Figure 1. A short stem-loop in the Hoxa9 5’ UTR is sufficient for recruitment of the ribosome.
(A) Model of functional RNA elements in the Hoxa9 5’ UTR that regulate the translation of subsets of Hoxa mRNAs in the embryo (Xue et al., 2015). TIE, translation inhibitory element; IRES, internal ribosome entry site.
(B) Schematic of the topology of regulatory elements in the mouse Hoxa9 5’ UTR. The 180 nucleotides (nt)-long Hoxa9 IRES-like RNA element (a9 IRES180) harbors the P3 and P4 stem-loops and resides 130 nt upstream of the AUG (native spacer).
(C) Secondary structure model of a9 IRES180, a zoomed-in view of the P4 stem-loop (red), and substitution mutations mapped onto the P4 structure. Nts mutated in P4(M5) (green). Numbers refer to nt positions in the Hoxa9 5’ UTR. Active P4 mutants (normalized Fluc/Rluc < 0.5 A.U.) are labeled “+”, moderately active mutants (Fluc/Rluc < 0.5, > 1.0 A.U.) are labeled “+/–”, and inactive mutants (Fluc/Rluc > 0.5 A.U.) are labeled “–”. Yellow: Sequence critical for IRES-like activity. See also Figure S1.
(D) Spacer sequence requirement for a9 IRES-like element activity is tested by inserting spacers of different lengths downstream of an IRES-like element in a bicistronic reporter mRNA plasmid (pRF). Rluc, renilla luciferase; Fluc, firefly luciferase.
(E) Bicistronic reporter genes were transiently transfected into mouse C3H/10T1/2 cells and expressed from plasmids. Cells from the same transfection were split in half for protein and mRNA analysis. Relative luciferase activity is expressed as a Fluc(IRES)/Rluc(cap-initiation) ratio normalized to respective Fluc/Rluc mRNA levels. Average IRES-like activity ± standard error of the mean (SEM), n = 4–15. pRF and actin 5’ UTR serve as negative controls, HCV IRES as an IRES control. a9 IRES FL: FL, full-length; pRF (vector), no insert in the intergenic region; A.U., arbitrary units.
(F) Bicistronic reporter mRNAs were transiently expressed as described in (E). Average IRES-like activity normalized to respective Fluc/Rluc mRNA levels ± SEM, n = 4–15.
(G) Bicistronic reporter mRNAs were transiently expressed as described in (E). actin(inv) serves as a spacer sequence control. Average Fluc/Rluc IRES-like activity ± SEM, n = 3–8.
(H) Schematic of monocistronic “mini UTR” Fluc and control Rluc reporter mRNAs. IRES-like elements and spacer-derivatives were introduced into the Fluc 5’ UTR, and Fluc/Rluc luciferase activity was measured in transiently plasmid-transfected C3H/10T1/2 cells. A co-expressed Rluc reporter served as reference. Average Fluc/Rluc activity is normalized to respective globin/NupL1 mRNA levels ± SEM, n = 3–7; -, TIE alone; ns, not significant.