ABSTRACT
The second intron ([beta]IVS-II) of the human [beta]-globin gene is essential for the accumulation of stable cytoplasmic mRNA and is implicated in promoting efficient 3'-end formation. This report presents quantitative comparisons between [beta]IVS-II mutants at physiological levels of expression from within a natural chromatin context in vivo which further defines it's function. In marked contrast to a [beta]-globin gene lacking a second intron, two mutants defective in splicing (small size or a splice donor mutation), still undergo essentially normal levels of 3'-end formation and in the absence of exon skipping. Therefore, 3' cleavage of [beta]-globin transcripts requires the presence of [beta]IVS-II sequences, but not the splicing reaction. The placement of [beta]IVS-II in the IVS-I position did not reduce the efficiency of 3' cleavage indicating that the distance between the necessary element(s) in this intron and the polyadenylation recognition site is not a crucial factor. Subsequent placement of [beta]IVS-I in the intron II position, reduced the efficiency of 3'-end formation to only 16% of normal. A direct replacement of intron II by the heterologous introns [beta]IVS-I or [alpha]-globin IVS-II, only partially substitute (16 and 30% respectively) for [beta]IVS-II. Hybrid introns show that efficient 3'-end formation is strongly enhanced by the presence of the terminal 60 nt of [beta]IVS-II. These data imply that the last intervening sequence of multiple intron containing genes is a principal determinant of the efficiency of 3'-end formation and may act as a post-transcriptional regulatory step in gene expression.
The protein coding regions of most eukaryotic genes are interrupted by intervening sequences (IVS) or introns. These intronic sequences are subsequently removed or spliced from the primary transcript as part of the maturation process to mRNA (1). Interestingly however, the removal of introns from genes and their subsequent expression in both tissue culture cells (2-7) and transgenic mice (8,9), results in very low levels of mRNA which is not due to transcriptional insufficiency resulting from the absence of an intronic enhancer element.
The [beta]-globin gene which has a three exon-two intron structure is no exception in this regard. The transient expression in HeLa cells of rabbit [beta]-globin has shown that the inclusion of either IVS-I or IVS-II can restore expression of an intronless gene (10). In contrast we have demonstrated that physiological levels of expression of the human [beta]-globin gene from a natural chromatin context afforded by stable transfection in murine erythroleukaemia (MEL) cells under the control of the locus control region ([beta]LCR), can only be achieved in the presence of the second intron ([beta]IVS-II; 11). A [beta]-globin gene possessing only IVS-I does not result in detectable levels of cytoplasmic mRNA, although both introns are required for maximum accumulation of the mature transcript. Furthermore, our results indicated that the presence of [beta]IVS-II appeared to be necessary for correct and efficient 3'-end formation (11). Subsequently, others have also demonstrated that certain introns for a given gene are more crucial for the accumulation of mRNA, for example, the human genes for growth hormone (9), purine nucleoside phosphorylase (12) and triosephosphate isomerase (TPI; 13). In the case of the TPI gene, the last intron of this seven exon gene, is implicated in 3'-end formation (13). Experiments in cell-free systems in vitro (14) and transient transfection assays (15) also imply that the 3' splice acceptor intronic region is required for efficient 3'-end cleavage and polyadenylation. These effects may be mediated by either a direct interaction of the known splicing and polyadenylation factors or by as yet uncharacterised element(s) within the last intron. Upstream efficiency elements (USEs) that promote polyadenylation have been described for both viral (see 16) and cellular (17) genes. The effect of mutation of the AAUAAA polyadenylation signal on removal of upstream introns, is however, less clear. Analysis of AAUAAA mutants in vitro using cell-free systems, has shown a marked reduction in the efficiency of splicing of a proximal but not distal intron upstream from this element (18), whereas transient transfection experiments with TPI gene mutants have shown no effect on the efficiency of removal of the penultimate and final introns (19).
