Fig. 2: Further optimization of nuclear export, RNA stability and 5′ cap-independent translation of fluorescent INSPECT reporter modules.
From: Intron-encoded cistronic transcripts for minimally invasive monitoring of coding and non-coding RNAs
a, The INSPECT system was tested by transient transfection of HEK293T cells, followed by FACS analysis 48 h post-transfection. The effect of different genetic elements on the ability to express proteins from an intron (that is, the cumulative effect of nuclear export of the intron and translational efficiency of the intron-encoded protein) was validated by mScarlet-I fluorescence (readout at 586 nm), while detection of msfGFP signal indicated correct splicing of the exonic sequence (readout at 530 nm). b, A synthetic intron was nested within an msfGFP CDS (green fluorescence) on a plasmid system driven by the strong mammalian CAG promoter. c, In addition, an intron-encoded translational unit, IRES–mScarlet-I (red fluorescence; 3,795 bp from the splice donor to the splice acceptor) was inserted into the artificial intron already equipped with the INSPECT elements and offers the opportunity to insert additional genetic elements for nuclear export or RNA stability at the 5′ and 3′ end to enhance the INSPECT system. d, Representative results of FACS analysis readout at 530 nm (msfGFP; exonic signal; left) and 586 nm (mScarlet-I; intronic signal; right) of HEK293T cells transfected with the indicated INSPECT versions, including additional modifications. e, Median fluorescence intensity of cells transfected with the indicated constructs in d. Green represents msfGFP (exonic signal) and red represents mScarlet-I (intronic signal). The bars represent the median of three biological replicates with error bars representing s.d. Statistical significance was determined by one-way ANOVA with Bonferroni MCT (*P < 0.05; **P < 0.01; ***P< 0.001). Full statistical results are provided in Supplementary Table 1.
