Fig. 4

Repression by iSINEs is dependent on secondary structures and sequence in a species-independent manner. a The iSINE-containing 3′ UTR of the Znf708 gene was inserted downstream of the firefly luciferase ORF. To generate perfect complementarity, one Alu Sg was replaced by a duplication of the Alu Sc, giving rise to a perfect inverted SINE (piSINE). A perfect tandem SINE (pdSINE) and 1SINE were used as controls and made by flipping or deleting the second SINE, respectively. b Dual luciferase assays of different SINE configurations derived from the Znf708 3′ UTR demonstrate that the reduction of gene expression correlates with the extent of double-strandedness. c–f To evaluate whether the observed reduction in gene expression is specific for iSINEs or dependent on RNA structure alone, UTRs that resemble the secondary structure of the Znf708 UTR but with different sequence context were designed. The Znf708 analogues and respective controls were transfected into U2OS cells and gene expression was quantified using a dual luciferase assay. See Additional file 1: Figure S2 for minimum free energy structures of the Znf708 UTR and the designed Znf708 analogues. g To generate reporter constructs harboring mouse SINEs, B1 elements of the mouse car5b gene were used to replace the Alu elements in Znf708. h The B1-harboring reporter genes were transfected into U2OS cells and a dual luciferase assay was performed after 24 h. Standard deviations are indicated. Asterisks indicate p values calculated with Student’s t-test: *p < 0.05, **p < 0.005, ***p < 0.0005