Evolutionary conservation of a molecular machinery for export and expression of mRNAs with retained introns

Functional analysis of the zebrafish CTE. (A) Expression of p24 from CTE reporter plasmid pCMVGagPol-DrCTE(1X) (HR4402) in the presence of human Nxf1 (HsNxf1) and mouse Nxt1 (MmNxt1). 293T cells (1.2 × 10⁶) were transfected with 1.8 µg pCMVGagPol-DrCTE (HR4402) and 90 ng of a plasmid expressing secreted alkaline phosphatase (pCMV-SEAP [HR1831]) to control for transfection efficiency, with or without plasmids expressing HsNxf1 and MmNxt1 as indicated in the figure. At 60 h post-transfection, supernatants were collected and analyzed for p24 levels and SEAP activity. p24 values shown in the figure have been normalized for SEAP activity. The data represent the average from two independent transfections. (B) Titration analysis of the Nxt1 protein on the function of the duplicated zebrafish CTE in the GagPol reporter construct. 293T cells (1.2 × 10⁶) were transfected with 1.8 µg pCMVGagPol-DrCTE(2X) (HR4466), 90 ng of pCMV-SEAP(HR1831), 125 ng pCMV-HsNxf1 (HR3704), and increasing amounts of pCMV-MmNxt1 (HR2415), as indicated in the figure. Transfection, p24, and SEAP analyses are the same as described in A.