FIG. 3.

Endogenous miRNAs can act as siRNAs on synthetic modified mRNAs with a miR target site. (a) Schematic representation of Luc mRNA with a miRts in the 3′ UTR. (b, c) Endogenous miRNAs can act as siRNAs on modified mRNA and mediate mRNA cleavage. (b) RLUs 24 h after Jurkat cells were EP with 1-methyl-pseudouridine (m¹Ψ) mRNA encoding Luc. Luminescence from cells with 142ts-containing mRNAs was compared with cells with CTRL mRNA, and P values were generated by Prism using the unpaired, two-tailed t-test. ****P < 0.0001. (c) Luc transcript levels normalized to HPRT transcript at various time points after EP. Luc transcript was assessed using three different primer pairs (Pf1-Pr1, Pf2-Pr2, and Pf3-Pr3) depicted in the schematic representation in (a). All values were normalized to Luc relative to HPRT transcript 5 min after electroporation. Bar graph showing percent total reads obtained across the Luc transcript after 5′-phosphate sequencing from total RNA extracted from cells 30 min post-EP. A zoomed-in view of the boxed region in the transcript is shown in the inset. miR142 sequence is shown in blue. (d) Schematic representation of Epo encoding mRNA with a miRts in the 3′ UTR. (e) Endogenous miRNAs trigger mRNA decay and protein suppression for modified mRNA for multiple open reading frames. Serum Epo levels 24 h after Jurkat cells were EP with m¹Ψ mRNA encoding Epo. Epo expression from cells with 142ts-containing mRNAs was compared with cells with CTRL mRNA, and P values were generated by Prism using the unpaired, two-tailed t-test. **P < 0.01. Epo transcript levels normalized to HPRT at various time points after EP. Epo transcript was assessed using the primer pair (Pf-Pr) depicted in the schematic representation in (d). All values were normalized to Epo relative to HPRT transcript 5 min after electroporation. HPRT, Hypoxanthine phosphoribosyltransferase; siRNA, small interfering RNA; EP, electroporated; Epo, erythropoietin.