Figure 1.

STAU1 increases the production of EGFP from EGFP-IRAlus mRNAs by augmenting nuclear mRNA export to the cytoplasm. (A) Diagram of the pEGFP-IRAlus reporter plasmids. (Black box) EGFP translational reading frame; (diverging arrows) 3′ UTR IRAlus. (B) Western blotting using lysates of HEK293T cells (8 × 10⁶ per 100-mm dish) that had been transiently transfected with 2.5 μg of pmRFP and 2.5 μg of the designated pEGFP reporter, 1 d later with 50 nM control siRNA or STAU1 siRNA, and 2 d later with 10 μg of pcDNA3-HA (−) or pcDNA3-STAU1⁵⁵-HA₃ (+). EGFP levels were normalized to the level of mRNA from which they derived (Supplemental Fig. S1C), and the normalized level in the presence of control siRNA + pcDNA3-HA is defined as 100. STAU1⁵⁵ and STAU1⁶³ are, respectively, 55-kDa and 63-kDa isoforms of STAU1 that are the products of alternative splicing (Wickham et al. 1999). Here and elsewhere, each bracket to the left denotes blots loaded so the amounts of reference protein, marked with a solid diamond (♦), were comparable. (C) Western blotting of lysates of HEK293T cells (1.8 × 10⁷ per 150-mm dish) that had been transiently transfected with 5 μg of pmRFP and 5 μg of the specified pEGFP reporter, 1 d later with 50 nM control siRNA or STAU1 siRNA, and 2 d later with pcDNA3-HA (−) or 15 μg (+) or 25 μg (++) of pcDNA3-STAU1⁵⁵-HA₃ (where pcDNA3-HA was added to keep the total micrograms of introduced plasmid constant). See Supplemental Table S3 for quantitations. (D) Western blotting of nuclear (N) and cytoplasmic (C) fractions prepared from cells analyzed in C. (E) Histograms of RT-qPCR quantitations (Supplemental Fig. S1D,I; Supplemental Table S2) of samples shown in C and D, where the level of each EGFP reporter mRNA was normalized to the level of mRFP mRNA, and the levels of cellular mRNAs were normalized to the level of GAPDH mRNA in total cell (T) and cytoplasmic (C) extracts or the level of U6 snRNA in nuclear (N) extracts. The normalized level in the presence of control siRNA + pcDNA3-HA is defined as 100. Note that the level of cytoplasmic LIN28 mRNA was normalized to the level of total cell LIN28 mRNA, since its cytoplasmic level in the absence of STAU1⁵⁵-HA₃ was undetectable. The left lanes below wedges analyzed serial threefold dilution of total cell protein. All results are representative of three independently performed experiments. Additionally, they recapitulate quantitations of blots that analyzed larger amounts of pEGFP-IRAlus-derived proteins. (*) P < 0.05; (**) P < 0.01; n ≥ 3.