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Originally published In Press as doi:10.1074/jbc.M002969200 on June 23, 2000

J. Biol. Chem., Vol. 275, Issue 39, 30248-30255, September 29, 2000
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Length Increase of the Human alpha -Globin 3'-Untranslated Region Disrupts Stability of the Pre-mRNA but Not That of the Mature mRNA*

Pierre R. Provost and Yves TremblayDagger §

From the Laboratory of Ontogeny and Reproduction, Centre Hospitalier Universitaire de Québec, Pavillon CHUL, and the Dagger  Department of Obstetrics and Gynecology, Laval University, Québec G1V 4G2, Canada

Received for publication, April 7, 2000, and in revised form, June 6, 2000

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Polyadenylation increases the stability of mRNA molecules. By studying the effect of the length of 3'-untranslated region (UTR) on mRNA levels, we have found that alpha -globin pre-mRNA is stabilized by a mechanism that does not modulate the half-life of mature mRNA. The insertion of DNA fragments of various unrelated sequences into the 3'-UTR of the human alpha -globin gene strongly reduces mRNA abundance upon transfection into choriocarcinoma JEG-3 cells. We found an inverse relationship between mRNA levels and the length of the introduced fragments. In fact, mRNA levels as low as 1% were observed after inserting a 477-nucleotide (nt) fragment, whereas inserting a fragment of 86 nt at the same position had no effect on mRNA accumulation. DNA insertion induced no change in transcription rate or in half-life of mature mRNA. Semi-quantitative reverse transcription-polymerase chain reaction revealed that inserting a 477-nt fragment in the 3'-UTR resulted in decreased levels of nuclear pre-mRNA in proportion to that observed for mature mRNA. In contrast, the insertion of the 477-nt exogenous DNA in the last intron had no effect on mRNA levels despite the presence of intronic sequences in the pre-mRNA. This shows that the reduction of pre-mRNA level was not due to the insertion of putative ribonuclease cleavage sites or the insertion of a segment DNA that reduces the elongation efficiency. Taken together, our results strongly support the existence of a pre-mRNA stabilizing mechanism that can be disrupted by increasing the length of the 3'-UTR. The fact that the half-life of mature mRNA is not affected by DNA insertion is compatible with a pre-mRNA-specific stabilizing mechanism that acts specifically before polyadenylation.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Before reaching cytoplasm, most vertebrate primary transcripts are processed at their 5' end by capping (1) and at their 3' end by polyadenylation (2), and they are spliced to remove introns (3, 4). Pre-mRNA 3'-end processing is a two-step process that involves endonucleolytic cleavage and addition of 50-250 adenosine residues. 3'-End formation requires the presence of a polyadenylation signal (A(A/U)UAAA) (5) and a G/U-rich sequence (6) localized upstream and downstream of the cleavage site on pre-mRNAs, respectively.

RNA 3'-end formation and splicing can occur independently in vitro (7-9). However, when introns were removed from genes, very low levels of mRNA accumulated upon transfection of culture cells (10-15) and in transgenic mice (16, 17). In fact, the presence of introns in pre-mRNAs increases levels of mature mRNA up to 50-fold by enhancing 3'-end formation (15, 18-22). It is now generally accepted that this effect depends on the splice acceptor site of the last intron (15, 18, 19, 23-28). Little or no effect on 3'-end formation was observed by deleting the donor site of the last intron indicating that the intron removal per se is not essential in 3'-end formation. This conclusion was reinforced by the observation that pre-mRNAs transcribed from two human beta -globin genes defective in splicing were cleaved and polyadenylated as the normal beta -globin pre-mRNA (29). A favored model to explain the role of the acceptor site is that spliceosome assembly at the 3' splice site of the last intron may facilitate cleavage and polyadenylation processes (18, 19, 25, 26). We reasoned that if the enhancing effect of the intron on 3'-end formation involves an interaction between elements present in the two regions, then the length between the last intron and the polyadenylation site should influence the efficiency of 3'-end formation and, hence, the steady state levels of mRNA.

To study the interaction between a cis-acting element of the polyadenylation region and other element(s) located further upstream, we have increased the length of the terminal exon of the alpha -globin gene, more precisely, the length of its 3'-untranslated region (UTR),1 and analyzed mRNA steady state level. This was performed by inserting DNA fragments of various length and sequences. Co-transfection of the resulting clones with the parental plasmid into a choriocarcinoma cell line, JEG-3, that does not express the endogenous globin genes revealed that the length of the 3'-UTR strongly influences the level of alpha -globin mRNA. We succeeded in linking this effect to the disruption of pre-mRNA stability with no change in mature mRNA decay.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Recombinant Plasmids-- To prepare the pSV-SPORT-1-Delta plasmid vector, pSV-SPORT-1 (Life Technologies, Inc.) was digested by NheI and HpaI to remove a 380-bp fragment bearing the small t intron and one ATTTA putative regulatory element, blunted, and re-circularized. Plasmid SV-alpha GL was prepared by inserting the 1119-bp HinfI-HinfI fragment of the plasmid pUCalpha (30) at the EcoRI site of pSV-SPORT-1-Delta , downstream of the SV40 early promoter that encompasses the 72-bp direct repeats and the 21-bp GC-rich sequences. The pUCalpha plasmid contains the 1.5-kilobase pair PstI-PstI fragment of the alpha 1-globin gene. The HinfI-HinfI fragment contained the alpha 1-globin gene sequence from the 18th base upstream of the initiation codon to the 101st base downstream of the poly(A) signal and 209 bases of pUC19. All of the clones presented were confirmed by DNA sequencing.