We have mapped and characterised the [beta]IVS-II requirement for human [beta]-globin gene expression. We have continued to use the human [beta]-globin LCR to drive physiological levels of expression in MEL cells in vivo to provide quantitative comparisons between different intron mutants expressed from within a natural chromatin context. This also avoids complications that can arise from transient transfection and cell-free functional assay systems. The analysis of genes harbouring splice donor and [beta]IVS-I/[beta]IVS-II hybrid intron mutants, clearly shows that full, normal levels of 3'-end formation of [beta]-globin transcripts are produced by the presence of the 3' 60 nt of [beta]IVS-II and that this occurs completely independently of the splicing reaction and aberrant exon skipping. In addition, the position of [beta]IVS-II within the gene is not crucial to promote efficient 3' cleavage. The inability of heterologous introns to substitute [beta]IVS-II in this process, implies that the second intron of the [beta]-globin gene possesses unique structural features which allow it to strongly enhance the efficiency of 3'-end formation.
The maintenance, stable transfection by electroporation and induction to terminal erythroid differentiation (with 2% DMSO for 4 days) of the APRT negative MEL cell line C88, were all as previously described (20). All plasmids were linearised with PvuI prior to transfection.
The [beta]IVS-I containing and wild-type [beta]-globin gene constructs in the micro-locus LCR expression cassette were as described (11). The deletion mutant series of [beta]IVS-II was produced as subclones within pBluescript (Stratagene, La Jolla, CA, USA) between the exon II BamHI and exon III EcoRI sites which are located 18 and 50 bp from the start and end of this intron respectively (see Fig. 1A) using the restriction enzymes indicated. This generated the series [Delta]RS, [Delta]RH and [Delta]HH. The [Delta]89 and the donor mutant (GT -> AC) DM[Delta]89 introns (Fig. 2), were generated as synthetic oligonucleotides starting at the exon II BamHI site and extending to a SmaI linkered HinfI site 32 bp downstream of the 5' splice donor. These oligonucleotides were then ligated to the HinfI site 57 bp upstream of the 3' splice acceptor site. The exchange of [beta]IVS-II with [beta]IVS-I ([beta]I/I) or [alpha]-globin IVS-II ([beta]I/II[alpha]), as well as the [beta]IVS-I and [beta]IVS-II hybrid introns (5'I/3'II; 5'II/3'I) were generated by ligating together overlapping synthetic oligonucleotides of 30-40 bp in length and which spanned between the exon-II BamHI and exon-III EcoRI sites as before (Fig. 4A). The 5'I/3'II intron consisted of the 5'-half of [beta]IVS-I (75 bp) linked to the last 60 bp of [beta]IVS-II. The 5'II/3'I intron has the reverse configuration, namely the first 70 bp of [beta]IVS-II linked to the 3'-half (75 bp) of [beta]IVS-I. The placement of the [Delta]89 intron in the IVS-I position was again by ligating overlapping oligonucleotides between the exon I BspMI and the exon II AccI sites. The BspMI and AccI sites are respectively 12 bp 5' and 3' of the intron I borders. All these intron mutants were incorporated into a human [beta]-globin gene (also as a subclone in pBluescript) either with or without IVS-I. This [beta]-globin gene extended from the SnaBI site at -265 from the start of transcription to a SalI linkered MnlI site 45 bp past the poly(A)-addition site (4) in the case of the [beta]IVS-II deletion series (Figs 1A and 2), or to the BglII site 1815 bp past the poly(A)-addition site for all other constructs (Fig. 4A). The [Delta]RS deletion mutant was built into [beta]-globin genes extending to both the MnlI and BglII sites. The 3' truncation to the MnlI site did not affect the efficiency of polyadenylation (data not shown). Finally, genes were cloned between the ClaI and KpnI sites of the [beta]LCR expression vector (11). The [beta]LCR confers gene copy number dependent expression once these constructs are stably transfected in MEL cells.
