Nucleic Acids Research, 1993, Vol. 21, No. 25 5901-5908 Stimulation of gene expression by introns: conversion of an inhibitory intron to a stimulatory intron by alteration of the splice donor sequence Martin Korb+, Yunbo Ke and Lee F.Johnson* Departments of Molecular Genetics and Biochemistry, The Ohio State University, Columbus, OH 43210, USA Received September 16, 1993; Revised and Accepted November 18, 1993 GenBank accession no. L11364 ABSTRACT Efficient expression of many mammalian genes Introns have been shown to be directly responsible for a variety depends on the presence of at least one intron. We of qualitative changes in gene expression. For example, previously showed that addition of almost any of the alternative splicing of introns from primary transcripts can give introns from the mouse thymidylate synthase (TS) gene rise to multiple mRNA (and protein) species from a single gene to an intronless TS minigene led to a large increase In (reviewed in 3). expression. However, addition of intron 4 led to a Introns can also have quantitative effects on gene expression. reduction In minigene expression. The goal of the Some introns contain promoter or enhancer elements and present study was to determine why TS intron 4 was stimulate expression by increasing the rate of gene transcription unable to stimulate expression. Insertion of intron 4 (4-12). Regulating the efficiency of intron splicing also appears into an intron-dependent derivative of the ribosomal to play an important role in the autogenous regulation of yeast protein L32 gene did not lead to a significant increase ribosomal protein genes (13), and in the growth-regulated in expression, suggesting that its inability to stimulate expression of several mammalian genes for S-phase proteins expression was due to sequences within the intron. (14-18). Studies with chimeric genes or with intronless Deleting most of the interior of intron 4, improving the derivatives of normal genes have shown that addition of an intron putative branch point, removing purines from the to an intronless gene can lead to a large increase in the level of pyrimidine stretch at the 3' end of the intron, or expression (19-25). In many cases, the increase is not due to removing possible alternative splice acceptor or donor a change in the rate of transcription and is observed even when sites within the intron each had little effect on the level foreign or chimeric introns are inserted into the intronless genes. of expression. However, when the splice donor Buchman and Berg (26) showed that sequences located in the sequence of intron 4 was modified so that it was vicinity of the intron/exon boundaries are responsible for the perfectly complementary to Ul snRNA, the modified stimulatory effects of some introns. These and earlier studies are intron 4 stimulated expression approximately 6-fold. consistent with the possibility that the splicing reaction itself is When the splice donor site of TS intron 1 (a stimulatory required for efficient production of a variety of mRNA species. intron) was changed to that of TS intron 4, the modified We have been studying the effects of introns on the expression intron 1 was spliced very inefficiently and lost the ability of the mouse thymidylate synthase (TS) gene. The mouse TS to stimulate mRNA production. Our observations gene is 12 kb in length and consists of a 1 kb open reading frame support the idea that introns can stimulate gene that is interrupted by 6 introns (27). Intronless derivatives of the expression by a process that depends directly on the mouse TS gene direct the production of small amounts of normal splicing reaction. TS enzyme and mRNA when transiently or stably transfected into TS(-) hamster V79 cells (28). However, when introns 5 and 6 were included in the minigene at their normal locations INTRODUCTION in the coding region, expression increased approximately 8-fold. The primary transcripts of almost all mammalian genes are The stimulatory effect is not intron-specific since inclusion of interrupted by introns which must be removed during the introns 1 and 2, 3 and 4 or intron 3 alone also led to a 3-20-fold processing reactions that lead to the formation of mature mRNA. increase in the level of expression (29,30). The effects of the Much progress has been made in understanding the biochemistry introns are not additive; rather, the level of expression of the of the splicing reaction (1,2). However, information about the minigene appears to be determined by the intron providing the biological significance of introns has emerged more slowly. highest degree of stimulation. Deletion of almost all of the interior * To whom correspondence should be addressed + Present address: Fred Hutchinson Cancer Research Center, 1124 Columbia Street, Seattle, WA 98104, USA 5902 Nucleic Acids Research, 