The 3beta -HSD-1-PvuII-EcoRI (477 bp) and -FokI-EcoRI (370 bp) fragments were prepared from the type 1 3beta -hydroxysteroid dehydrogenase (3beta -HSD-1) cDNA clone 36 (31), blunted, and introduced at the BalI or the NarI site of SV- alpha GL to produce SV-alpha GL/3beta -370-S, -A,SV-alpha GL/3beta -477-S, -A (Fig. 1A), and SV-alpha GL/3beta /INTRON (Fig. 3A). The 477-bp fragment was also introduced in the BalI site of pUCalpha to produce pUCalpha /3beta -477-S, and -A. These 3beta -HSD-1 fragments did not contain the 3beta -HSD-1 poly(A) signal. The sense (-S) and antisense (-A) orientations refer to the conventional 5' right-arrow 3' transcription of the 3beta -HSD gene. The 3beta -HSD-1 fragment of 370 bp was further digested by MaeI (Fig. 1A), and the two smaller fragments were inserted into the BalI site of SV-alpha GL to produce SV-alpha GL/3beta -86-S, -A, SV-alpha GL/3beta -286-S, and -A. Clone SV-alpha GL/17beta was constructed by inserting the EcoRI-StuI fragment of 295 bp, originating from the 5'-end of the 17beta -HSD-1 cDNA clone (32, 33), into the BalI site of SV-alpha -GL in the sense orientation.

Substitution of the 3'-UTR of the alpha -globin gene by that of SV40 present in vector sequences was performed by deletion. SV-alpha GL was digested by BstXI and NotI to remove the 3'-UTR, a part of exon 2, and the entire intron 2 and exon 3 of alpha -globin. The resulting fragment was ligated to a BstXI- and NotI-digested PCR product containing the alpha -globin sequence from BstXI to the stop codon inclusively, followed by a NotI site. The 5' primer sequence containing the BstXI site of alpha -globin was 5'-CTGACCAACGCCGTGGCG-3'. The 3' primer sequence containing the NotI site joined to alpha -globin sequences that begin at the stop codon and extend into the open reading frame (ORF) was 5'-GGGGCGGCCGCTTAACGGTATTTGGAG-3'. The resulting DNA was named SV-alpha GL-Delta 3'UTR-A (Fig. 4A). The 3'-UTR of this clone contains no alpha -globin sequences and starts at the first nucleotide of the NotI sequences and extends over the poly(A) signal present in the vector sequences. SValpha GL was also digested by BalI and NotI, blunted, and ligated in the presence (SV-alpha GL-Delta 3'UTR-B/3beta -477-S) or in the absence (SV-alpha GL-Delta 3'UTR-B) of the 3beta -HSD 477-bp fragment (Fig. 4A). The 3'-UTR of these two clones contains the first 16 bases of the alpha -globin 3'-UTR. PCRs were performed with the Pwo enzyme (Roche Molecular Biochemicals), which has proofreading activity.

Other DNA clones were prepared as follows. The first step in the construction of clone SV-alpha GL Delta in1 was performed by digestion of SV-alpha GL by ClaI and BstXI to remove all alpha -globin sequences upstream of the BstXI site and a part of the vector sequences. This fragment was ligated to a ClaI-BstXI-digested PCR fragment produced from SV-alpha GL. The 5' primer contained vector sequences encompassing the ClaI site (5'-ACAGCATCGATGCTGTGG-3'), whereas the 3' primer was composed of a BstXI site, followed by alpha -globin sequences encompassing the first codon (5'-CGCCCACGGCGTTGGTCAGCACCATGGTGGGTTC-3'). In the resulting clone, the third codon was joined to the 68th codon, and the sequence remained in frame (Fig. 9A). The clone SV-alpha GL Delta in2 was prepared by digestion of SV-alpha GL by BstXI and NotI to remove the alpha -globin sequence downstream of the BstXI site and part of the downstream vector sequences. The resulting fragment was ligated to a PCR product digested by BstXI and NotI. The 5' primer was composed of a BstXI site, followed by the alpha -globin sequence starting two nucleotides before the stop codon and extending into the 3'-UTR (5'-GGGCCAACGCCGTGGGTTAAGCTGGAGCCTCGG-3'). The 3' primer encompassed the NotI site of the vector (5'-GGGAGCGGCCGCCGACTAGTG-3'). In the resulting clone, the 71st codon was followed by a new glycine residue and the stop codon (Fig. 9A). The clone SV-alpha GL Delta SmaI-NarI was produced by circularization of a DNA fragment prepared by a total NarI digestion, followed by a partial SmaI digestion of SV-alpha GL to remove the 340-bp fragment containing exon 2 and part of introns 1 and 2. The 3beta -HSD-1-PvuII-EcoRI fragment of 477 bp was inserted into the BalI site of these clones to produce clones SV-alpha GL Delta in1/477, SV-alpha GL Delta in2/477, and SV-alpha GL Delta SmaI-NarI/477.

SV-alpha GL-cDNA was constructed as follows. First, human blood was collected and centrifuged to remove serum and the white interface. The erythrocyte-enriched pellet was subjected to RNA extraction as described below, and first strand cDNA synthesis was performed using a Ready-To-Go T-Primed First-Strand Kit (Amersham Pharmacia Biotech). Then, an aliquot of this reaction was subjected to PCR using a 5' primer composed of a PstI restriction site, followed by sequences present in SV-alpha GL, starting immediately downstream the PstI site of the Multiple Cloning Site (MCS) and ending at the 18th base of the alpha -globin gene (5'-GGGCTGCAGGTACCGGTCCGGAATTACTCAGAGAGAACCCACC-3') and a 3' primer composed of alpha -globin 3'-UTR sequences localized downstream the BalI site (5'-TTCAAAGACCACGGGGGTAC-3'). The resulting PCR fragment was then digested with PstI and BalI. Second, SV-alpha GL was digested by BalI and then partially digested by PstI. The resulting molecules were ligated to the PCR product. Molecular screening permitted isolation of the SV-alpha GL-cDNA clone that contains exactly the same sequences as SV-alpha GL but without introns. The 3beta -HSD-1 fragment of 477 nt was then inserted into the BalI restriction site to obtain SV- alpha GL-cDNA/477.

