Fig. 1: SINEB2 RNAs from different mouse antisense lncRNAs can act as SINEUP effector domain and upregulate GFP protein expression in HEK293T/17.

a Schematic representation of the sense/antisense EGFP system used to validate the effect of different SINEB2 RNAs on translation. BD, binding domain; ED, effector domain. b SINEUP construct design to verify independent and combined effects of two SINEB2 elements from the same antisense lncRNA. c Western blot-based EGFP band intensities normalized to ACTB expression as a measurement of SINEUP-induced protein fold change observed in the HEK293T/17 cells. Empty vector, Direct AS Uchl1 SINEB2, and ΔAS Uchl1 SINEB2 (SINEB2 deletion) were used as controls. N = 3 biologically independent experiments. d Western blot images with anti-GFP and anti-ACTB antibodies. AS, antisense; Direct, direct orientation of SINEB2; Δ, deletion; a and b, two different SINEB2 elements from the same antisense lncRNA; ab and ba, the two SINEB2 elements in original and reversed order, respectively; AS Uchl1 SINEB2–RS, antisense Uchl1 SINEB2 construct without any restriction enzyme sites around the SINEB2 elements. e GFP protein fold changes in HEK293T cells after co-transfection with sense EGFP and miniSINEUP. Western blot images and corresponding GFP band intensities normalized to ACTB expression level. Empty vector = negative control, and antisense Uchl1 SINEB2 containing miniSINEUP (AS Uchl1) = positive control. Sample labels indicate names of antisense lncRNAs from which the SINEB2 sequences were isolated, which represent four sub-families (based on RepeatMasker annotation): B3, B3A, Mm2, and Mm1t. SINEB2 elements are from the B3 subfamily unless specifically stated. AS Uchl1 + AS Uxt-b = combination of AS Uchl1 SINEB2 and the second SINEB2 of AS Uxt; 3× indicates three repeats of SINEB2; and SINEB2/B3 consensus is the B3 subfamily consensus sequence taken from the RepBase database. N = 5 biologically independent experiments. In (c) and (e), the data are presented as mean values ± SEM, data points for biological replicates are depicted by blue dots and significant P values calculated by two-tailed Student’s t-test in comparison to empty vector are noted on the charts. Source data are provided as a Source Data file.