1993, Vol. 21, No. 25 of introns 5 and 6 had no effect on their ability to increase TS Sc Su Sm gene expression, which argues against the presence of a 13 transcriptional enhancer sequence within the introns. Sc Su Sm Unexpectedly, inclusion of intact intron 4 alone at its normal I position in the TS coding region led to a decrease in the level A 123 of expression relative to that observed with the intronless TS *243 minigene (29). TS intron 4 represents a highly unusual example * 83 of an intron that is unable to stimulate expression of an intron- Ild2 A 123-Ild2 dependent gene. When another intron was included with intron 14 A 123-14 4 in the TS minigene, expression was elevated relative to the intronless minigene. This indicates that the inhibitory effect of intron 4 is recessive to the stimulatory effect of other introns. Figure 1. Intronless and intron-containing rpL32 minigenes. The construction The goal of the present study was to determine why intron 4 of the minigenes is described in Materials and methods. The black boxes represent is unable to stimulate expression. Analyses of this unusual intron the exons and the lines represent the intron or the 5' and 3' flanking regions. The A12 minigene retains intron 3 (13) of the rpL32 gene at its normal location. might provide unique insight into the mechanisms by which The A123 minigene lacks all three introns. The TS introns IId2 or I4 were inserted introns stimulate and inhibit gene expression. Our approach was at the SmaI site (Sm) in exon 4. The end-labeled (*) probe (243 nucleotides) that to attempt to convert the intron into a stimulatory intron by was used in the S1 nuclease protection assay and the 83 nucleotide fragment deleting sequences within the intron that may be responsible for protected by rpL32 mRNA are indicated. Abbreviations: Sc, Sacl; Sm, SnaI; inhibiting expression or by making specific changes in sequences Su, Sau3A. that are important for splicing. Our results suggest that the inhibitory effect of TS intron 4 is due to inefficient splicing of the intron which is caused by inefficient recognition of the splice SphI-EcoRI fragment that contains the internally deleted TS donor sequence by Ul snRNA. intron 1 from the pTI1d2T (see below), blunt-ending the fragment with T4 DNA polymerase, and ligating the fragment MATERIALS AND METHODS into the SmaI site in rpL32A 123. The SphI site is 22 nucleotides upstream of intron 1, while the EcoRI site, which was created Tissue culture and transfection by site directed mutagenesis (18), is 8 nucleotides downstream TS(-) V79 chinese hamster fibroblasts (31) and COS-l cells (32) of the intron 1/exon 2 boundary. A123-14 was constructed by were cultured in Dulbecco's modified Eagle's medium (GIBCO) ligating a blunt-ended Pvull-Bgll fragment containing the entire supplemented with 10% NuSerum (Collaborative Research). The TS intron 4 into the SmaI site of rpL32A 123. The Pvull site is medium for the V79 cells was also supplemented with 50 ,iM 73 nucleotides upstream of intron 4, while the Bgll site is 4 thymidine. Cells were transiently transfected by the calcium nucleotides downstream of the intron 4/exon 5 boundary. phosphate method (28,29). Routinely, 12 pmole of the test plasmid and 3.0 jig of control plasmid [pSV2cat, pSI56S, or Si nuclease protection assays TI56G(d)] were used per 10 cm tissue culture dish. The cultures Whole cell RNA was isolated using the guanidinium were harvested for protein or RNA analysis two days after isothiocyanate procedure (33). The RNA was ethanol precipitated transfection. 3 times to remove any residual guanidinium salt. Total cytoplasmic RNA was isolated by lysing the cells with buffer Minigenes containing Nonidet P-40, removing the nuclei by centrifugation The TS minigenes used in these studies contained 1 kb of 5' and purifying the cytoplasmic RNA by phenol-chloroform flanking DNA and 0.25 kb of 3' flanking DNA linked to the extraction (34). In some experiments, poly(A)+ RNA was first and last exons of the TS coding region, respectively. TS isolated by oligo(dT) -cellulose column chromatography (35). minigenes that are cloned into pUC18 are designated with the S1 nuclease protection assays were performed with total prefix 'p' whereas those cloned into pBS(+) (Stratagene) have cytoplasmic RNA or with poly(A) + RNA as described the prefix 'bs'. The intronless minigene (pTTT) has been previously (29,36) using the end-labeled probes described in each described (30). pTI1T contains intact intron 1 at its normal figure. The reaction products were analyzed on a 6% location in the coding region (18). In some cases, the minigenes polyacrylamide sequencing gel and quantitated with a Betascope were tagged with a 57 bp deletion between two adjacent BamHI 603 beta scanner (Betagen). sites in exon 3. Such minigenes were indicated with the suffix '(d)'.