DNA extractions were performed as described by Sambrook et al. (34) with two rounds of purification on cesium chloride (CsCl) gradients. Three different DNA preparations of each of the SV-alpha GL, SV-alpha GL/3beta /370-S, and SV-alpha GL/3beta /477-S plasmids were used for transfections into JEG-3 cells without variation in their relative levels of expression.

Cell Culture and Transfections-- COS-7 cells and the choriocarcinoma cell line JEG-3 (ATCC HTB-36) were grown in Dulbecco's modified Eagle's medium containing 25 mM glucose, 25 mM HEPES, 50 units/ml penicillin, 50 µg/ml streptomycin, and 5% (v/v) (JEG-3) or 10% (v/v) (COS-7) heat-inactivated fetal bovine serum (HyClone, Logan, UT). Cells were cultured as described previously (35-37). Cells were plated 18-24 h before transfection at a concentration of 2 × 106 cells per dish (10 cm) and transfected by the calcium phosphate procedure (38) using 20 µg per plasmid per dish.

RNA Extraction and Hybridization-- Cellular RNA was extracted with Tri Reagent (39, 40) from pools of four dishes. RNAs were purified on CsCl gradients (41). The RNA samples were glyoxalized, electrophoresed, and gels transferred to Nytran Plus membranes (Schleicher & Schuell). Filters were hybridized at 42 °C and washed under high stringency conditions (33). Autoradiographs were quantified by densitometric scanning (Amersham Pharmacia Biotech RAS image analyzer system). The alpha -globin DNA probe was prepared using the 1119-bp HinfI-HinfI fragment of the alpha -globin gene. Clones with deletions in the ORF and those with a DNA insertion into the BalI site were also hybridized with a BalI-PstI 183-bp probe encompassing the 3'-UTR but lacking the open reading frame (ORF). Their levels of expression relative to SV-alpha GL were the same as for the 1119-bp probe (data not shown). The gamma -actin cDNA probe was composed of a 2-kilobase pair cDNA fragment (42). All of the probes were labeled by random priming (43).

Nuclear Runoff Assay-- After transfection with the selected plasmids, cells were harvested in cold phosphate-buffered saline and lysed in cold Nonidet P-40 lysis buffer (10 mM Tris (pH 7.5), 10 mM NaCl, 3 mM CaCl2, 0,5% Nonidet P-40) for 5 min on ice. Nuclei were collected by centrifugation and used for nuclear RNA extraction (see below) or resuspended in storage buffer (50 mM Tris (pH 8.3), 40% glycerol, 5 mM MgCl2, 0.1 mM EDTA) and frozen in liquid nitrogen until nuclear runoff assays. Reactions were performed by adding 200 µl of nuclear suspension (1 × 107 nuclei) to 200 µl of reaction buffer (10 mM Tris (pH 8.0); 5 mM MgCl2; 0.3 M KCl; 10 mM dithiothreitol; 1 mM each of ATP, CTP, and GTP) and 100 µCi of [alpha -32P]UTP (3000 Ci/mmol) (NEN Life Science Products) for 40 min at 30 °C. Then, digestion with deoxyribonuclease (DNase) I (620 units per reaction) and proteinase K (0.2 mg) were performed for 20 min at 37 °C and 30 min at 42 °C, respectively. Newly synthesized RNAs were recovered by trichloroacetic acid precipitation and filtration on glass microfiber FG/A filters (VWR, Mississauga, Ontario, Canada). RNA probes were hybridized to SV-alpha GL-Delta 3'UTR-B and gamma -actin plasmid DNA, previously linearized, denatured, and immobilized on Hybond-XL membranes (Amersham Pharmacia Biotech) using a dot blot manifold (Life Technologies, Inc.) (5 µg of DNA per dot). Hybridizations were performed in 50 mM PIPES (pH 7.0), 0.5 M NaCl, 2 mM EDTA, 0.4% SDS, 33% formamide, 200 µg/ml sheared and denatured salmon sperm DNA, and 1× Denhardt's (100 × Denhardt's: 2% bovine serum albumin, 2% Ficoll, 2% polyvinylpyrrolidone) for 72 h at 42 °C. After hybridization, membranes were washed 4 times for 30 min at 65 °C in 2× SSC, 0.1% SDS (20× SSC: 3 M NaCl, 0.3 M sodium citrate), treated with ribonuclease A (10 ng/ml in 2× SSC) for 30 min at 37 °C, with proteinase K for 30 min at 37 °C, 2 times for 30 min at 65 °C in 2× SSC, 0.1% SDS, and exposed to Kodak XAR film. Quantification was performed using a PhosphorImager.