The extraction of total RNA from MEL cells was by selective precipitation in the presence of 3 M LiCl and 6 M urea (20). Nuclear and cytoplasmic RNA fractions from uninduced and induced MEL cells were prepared as follows. The cells were washed twice in phosphate buffered saline and resuspended at 2-5 × 107 cells/ml in lysis buffer (10 mM Tris-HCl, pH 7.5, 10 mM NaCl, 3 mM MgCl2, 0.1% Triton X-100, 0.5 mM dithiothreitol (DTT), 0.1 M sucrose). The cells were gently lysed with a Dounce homogeniser (5-10 strokes of the pestle). The lysed cell mixture was then diluted with an equal volume of 0.25 M sucrose in lysis buffer and layered over half a volume of 0.33 M sucrose (in 10 mM Tris HCl, pH 8, 5 mM MgCl2 0.5 mM DTT) in a 12 ml centrifuge tube. This was then spun at 800 g for 5 min in a Beckman J6 centrifuge. All the above manipulations were carried out at 4°C. The cytoplasmic upper layer was then transferred to a 15 ml screw capped polypropylene tube and made 1% with SDS, 0.5 mg/ml proteinase K and incubated at 37°C for at least 1 h. The mixture was then extracted twice with an equal volume of buffer (10 mM Tris-HCl, 1 mM EDTA, pH 7.5) saturated phenol-chloroform (1:1 v/v). The final aqueous phase was made 0.3 M with sodium acetate and the RNA precipitated by addition of 2.5 vol of 100% ethanol and storage at -20°C. The RNA was collected by centrifugation (3000 r.p.m., 10 min, 4°C, Beckman J6 centrifuge), and washed once with 70% (v/v) ethanol. The remaining supernatant was carefully removed by suction and the pelleted nuclei resuspended in 5 ml of ice cold lysis buffer. The suspension was transferred to a 25 ml screw capped, polystyrene tube (Sterilin) and the nuclei re-pelleted by centrifugation at 800 g as described above. After removal of the supernatant, the nuclei were lysed in 3 ml of 3 M LiCl, 6 M urea and RNA recovered as described for the total RNA preparation. All final total, nuclear and cytoplasmic RNA pellets were dissolved in S1-nuclease hybridisation buffer at 1-5 mg/ml and stored at -20°C.
Human [beta]-globin and mouse [beta]maj-globin mRNA was quantified in the various RNA fractions by an S1-nuclease protection assay using double stranded, end-labelled DNA probes (21). The nature of the human [beta]-globin probes used are illustrated in Figures 1-4. The [beta]IVS-II deletion series of genes (Fig. 1) including the donor mutant of [Delta]89 (DM[Delta]89; Fig. 2) which terminate at the MnlI site 45 bp past the poly(A)-addition site, were analysed with probes that extend to a natural PstI site in the 3' flanking region of the human [beta]-globin gene (Figs 1D and 2B). This probe therefore mismatches with transcripts from these genes after the MnlI site that are not correctly 3' cleaved. This allows detection of 3' uncleaved RNA as 257 nt S1-nuclease protected products (Fig. 2A). In order to detect the 3' uncleaved as well as the correctly cleaved transcripts from the intron exchange and hybrid intron constructs (Fig. 4A), probes were prepared from a pBluescript sub-clone of an intronless human [beta]-globin gene which terminates at the MnlI site 45 bp past the poly(A)-addition site (Fig. 4D). As these constructs extend to the BglII site 1815 bp past the poly(A)-addition site, uncleaved transcripts from these genes will mismatch with this probe after the MnlI site and, as before, give an S1-nuclease protected product of 257 nt (Fig. 4C). The mouse [beta]maj-globin probe is a 700 bp HindIII-NcoI fragment from the 5'-half of this gene and includes IVS-I (22). This [beta]maj-globin probe therefore gives a 96 nt S1-nuclease protected product from processed mRNA and a 336 nt product from unprocessed transcripts (Figs 2A, 3A and 4C). The products from the S1-nuclease digestion were resolved on a 6% polyacrylamide gel in the presence of 8 M urea, dried, quantified by PhosphorImager (Molecular Dynamics) analysis and exposed to Kodak XAR 5 or Fuji X-ray film to obtain a hard copy of the results.
