Nucleoplasmic accumulated mRNAs upon NXF1 depletion had passed through NSs and recruited export factors. (A) Western analysis was used to examine the knockdown (KD) efficiency of NXF1 and UAP56. Note that to achieve efficient mRNA nuclear retention and to avoid extensive cell death, the cells were treated with UAP56 and URH49 siRNAs for 36 h. (B and C) Confocal microscopic images show the distribution of polyA RNAs (B) or the endogenous HSPA1A mRNA (C) and NSs (SRSF2) in HeLa cells treated with Cntl or UAP56/URH49/NXF1 siRNAs for 36 h. The green and red lines in the graphs demonstrate the RNA and SRSF2 IF signal intensities, respectively. Colocalization indexes are shown on the right. (D and E) Confocal microscopic images show the distribution of polyA RNAs (D) or the endogenous (Endo.) HSPA1A mRNA (E), UAP56, and NSs (SRSF2) in HeLa cells treated with Cntl or NXF1 siRNA for 48 h. The green, red, and blue lines in the graphs demonstrate the RNA, UAP56, and SRSF2 signal intensities, respectively. Colocalization indexes are shown on the right. Data represent the mean ± SD from three independent experiments; n = 10. (F) mRNAs derived from intron-containing genes associate with NSs dependent on splicing, whereas naturally intronless mRNAs enter these subnuclear structures via SR proteins that are bound on ESEs. Aberrant mRNAs such as cDNA transcripts cannot enter NS due to lack of splicing. In NSs, mRNA export factors are efficiently recruited due to their high concentration, resulting in efficient nuclear export of spliced and intronless mRNAs. Aberrant mRNAs have a limited chance to get access to mRNA export factors and are mostly retained in the nucleus. Bars, 20 µm. Statistical analysis was performed using an unpaired t test. ***, P < 0.01. Molecular masses are given in kilodaltons.