Pre-mRNA Analysis by Reverse Transcription-PCR (RT-PCR)-- After transfection of JEG-3 cells with plasmids SV-alpha GL-Delta 3'UTR-B and/or SV-alpha GL-Delta 3'UTR-B/3beta -477-S, nuclear RNA was extracted with Tri Reagent from isolated nuclei prepared as described above, and purified on CsCl gradients (41). After DNase I treatment and purification, each 5-µg RNA sample was subjected to RT using the First-Strand cDNA Synthesis Kit (Amersham Pharmacia Biotech) and a primer starting at the 174th nt and ending at the 157th nt downstream from the SV40 polyadenylation signal used by these recombinant alpha -globin genes (5'-CAGTGAGCGAGGAAGCGG-3'). Only 2% of the RT reactions were used in PCR reactions in the presence of the same 3' primer and the following 5' primer corresponding to sequences within the intron 2 of the alpha -globin gene, 5'-GGAGCGATCTGGGTCGAG-3'. PCRs were performed with the Expand High Fidelity PCR System (Roche Molecular Biochemicals) as follow: 5 min at 94 °C; 10 min at 72 °C; 28 cycles (1 min at 94 °C; 1 min 15 s at 55 °C; 1 min 45 s at 72 °C); 10 min at 72 °C.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

An alpha -globin DNA, beginning in the 5'-UTR sequences and extending over the polyadenylation site, was placed under the control of the SV40 early promoter (Fig. 1A). Except when indicated, all plasmid constructs were derived from this parental clone, named SV-alpha GL.


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  		Fig. 1.   Negative effect of insertion of DNA into the 3'-UTR on levels of alpha -globin mRNA. A, SV-alpha GL contains the 1119-bp HinfI-HinfI segment of human alpha 1-globin gene including the poly(A) signal. Exons correspond to boxes; introns and 3'-flanking sequences are shown by thin lines. Wavy lines and boxes represent vector sequences, and thick lines refer to 3beta -HSD-1 cDNA fragments. Black boxes correspond to the ORF of alpha 1-globin, and 5'- and 3'-UTR are represented by open boxes. All 3beta -HSD-1 DNA sequences originate from 3'-UTR, except the small PvuII to TGA segment that is from the ORF. B, plasmids SV-alpha GL/3beta -370-S (lane 1) and SV-alpha GL/3beta -477-S (lane 2) were co-transfected with SV-alpha GL in JEG-3 cells. Cellular RNA was extracted 48 h later and Northern blot probed with alpha -globin DNA. Positions corresponding to each mRNA and their relative levels are shown. C, plasmids SV-alpha GL/3beta -370-S and -A (lanes 1 and 2, respectively), and clones SV-alpha GL/3beta -477-S and -A (lanes 3 and 4, respectively) were co-transfected with SV-alpha GL in JEG-3 cells. This experiment was performed as described in B. Arrows correspond to the orientation of DNA inserts into alpha -globin sequences. D, plasmids SV-alpha GL/3beta -370-S (lane 1) and SV-alpha GL/17beta (lane 2) were co-transfected with SV-alpha GL in JEG-3 cells. This experiment was performed as described in B.

To study specifically the effect of the length of the 3'-UTR on alpha -globin mRNA levels, transfections were performed in non-erythroid JEG-3 cells to eliminate possible effects of putative tissue-specific elements. The endogenous alpha -globin gene contains a CpG island extending both upstream and downstream from the transcription start site (44). As it has been suggested that CpG island DNA may increase transcription specifically upon integration in the genome, we performed transient transfections.

Two overlapping fragments originating from the type 1 3beta -HSD cDNA clone were inserted in the unique BalI restriction site of the alpha -globin gene localized 14 bases downstream of the stop codon (Fig. 1A). The resulting clones, containing an insertion of DNA of 477 bp (SV-alpha GL/3beta -477-S) or of 370 bp (SV-alpha GL/3beta -370-S) in sense orientation, were each co-transfected with SV-alpha GL into JEG-3 cells. Their relative transient expression levels were analyzed by Northern blot. Although the insertion of the 370-bp 3beta -HSD-1 fragment reduced the level of mRNA to 15% that of parental alpha -globin mRNA, the effect of the 477-bp 3beta -HSD-1 fragment was more pronounced, leading to an mRNA level 1% that of alpha -globin mRNA (Fig. 1B). Similar results were observed in COS-7 cells (data not shown) indicating that the effect of DNA insertion is not specific to choriocarcinoma cells.

Effect of the Sequence and the Length of Exogenous DNA Inserts on alpha -Globin mRNA Levels-- To define the properties of putative inhibitory inserts, we first studied the same DNA fragments inserted in both orientations. Both orientations of the 477- and the 370-bp 3beta -HSD-1 segments decreased mRNA levels to the same extent (Fig. 1C), showing that the effect of DNA insertion is orientation-independent. To determine whether the sequence of the DNA insert is important, a 295-bp cDNA segment originating from the ORF of the 17beta -HSD-1 cDNA was cloned into the same BalI restriction site of SV-alpha GL. The sequence identity between the 3beta -HSD-1 3'-UTR and 17beta -HSD-1 cDNA fragments was less than 5%. Co-transfections with SV-alpha GL revealed that the 17beta -HSD-1 fragment reduced mRNA levels to approximately that of the 370-bp 3beta -HSD-1 insert (Fig. 1D). Thus, the inhibitory effects of DNA insertion are independent of the sequence of the DNA insert.

To determine whether the length of the DNA insert correlates with mRNA levels, we introduced two DNA fragments of 286 and 86 bp in both orientations into the BalI site of the alpha -globin gene. The four clones were each co-transfected with SV-alpha GL in JEG-3 cells. Clones containing the 477- or the 370-bp 3beta -HSD-1 fragment in both orientations were included as controls. An example of a Northern blot with clones containing an insertion in the sense orientation is shown in Fig. 2A. The 86-bp 3beta -HSD-1 fragment had no effect on mRNA levels in both sense and antisense orientations (Fig. 2B). This observation demonstrates that DNA insertion does not destroy any putative alpha -globin regulatory element encompassing the BalI site. Thus, the insertion of DNA per se does not explain the above observations. Moreover, a negative correlation was found between the relative expression of plasmids and the length of the DNA inserts (Fig. 2C). Therefore, the effects of DNA inserts on mRNA levels depend of the length of these inserted fragments.


