Pmirglo luciferase expression vector

The dual luciferase assays were used to verify the function of the m⁶A site of PxJHE gene in HKT‐293T cells as described elsewhere.^[40 (link)
^] The pmirGLO luciferase expression vector (Promega) with a firefly luciferase (F‐luc) and a Renilla luciferase (R‐luc) was used to construct the fluorescent reporter plasmid as described elsewhere.^[41 (link)
^] Wild‐type PxJHE fragment (PxJHE‐wt) contained CDS and 3′‐UTR of PxJHE and mut‐type PxJHE fragment (PxJHE‐mut) was made by replacing the adenosine bases (A) with cytosine (C). The fragment of PxJHE‐wt and PxJHE‐mut was attached to the pmirGLO vector by In‐Fusion HD Cloning Kit (TaKaRa) according to the instructions. The HKT‐293T cells were seeded in a 24‐well plate using DMEM medium with 10% FBS and 1% antibiotics, and cells were cultured in a CO₂ incubator at 37 °C. Transfection experiments were performed when the cell density was ≈ 80%. 200, 400, 600, and 800 ng plasmid of PxJHE‐wt or PxJHE‐mut were transfected into HKT‐293T cells cultured in DMEM medium with 10% FBS using Lipofectamine 3000 (Invitrogen). After 48 h, the cells were lysed, and the fluorescence activity was detected using the Dual‐Glo Luciferase Assay System (Promega). The effect of the m⁶A site on PxJHE mRNA was evaluated by firefly luciferase activity, and firefly luciferase activity was normalized to Renilla luciferase activity.

pmirGlo luciferase expression vector (Promega) containing a firefly luciferase (F‐luc) and a Renilla luciferase (R‐luc) was used to construct the reporter plasmid. The F‐luc‐6 × Phe(TTT) reporter plasmid was obtained by inserting TTTTTTTTTTTTTTTTTT before the F‐luc coding region; the F‐luc‐6 × Phe(TTC) reporter plasmid was obtained by inserting TTCTTCTTCTTCTTCTTC before the F‐luc coding region. The F‐luc genes with all the Phe codons using either TTT or TTC were synthesized by TsingKe (Beijing, China) and constructed into the reporter plasmids. Basic setting: 40 ng of reporter plasmids (pmirGlo empty vector or pmirGlo mutated vector) was transfected into WT HEK293T cells and ftsj1 knockout cells in a 24‐well plate using Lipofectamine 2000. After 24 h, the cells in the 24‐well plate were assayed by Dual‐Glo Luciferase Assay System (Promega). R‐luc was used to normalize F‐luc activity to evaluate the translation efficiency of the reporter. We employed the pmirGlo mutated reporter normalized to the pmirGlo empty vector.

For function analysis of tRNA‐LysCTT, dual reporter plasmid, including firefly luciferase (F‐luc) coding sequence and Renilla luciferase (R‐luc) coding sequence, was established on the basis of pmirGlo luciferase expression vector (Promega, Madison, WI, USA). Five repeats of AAG codon sequence (5X AAG) were introduced into the upstream of F‐luc coding sequence. Five hundred nanograms of 5X AAG (Lys) or control reporter plasmids were transfected into Huh7 or MHCC97H cells using Lipofectamine 3000 in a six‐well plate. After 48 h, the cells were assayed by Dual‐Glo Luciferase Assay system (Promega) in a 96‐well plate. R‐luc was used for the normalization of F‐luc activity.