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. 2003 May;9(5):607-17.
doi: 10.1261/rna.5250403.

A quantitative analysis of intron effects on mammalian gene expression

Ajit Nott  1 , Shlomo H Meislin, Melissa J Moore
Affiliations

A quantitative analysis of intron effects on mammalian gene expression

Ajit Nott et al. RNA. 2003 May.

Abstract

In higher eukaryotes, intron-containing and intronless versions of otherwise identical genes can exhibit dramatically different expression profiles. Introns and the act of their removal by the spliceosome can affect gene expression at many different levels, including transcription, polyadenylation, mRNA export, translational efficiency, and the rate of mRNA decay. However, the extent to which each of these steps contributes to the overall effect of any one intron on gene expression has not been rigorously tested. Here we report construction and initial characterization of a luciferase-based reporter system for monitoring the effects of individual introns and their position within the gene on protein expression in mammalian cells. Quantitative analysis of constructs containing human TPI intron 6 at two different positions within the Renilla luciferase open reading frame revealed that this intron acts primarily to enhance mRNA accumulation. Spliced mRNAs also exhibited higher translational yields than did intronless transcripts. However, nucleocytoplasmic mRNA distribution and mRNA stability were largely unaffected. These findings were extended to two other introns in a TCR-beta minigene.

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Figures

FIGURE 1.
FIGURE 1.
Schematic representation of TPI/Renilla luciferase reporter constructs. (A) Human triose phosphate isomerase (TPI) exons 6 and 7 were cloned in frame at both ends of the Renilla luciferase ORF (no intron). TPI intron 6 was inserted at either end to generate the 5′ and 3′ intron constructs. (B) Increasing the amount of transfected 5′ intron plasmid elicits a linear increase in mRNA levels. RPA was performed 24 h post-transfection with probe K (Fig. 2C ▶). Two-hundred-fifty nanograms (lanes 1,3,5,7,9,11) or 1 μg (lanes 2,4,6,8,10,12) total RNA was used for RPA. (C) Renilla luciferase activity increases linearly with increasing mRNA levels. mRNA levels are represented relative to 100 ng DNA transfected. (LU) Luminescence units.
FIGURE 2.
FIGURE 2.
Introns enhance both luciferase expression and mRNA accumulation in transiently transfected HeLa cells. (A) Cell lysate luciferase activity measured 42 h post-transfection. Activities shown are an average of three independent experiments; error bars represent standard deviation. (B) Time course of luciferase expression in living cells. Transfected HeLa cells were monitored for luciferase activity from 4 to 46 h post-transfection. (C) Analysis of total TPI/Renilla mRNA levels 42 h post-transfection. RPA reactions contained 250 ng or 1 μg total RNA using probe A and probe C (schematized in Fig. 1A ▶). Bands corresponding to pre-mRNA and spliced mRNA are as indicated. Doublet observed with probe A is due to partial homology in the noncomplimentary tail of the probe. Zeocin mRNA, which is transcribed from the same plasmid as TPI/Renilla mRNA was used as a control. (UT) Untransfected. (D) Quantitation of mRNA levels. TPI/Renilla mRNA levels were normalized to zeocin mRNA and plotted relative to the no-intron control. Relative mRNA levels reflect an average of three independent experiments; error bars represent standard deviation.
FIGURE 3.
FIGURE 3.
Splicing does not affect mRNA stability. (A) Time course of TPI/Renilla mRNA decay after treatment of transfected cells with actinomycin D. RPAs were performed on 4 μg total RNA using probe C (Fig. 1A ▶) at times indicated following actinomycin D addition. The graph represents a quantitative analysis of the inset gel. (B) Time course of TPI/Renilla and GFP mRNA decay after transcription repression with 1 μg/mL doxycycline. Constructs expressed both mRNAs in opposing orientations as schematized at top. RPAs were performed on 2 μg total RNA using TPI/Renilla probe B (Fig. 1A ▶) and a GFP-specific probe at times indicated following doxycycline addition. Right-most lane monitors mRNA levels at the last time point in the absence of transcriptional repression.
FIGURE 4.
FIGURE 4.
Uncoupling transcription and 3′ end formation reduces mRNA accumulation from the 5′ intron construct, but has little effect on 3′ intron mRNA. (A) Efficiency of ribozyme (rz) self-cleavage. A probe spanning the ribozyme sequence (probe R) was hybridized to 1.5 μg (lanes 1,3,5) or 6 μg (lanes 2,4,6) total RNA from transfected cells. No intron plasmid DNA was used to obtain protected fragments corresponding to uncleaved mRNA (lane 7). (B) Comparison of mRNA levels from cells transfected with either the parental constructs (Fig. 1A ▶) or their ribozyme-containing equivalents. RPAs were performed with probe B on 1.5 or 6 μg total RNA. Lower panel shows endogenous cyclophilin mRNA levels. (C) Summary of TPI/Renilla mRNA levels for each construct relative to the no-intron/no-ribozyme control. Numbers represent the average of two independent experiments and error bars indicate standard deviations.
FIGURE 5.
FIGURE 5.
Splicing does not influence the nuclear/cytoplasmic distribution of TPI/Renilla mRNA. HeLa cells were transfected with varying amounts of the three plasmids in Figure 1A ▶ to achieve similar in vivo mRNA levels, and nuclear and cytoplasmic RNAs were fractionated as described (see Materials and Methods). RPA was performed on equal volumes of isolated RNA with TPI/Renilla probe B (Fig. 1A ▶) or one specific to endogenous cyclophilin mRNA. (n) nuclear; (c) cytoplasmic.
FIGURE 6.
FIGURE 6.
Analysis of overall gene expression, mRNA levels and nucleocytoplasmic distributions for TCR-β minigene constructs. (A) Schematic representation of TCR-β constructs containing two introns, intron 2 alone, intron 1 alone, or no intron. (B) Western blot analysis of total protein extracted from HeLa cells 42 h post-transfection with anti-FLAG™ and anti-hPrp18 antibodies. Note that the relative TCR-β protein level indicated for lane 4 (<1.8) only represents an upper estimate, as no specific Western signal could be detected in this lane. (C) RPA of 500 ng or 2 μg total RNA with probes E and F (schematized in A), and probes specific to endogenous cyclophilin mRNA as well as plasmid-encoded zeocin mRNA (for determination of transfection efficiencies). (D) Nucleocytoplasmic distribution of TCR-β and cyclophilin mRNAs. (n) Nuclear; (c) cytoplasmic.
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