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  		Fig. 2.   Effect of the length of DNA inserts on levels of alpha -globin mRNA. Plasmids SV-alpha GL/3beta -477-S, -A, SV-alpha GL/3beta -370-S, -A, SV-alpha GL/3beta -286-S, -A, and SV-alpha GL/3beta -86-S, -A, were each co-transfected with SV-alpha GL in JEG-3 cells. Samples were processed as in Fig. 1. A, lanes 1-4 correspond to co-transfection of SV-alpha GL and the clones with the sense orientation of the 477-, 370-, 86-, and 286-bp inserts, respectively. B, relative mRNA levels of alpha -globin clones bearing DNA fragments of various lengths and orientations. The value of SV-alpha GL mRNA was arbitrarily fixed at 100%. Arrows indicate the orientation of inserts within the alpha -globin sequence. C, relative expression versus length of DNA inserts in nucleotides for clones bearing a 3beta -HSD-1 DNA fragment into the restriction site BalI of the alpha -globin gene.

The Effect of Exogenous DNA Insert Is Function of Its Localization in the alpha -Globin Sequences-- We also examined whether exogenous DNA alters mRNA levels when introduced into the last intron. The SV-alpha GL/3beta /INTRON clone was constructed in which the 477-bp 3beta -HSD-1 fragment was inserted into the first half of the second intron of the alpha -globin gene (Fig. 3A). Messenger RNA from SV-alpha GL/3beta /INTRON and SV-alpha GL had similar lengths, indicating that RNA splicing occurred correctly, albeit the presence of an insert into intron 2 (Fig. 3B). No difference in mRNA levels was observed between the clones after readjustment with gamma -actin mRNA for the amount of RNA loaded per lane. These results demonstrate that the strong effect observed on alpha -globin mRNA levels by insertion of DNA fragments depends on its localization in the alpha -globin sequence. Moreover, because the presence of the 477-nt insert in the transfected plasmid does not necessarily correlate with low steady state mRNA levels, the decrease observed with SV-alpha GL/3beta -477-S and -A compared with the parental plasmid is not an artifact caused by unequal transport of individual plasmid during transfection.


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  		Fig. 3.   The effect of insert DNA depends on its localization in the alpha -globin sequences. A, the structure of SV-alpha GL DNA is shown on top. Symbols were described in Fig. 1. Clone SV-alpha GL/3beta /INTRON contains the 477-bp PvuII-EcoRI fragment from 3beta -HSD-1 cDNA inserted at the indicated position in the antisense orientation (arrow). B, plasmids SV-alpha GL and SV-alpha GL/3beta /INTRON were transfected separately in duplicate in JEG-3 cells. Samples were processed as described in Fig. 1 using the alpha -globin probe and the labeled gamma -actin fragment.

alpha -Globin-specific Regulatory Element Downstream from the Site of DNA Insertion-- The fact that the length of DNA inserts determine the extent of the effect suggests that an interaction between two hypothetical cis-acting elements localized on each side of the BalI site is disrupted by DNA insertion. The next experiment was designed to determine whether an alpha -globin-specific regulatory element may be found downstream from the site of insertion. Sequences downstream from the stop codon of SV-alpha GL (Fig. 4A, SV-alpha GL-Delta 3'UTR-A) were deleted by joining the stop codon directly to the vector sequence, which contains its own polyadenylation signal originating from SV40. The replacement of the alpha -globin 3'-UTR by the 3'-UTR of the vector in both JEG-3 and COS-7 cells had no or only a weak effect on mRNA accumulation (Fig. 4B). Thus, in JEG-3 and COS-7 cells, no alpha -globin-specific regulatory element can be mapped within the 3'-UTR by substitution.


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  		Fig. 4.   The effect of DNA insertion cannot be explained by the presence of an alpha -globin-specific regulatory element in the 3'-UTR. A, the structure of SV-alpha GL DNA is shown on top. Symbols were described in Fig. 1. The structures of the inferred SV-alpha GL and SV-alpha GL-Delta 3'UTR-A and -B mRNAs are shown by thinner boxes. For mRNA from SV-alpha GL-Delta 3'UTR-B/3beta -477, only the position of the 477-nt insert relative to the SV-alpha GL-Delta 3'UTR-B mRNA is indicated. The junctions between exons on mRNAs are indicated by vertical lines. B, plasmids SV-alpha GL (lanes 1 and 3) or SV-alpha GL-Delta 3'UTR-A (lanes 2 and 4) were transfected separately in both JEG-3 and COS-7 cells. Samples were processed as in Fig. 1 and probed with alpha -globin and gamma -actin DNAs. C, plasmids SV-alpha GL/3beta -477-S and SV-alpha GL (lane 1), SV-alpha GL-Delta 3'UTR-B (lanes 3 and 4), and SV-alpha GL-Delta 3'UTR-B/3beta -477-S (lanes 3, 5 and 6) were transfected separately or in combination in JEG-3 cells. Lane 2, mock control. Samples were processed as described in Fig. 1 using the alpha -globin probe. Lanes 1-5 were from a 4-h exposure, and lane 6 corresponds to 17 h of exposure.

