Fig. 1: Nested dual-luciferase system for optimizing nuclear export, RNA stability and 5′ cap-independent translation of INSPECT.
From: Intron-encoded cistronic transcripts for minimally invasive monitoring of coding and non-coding RNAs
a, General workflow for optimization of the INSPECT reporter system via dual-luciferase assay. b, The synthetic intron was nested within a FLuc–PEST CDS on a plasmid system driven by the mouse Pgk1 promoter. Furthermore, the translational unit IRES_NLuc–PEST was inserted into the artificial intron, which is composed of two highly efficient splice sites (splice donor (SD) and splice acceptor (SA)) for the insertion of additional genetic elements for nuclear export and cap-independent translation at the 5′ and 3′ end. Consequently, the NLuc signal represents effective nuclear export and translation of the intron-encoded cistronic transcript, while detection of the FLuc signal indicates correct splicing of the exonic sequence. NES, nuclear export signal; p(A), poly(A). c, The reporter system also features a Cre recombinase-inducible KO switch encoded by an inverted triple poly(A) signal flanked by two heterospecific loxP pairs. Upon transfection of Cre recombinase, the poly(A) sites are inverted, together with an upstream splice acceptor leading to KO of the gene. BP, branch point; SA, splice acceptor. d,e, Results of the dual-luciferase assay schematized in a, for different variants of nuclear export and RNA stabilization elements inserted at either the 5′ site or the 3′ site (relative to IRES_NLuc–PEST) as tandem repeats (the numbers of repeats are indexed as subscripts) (d), or at both insertion sites in parallel (e). RLU values are normalized to the negative control without nuclear export elements. CTE, from the Mason–Pfizer monkey virus; CTE*, a variant of CTE with cryptic splice donor-like motifs eliminated; CTE**, a variant of CTE with cryptic splice donor-like motifs replaced by a different sequence from another virus strain; RTE, m26 mutant of an RNA transport element with homology to rodent intracisternal A particles; triplex: triple helix-forming RNA from mouse MALAT1 lncRNA for 3′ end stabilization. f, The combination of 5′ CTE2 with 3′ CTE2** was compared in the context of different IRES sequences from either ECMV or the human gene VCIP. ‘Cre:’ indicates the co-transfection of a plasmid expressing Cre recombinase, which recognizes the heterospecific loxP and lox2272 to activate the KO switch (see schematic in c). In e,f, the grey shading indicates the INSPECT reporter of choice for further experiments. In d–f, the bars represent the mean of three biological replicates, with error bars representing s.d. Selected results of Bonferroni MCT after one-way ANOVA analysis are shown (*P < 0.05; **P < 0.01; ****P < 0.0001). For clarity, not all statistical comparisons are graphically presented, but full statistical results are given in Supplementary Table 1.
