Exon junction complexes mediate the enhancing effect of splicing on mRNA expression

Abstract

Intron-containing genes are generally expressed more effectively in human cells than are intronless versions of the same gene. We have asked whether this effect is due directly to splicing or instead reflects the action of components of the exon junction complex (EJC) that is assembled at splice junctions after splicing is completed. Here, we show that intron removal does not enhance gene expression if EJC formation is blocked. Conversely, RNA tethering of the EJC components SRm160 or RNPS1 boosts the expression of intronless mRNAs but not of spliced mRNAs. Splicing and RNPS1 tethering are shown to enhance the same steps in mRNA biogenesis and function, including mRNA 3' end processing and translation. Together, these data argue that the EJC is primarily responsible for the positive effect of splicing on gene expression.

Fig. 1.

The positive effect of splicing on β-globin gene expression depends on EJC recruitment. (A) Western analysis of the level of hemagglutinin-β-globin expression observed in 293T cells transfected with the indicated expression plasmids. Cells were transfected with 400 ng of each β-globin expression plasmid and 100 ng of pCMV/I-β-gal, which served as an internal control. This panel shows a representative experiment with quantitative data derived from three independent experiments, complete with standard deviation, given below each lane. (B) RPA of nuclear (N) and cytoplasmic (C) RNA fractions derived from 293T cells transfected with pCMV/I/Δ1+2/GLB or pCMV/SX/Δ1+2/GLB, or mock transfected. The probe used traverses the 3′ splice site of the 5′ UTR intron and therefore measures the level of expression of both unspliced (U) and spliced (S) mRNA.

Fig. 2.

Tethered EJC proteins can activate expression of an intronless gene. (A) 293T cells were transfected with 100 ng of the pCMV/CAT/B or pCMV/CAT indicator plasmid and 300 ng of a plasmid encoding an N-peptide fusion protein, or pBC12/CMV as a negative (NEG) control. Cells were also cotransfected with 25 ng of pCMV/I-β-gal as an internal control. Induced CAT activities were measured at 48 h posttransfection and adjusted for minor differences in the β-gal internal control. Data shown represent the average of three experiments with standard deviation indicated. (B) This assay was performed as shown for A except that cells were transfected with 20 ng of the pDM128/B or pDM128/PL indicator plasmid. (C) This assay was performed as shown in A by using either the intronless pCMV/CAT/B or the intron-containing pCMV/I-CAT/B indicator plasmid. (D) Schematic representation of the indicator plasmids used in A–C.

Fig. 3.

Comparison of the effect of splicing and EJC protein tethering on intronless mRNA expression. (A) Western analysis of the effect of the N-RNPS1 or N-SRm160 fusion protein on the level of β-globin expression from an intronless expression plasmid (pCMV/Δ1 + 2/GLB) lacking (–B) or containing (+B) box-B binding sites. This panel shows a representative result with quantitative data drawn from three independent experiments summarized at the bottom. The pCMV/I-β-gal plasmid served as the internal control. (B) RPA of nuclear (N) and cytoplasmic (C) RNA fractions obtained from 293T cells transfected with the indicated indicator and effector plasmids. The probe used traverses the genomic preproinsulin 3′ polyadenylation site used and can therefore quantitate the level of β-globin transcripts that are (PA⁺) or are not (PA^–) appropriately 3′ end processed. The effector (pCMV/N-RNPS1 and pCMV/N-SRm160) and control (pBC12/CMV) plasmids use a genomic bovine growth hormone polyadenylation site. (C) Data obtained from three independent RPAs, performed as shown in B, and the average steady-state level of appropriately 3′ end processed nuclear β-globin mRNA as a percentage of the total nuclear β-globin mRNA level. (D) Relative level of cytoplasmic β-globin mRNA, detected as described for B. The mRNA levels shown are normalized to the culture transfected with pCMV/WT/GLB, which is arbitrarily set at 100.