Does insertion of DNA also decrease the abundance of mRNA when all sequences positioned downstream from the site of insertion in the 3'-UTR are replaced by heterologous 3'-UTR sequences? To answer this question, plasmids SV-alpha GL-Delta 3'UTR-B and SV-alpha GL-Delta 3'UTR-B/3beta -477-S were constructed (Fig. 4A). Results indicate that DNA insertion into the BalI site had similar effects on mRNA levels in both the alpha -globin and the vector 3'-UTR contexts (Fig. 4C). The only common features downstream from the BalI site between the two 3'-UTR might be the cis-acting elements involved in 3'-end formation. For this reason, if any cis-acting regulatory element positioned downstream from the site of insertion is disrupted by DNA insertion, this element might be included within sequences involved in 3'-end formation.

DNA Insertion and Promoter Activity-- We verified whether insertion of DNA in the 3'-UTR also reduces expression of the alpha -globin gene under the control of its promoter. The 3beta -HSD-1 477-bp fragment was inserted in both orientations at the BalI site of the plasmid pUCalpha , which contains the alpha -globin promoter and gene (30). Co-transfections in JEG-3 cells revealed that insertion of DNA in both orientations strongly reduced the expression of the alpha -globin gene under the control of its promoter (Fig. 5). We conclude that insertion of DNA had similar effects both in homologous and heterologous promoter contexts.


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  		Fig. 5.   The effect of DNA insertion is not promoter-specific. Plasmids pUCalpha /3beta -477-S (lane 1) and -A (lane 2) were each co-transfected with pUCalpha in JEG-3 cells. As control, plasmids SV-alpha GL/3beta -477-S and SV-alpha GL were also co-transfected (lanes 3 and 4). Experiments were performed as described in Fig. 1B. Lanes 1-3 were from an 18-h exposure, and lane 4 corresponds to 3 h of exposure.

Nuclear runoff experiments were performed using clones SV-alpha GL-Delta 3'UTR-B and SV-alpha GL-Delta 3'UTR-B/3beta -477-S. Although these two clones showed strong differences in mRNA accumulation (Fig. 4C), they produced signals of similar intensity in runoff assay (Fig. 6). This result indicates that insertion of DNA does not alter the rate of transcription.


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  		Fig. 6.   The presence of the 477-bp insert does not change the rate of transcription. Nuclear runoff assay was performed using nuclei isolated from JEG-3 cells transfected with SV-alpha GL-Delta 3'UTR-B (dots 1 and 2 in A and B) or SV-alpha GL-Delta 3'UTR-B/3beta -477-S (dots 3 and 4 in A and B). Dots 1-4 in A and dots 1 and 3 in B are SV-alpha GL-Delta 3'UTR-B DNA, and dots 2 and 4 in B are gamma -actin DNA. A and B are from two different series of experiments. A was of 1-h exposure at room temperature, and B was of 1-h exposure at -80 °C with a Lightning Plus intensifying screen. Substitution of SV-alpha GL-Delta 3'UTR-B DNA for SV-alpha GL-Delta 3'UTR-B/3beta -477-S DNA for dot blot also gave similar results (data not shown). C, transcription rate of plasmid SV-alpha GL-Delta 3'UTR-B (no DNA insert) and SV-alpha GL-Delta 3'UTR-B/3beta -477-S (+477) are presented after normalization to gamma -actin using values obtained by PhosphorImager scanning of membranes autoradiographed in B.

DNA Inserts Do Not Affect alpha -Globin Mature mRNA Half-life-- Is mature mRNA decay affected by insertion of DNA into the BalI site? We used two different approaches to examine this possibility. The first is based on the fact that two mRNAs with different half-lives, but transcribed from the same promoter, give a ratio close to 1.0 soon after transfection, but this ratio changes over time until the mRNAs eventually reach steady state levels. Each of the two clones, SV-alpha GL/3beta -477-S and -370-S, were co-transfected with SV-alpha GL, and their mRNA levels were analyzed at different times. We observed no variation in SV-alpha GL/3beta -370-S/SV-alpha GL or in SV-alpha GL/3beta -477-S/SV-alpha GL mRNA ratios over time (Fig. 7A). In fact, the ratios were independent of the establishment of mRNA steady state levels. Second, we did a time course experiment in the presence of actinomycin D (ActD) after co-transfection of SV-alpha GL/3beta -370-S and SV-alpha GL. SV-alpha GL/3beta -370 and SV-alpha GL mRNAs had very close t1/2 values, as evidenced by the stable mRNA ratio over time (Fig. 7B). These two observations indicate that the addition of an insert DNA into the BalI site of the alpha -globin gene did not alter mRNA decay.


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  		Fig. 7.   DNA insertion does not alter cytoplasmic mRNA half-life. A, plasmids SV-alpha GL/3beta -370-S (left) and SV-alpha GL/3beta -477-S (right) were co-transfected with SV-alpha GL in JEG-3 cells. t0 was fixed at 20 h after the addition of the calcium-phosphate precipitates. Northern blots were probed with alpha -globin DNA. B, plasmids SV-alpha GL/3beta -370-S and SV-alpha GL were co-transfected in JEG-3 cells. Cells were trypsinized, pooled, and re-plated after transfection to eliminate inter-variations. The next day, ActD (5 µg/ml) was added for 45 min, and RNA was extracted at different times. The t0 corresponds to the end of the ActD treatment. Samples were processed as described in A.

