measure the end-to-end distance by FRET, the 5′ and 3′ ends of mRNA were conjugated with donor (Cy3) and acceptor (Cy5) fluorophores, respectively, as indicated. b Average end-to-end distances of mRNAs and lncRNAs, which were folded in the presence of 100 mM KCl and 1 mM MgCl2, were determined by ensemble FRET measurements and plotted as a function of RNA length: yeast RPL41A mRNA (red), firefly luciferase mRNA (orange), rabbit β-globin mRNA (magenta), human ATP5J2 mRNA (green), HSBP1 mRNA (indigo), MIF mRNA (blue), MRPL51 mRNA (gray), GAPDH mRNA (brown), HOTAIR lncRNA (purple), and NEAT1_S lncRNA (dark green). The black line shows theoretically predicted end-to-end distance of unstructured RNA, assuming a freely jointed chain model. c FRET values were measured between fluorophores attached to the 5′ and 3′ ends of the following GAPDH and β-globin mRNAs: mRNA lacking poly(A) tail (blue); mRNA lacking poly(A) tail folded in the presence of a 50-nucleotide-long DNA oligonucleotide complementary to the 3′ end of mRNA (green); mRNA with poly(A) tail (pink). FRET could not be detected (n.d.) in GAPDH variants, which lacked poly(A) tail and contained 53 CA repeats introduced into the 5′ or 3′ UTR. Each FRET value represents the mean ± standard deviation (SD) of three independent experiments. A star indicates that FRET values are significantly different, as p-values determined by the Student t-test were below 0.05