Levels of Nuclear Pre-mRNA-- We next planned an experiment to show whether the effect of DNA insertion occurs before RNA processing. RT-PCR was performed on nuclear RNA isolated from JEG-3 cells transfected with SV-alpha GL-Delta 3'UTR-B and/or SV-alpha GL-Delta 3'UTR-B/3beta -477-S. To amplify specifically pre-mRNA, the 5' primer was located in the last intron, and the 3' primer was positioned 157 bases downstream from the polyadenylation signal (Fig. 8A). The presence of the 477-nt insert into the BalI site of the 3'-UTR strongly reduced levels of nuclear pre-mRNA (Fig. 8B). It should be noted that when plasmid DNA was added in a negative RNA sample before the DNase I reaction that preceded the RT-PCR, no amplification product was observed (data not shown). Therefore, the addition of an insert DNA into the BalI site resulted in a proportional decrease of pre-mRNA and mRNA levels, indicating that the effect of DNA insertion occurs before RNA processing.


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  		Fig. 8.   DNA insertion and pre-mRNA levels. JEG-3 cells were transfected with plasmids SV- alpha GL-Delta 3'UTR-B and/or SV-alpha GL-Delta 3'UTR-B/3beta -477-S, and nuclear RNA was extracted. A is a schematic representation of SV-alpha GL-Delta 3'UTR-B pre-mRNA, and the position of the 477-nt exogenous sequences (thick line) present in SV-alpha GL-Delta 3'UTR-B/3beta -477-S pre-mRNA is indicated. Black boxes correspond to ORF sequences; open box corresponds to 3'-UTR, thin line to intron 2, and wavy line corresponds to RNA sequences present in pre-mRNA but removed by endonucleolytic cleavage during the polyadenylation process. Arrows show position of the oligonucleotides used in RT-PCR. RT reaction was performed using 5 µg of nuclear RNA and the specific 3' primer, and semi-quantitative PCR conditions were obtained using only 2% of RT reactions. B, analysis of RT-PCR end products by Southern blot using the 1119-bp HinfI-HinfI alpha -globin probe. Because sequences localized downstream from the BalI site of these clones are not alpha -globin sequences, the alpha -globin probe hybridized specifically to a segment of the same length for the two RT-PCR products, which extend from the 5' primer to the BalI site. Fragments amplified from plasmids SV-alpha GL-Delta 3'UTR-B and SV-alpha GL-Delta 3'UTR-B/3beta -477-S were of 574 and 1051 bp, respectively. JEG-3 cells were transfected with SV-alpha GL-Delta 3'UTR-B (lanes 1-3) and/or SV-alpha GL-Delta 3'UTR-B/3beta -477-S (lanes 1, 4 and 5). The relative intensity of specific signals are the same than those observed with ethidium bromide staining. Relative levels of SV-alpha GL-Delta 3'UTR-B and SV-alpha GL-Delta 3'UTR-B/3beta -477-S mRNAs were similar to these observed in Fig. 4 as determined by Northern blot analysis of the corresponding cytoplasmic RNA (data not shown).

alpha -Globin Regulatory Elements in the ORF and the Introns-- We next examined the possibility that the effect of DNA insertion may be related to an alpha -globin-specific regulatory element localized in the ORF or in an intron. Messenger RNA levels were only slightly lowered by deletion of DNA regions encompassing the intron 1 (SV-alpha GL Delta in1) or intron 2 (SV-alpha GL Delta in2) of the alpha -globin gene (Fig. 9, A and B). When the 477-nt fragment was inserted in the BalI site of these two clones, a strong decrease in mRNA accumulation was observed (Fig. 9C). The segment SmaI-NarI was then removed from SV-alpha GL. This deletion overlaps the deletion of clones SV-alpha GL Delta in1 and SV-alpha GL Delta in2. Again, the addition of the 477-nt insert into the BalI site strongly reduced levels of mRNA accumulation (Fig. 9C). Therefore, we failed to associate the effect of DNA insertion to the presence of any single alpha -globin specific sequence between the 10th nt of the ORF to the stop codon.


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  		Fig. 9.   Effect of deletion of alpha -globin sequences on the effect of DNA insertion. A, the structure of SV-alpha GL clone is shown on top. Dotted lines correspond to deleted fragments. Other symbols were described in the legend of Fig. 1. The clone SV-alpha GL Delta SmaI-NarI is not illustrated, but positions of the restriction sites used for this construct are indicated. B, plasmids SV-alpha GL Delta in1 (lane 1) and SV-alpha GL Delta in2 (lane 2) were separately co-transfected with SV-alpha GL in JEG-3 cells. C, plasmids SV-alpha GL and SV-alpha GL/3beta -477-S (lane 1), SV-alpha GL Delta in1 and SV-alpha GL Delta in1/477 (lane 2), SV-alpha GL Delta SmaI-NarI and SV-alpha GL Delta SmaI-NarI/477 (lane 3), and SV-alpha GL Delta in2 and SV-alpha GL Delta in2/477 (lane 4) were co-transfected in JEG-3 cells. D, JEG-3 cells were transfected or co-transfected with the following plasmids: lane 1, SV-alpha GL; lanes 2 and 3, SV-alpha GL/3beta -477-S; lanes 4 and 5, SV-alpha GL-cDNA; lanes 6-8, SV-alpha GL-cDNA/477; lane 9, SV-alpha GL-cDNA and SV-alpha GL-cDNA/477; lane 10, mock. All lanes were from a 20-h exposure except lane 8 that was from a 50-h exposure. Samples of B-D were processed as in Fig. 1 except that the BalI-PstI alpha -globin probe of 183 bp containing no intron sequences was used in D instead of the alpha -globin 1119-bp HinfI-HinfI-labeled fragment.

Because common features exist between introns as donor and acceptor sites, it is possible that the effect of the DNA insertion into the BalI site persists as long as one intron is present in the gene. To study this possibility, both introns were removed (clone SV-alpha GL-cDNA, Fig. 9A). The resulting clone had mRNA levels similar to that observed after the insertion of the 477-nt fragment into the BalI restriction site of SV-alpha GL (Fig. 9D). However, the addition of the 477-nt insert into the BalI site of SV-alpha GL-cDNA also strongly reduced levels of mRNA accumulation (Fig. 9D). Therefore, the effect of DNA insertion is independent of the presence of intron sequences in the gene.

    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

We have clearly demonstrated that increasing the length of the 3'-UTR by insertion of DNA segments had a strong negative effect on alpha -globin mRNA level. In our model, neither the nucleotide sequence of the insert nor that of the 3'-UTR itself, excluding features common to all 3'-UTR, contribute to the observed effect. The only characteristic of the inserts that was related to the level of mRNA was their length.

We first postulated that increasing the length of the alpha -globin 3'-UTR might disrupt an interaction between the acceptor site of the last intron and the polyadenylation site, thereby reduce polyadenylation efficiency and levels of mRNA. It is now clear that the strong decrease in mRNA levels observed by increasing the length of the 3'-UTR was not due to the disruption of such a mechanism because the effect of the length of the 3'-UTR was observed in the absence of intronic sequences as well (clone SV-alpha GL-cDNA). In fact, insertion of DNA into the BalI site did not change the rate of transcription but decreased levels of pre-mRNA and mature mRNA proportionally. Therefore, our results strongly suggest that the alpha -globin pre-mRNA was actively protected from degradation in the nucleus and that the insertion of DNA into the BalI site, but not within intron 2, had disrupted this mechanism of stabilization. The decrease in mRNA amount was not due to insertion of putative ribonuclease sites present on the exogenous DNA insert, as demonstrated by the insertion of the same DNA segment within intron 2 with no effect on alpha -globin mRNA level. Interestingly, stabilization of alpha -globin pre-mRNA is achieved through a mechanism that seems to be inactive in mature polyadenylated mRNA, as witnessed by the absence of effect of DNA insertion on mature mRNA half-lives.

The negative correlation between RNA abundance and the length of DNA inserts strongly suggest the disruption of an interaction between two elements positioned on each side of the BalI restriction site used for DNA insertion. Substitution of 3'-UTR sequences downstream from the site of insertion did not change the effect of DNA insertion. Therefore, if a downstream cis-acting element is actually related to the effect of DNA insertion, it might be positioned within sequences related to polyadenylation. The upstream cis-acting element(s) that is aimed at interacting with this 3' element is unknown, but our results allow the conclusion that it is not localized within introns and that at least one copy of it might be localized between the beginning of exon 3 and the BalI site. This arises from the observation that no effect on mRNA abundance was observed when the 477-nt segment was introduced within the intron 2. However, the effect of DNA insertion in the BalI site was conserved even though all sequences localized between the fourth codon and the stop codon were sequentially deleted. Therefore, our results appear to be in agreement with reiterated 5'-cis-acting elements.

Increasing the length of the alpha -globin 3'-UTR resulted in destabilization of alpha -globin pre-mRNA but not mature mRNA, and this was true in both homologous and heterologous 3'-UTR contexts. Considering also the fact that alpha -globin is not normally expressed in JEG-3 and COS-7 cells, it is tempting to suggest that pre-mRNA stabilization could be a general mechanism of eucaryotic pre-mRNAs. Further investigation will help to elucidate this point, but some cases of modulation of gene expression, probably through the control of pre-mRNA decay, have yet been reported. For example, GM-CSF pre-mRNA accumulation was observed following treatment of murine B-cells with interleukin 1alpha , whereas decreased GM-CSF expression observed after treatment with interleukin 4 should be due to intranuclear destabilization of GM-CSF pre-mRNA (45). In another case, it was reported that decreased peptidylglycine alpha -amidating monooxygenase expression after 17beta -estradiol treatment might be largely due to intranuclear destabilization of the primary transcript (46). Therefore, pre-mRNA decay seems to be controlled at least for these genes.

We have demonstrated that alpha -globin pre-mRNA is stabilized and that this stabilization is responsible for an increase in alpha -globin mRNA of 100-fold or more. In addition, our results strongly suggest that this phenomenon is regulated by a mechanism that necessitates cooperation between two cis-acting elements localized each side of the BalI site. It is of considerable interest that this mechanism of RNA stabilization is specific to pre-mRNA, which would appear to represent a putative level of gene regulation.

    FOOTNOTES

* This work was supported by Medical Research Council of Canada Grant MT 14365 (to Y. T.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ Recipient of Senior Scholarship 990108-103 from the Fonds de la Recherche en Santé du Québec. To whom correspondence should be addressed: Laboratory of Ontogeny and Reproduction-CRBR, Room T-1-58, CHUL Research Center, 2705 Laurier Blvd., Québec G1V 4G2, Canada. Tel.: 418-656-4141 (ext. 6158); Fax: 418-654-2765; E-mail: yves.tremblay@crchul.ulaval.ca.

Published, JBC Papers in Press, June 23, 2000, DOI 10.1074/jbc.M002969200

    ABBREVIATIONS

The abbreviations used are: UTR, untranslated region; nt, nucleotide; PCR, polymerase chain reaction; RT-PCR, reverse transcription-PCR; bp, base pair; ORF, open reading frame; PIPES, 1,4-piperazinediethanesulfonic acid; ActD, actinomycin D; GM-CSF, granulocyte-macrophage colony-stimulating factor.

    REFERENCES
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ABSTRACT
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RESULTS
DISCUSSION
REFERENCES

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