As a first step towards mapping the region(s) within [beta]IVS-II which are essential for [beta]-globin mRNA accumulation, a series of deletion mutants of this intron were generated by the use of convenient restriction enzyme sites and oligonucleotide cloning (Fig. 1A; see Materials and Methods). All mutants preserved the consensus splice donor, acceptor and lariat branch point elements at the 5'- and 3'-ends of the intron. These intron mutants were built into a [beta]-globin gene both with and without IVS-I. These constructs were then cloned into the micro-locus, [beta]LCR expression cassette (11) and used to generate three large, independent pools of stable transfected MEL cells (20). The transfected pools were induced to undergo erythroid differentiation in the presence of 2% DMSO for 4 days and analysed for the expression of the transgene by an S1-nuclease protection assay using end labelled DNA probes (21). Figure 1 shows the analysis of total RNA from the transfected MEL cell pools with the constructs possessing IVS-I. The results in the absence of IVS-I, were identical except that the overall level of expression was only 30% compared with that observed with the complete gene (data not shown; see 11). The samples were simultaneously probed for the expression of the human [beta]-globin transgene and the endogenous mouse [beta]maj-globin which acts as an internal standard, allowing for the correction of variability in the degree of erythroid differentiation between samples (compare, for example, Fig. 1B and C lanes 9-11 and 13-15). Absolute quantitation was obtained by phosphorimage analysis. After correcting for differences in transfected gene copy number and variations in the degree of erythroid differentiation (data not shown), the results using a human 3'[beta](RI) probe (Fig. 1D) which detects all correctly 3' cleaved transcripts as 212 nt S1-nuclease protected products, show that levels of mRNA accumulation comparable (50-60% per gene copy) to that observed with the wild-type gene (WT; Fig. 1B, lanes 2 and 3), are obtained with all deletion constructs down to the 89 bp intron mutant ([Delta]89; Fig. 1B, lanes 13-15). The 63 bp [Delta]HH intron mutant (Fig. 1B, lanes 17-19) gives only 5%, whereas correctly terminated transcripts from a gene completely lacking in any [beta]IVS-II sequences ([Delta]), are undetectable in this experiment (Fig. 1B, lanes 21-23) as seen before (11).
The RNA from these pools was also analysed using a 3'[beta](Bam) probe (Fig. 1D) labelled to the same specific activity as 3'[beta](RI). The 3'[beta](Bam) probe starts 18 bp from the end of exon II and therefore only detects those transcripts that have undergone correct splicing at the IVS-II position. The results (Fig. 1C) show that all mutant introns with the exception of [Delta]HH are as efficiently spliced as the wild-type (WT) RNA. Presumably the small size of the [Delta]HH intron causes steric hindrances, which prevents the correct interaction between the various components of the splicing machinery which bind to the 5' splice donor (23) and 3' splice lariat branch point and acceptor sites (24,25).
In addition, transfections in murine fibroblast L-cells showed the same intron requirement for the accumulation of [beta]-globin mRNA as described above for MEL cells (data not shown). This demonstrates that the role of IVS-II is not due to the erythroid enhancer (26-28) or other tissue specific element present in this intron. Furthermore, we have shown that the placement of [beta]IVS-II downstream of the poly(A)-addition site or the substitution of the [beta]-globin 3'UTR and poly(A)-sequences with the terminator region of the human [beta]-interferon or murine histone H4 gene which lacks both introns and polyadenylation, did not rescue expression of an intronless [beta]-globin gene (data not shown). These data collectively confirm the unique and fundamental role for IVS-II in accumulation of [beta]-globin mRNA at an event downstream from initiation of transcription which is not due to tissue-specific regulatory elements.
Although the [Delta]HH intron mutant was not spliced efficiently (compare Fig. 1B and C, lanes 17-19), it nevertheless gave significant amounts of correctly 3' cleaved transcripts especially when compared to the situation when no [beta]IVS-II sequences were present (Fig. 1B and C; lanes 21-23). These data suggest that efficient 3'-end formation is dependent upon the presence of [beta]IVS-II but that the cleavage reaction takes place independently from those for splicing this intron. This supposition is further supported by the observation that the same results are obtained with a [Delta]HH intron containing gene lacking IVS-I and whose transcripts therefore do not undergo any splicing (data not shown).
In order to test this hypothesis further, a splice donor mutation (GT -> AC) which combines naturally occurring defects that give rise to [beta]0-thalassaemia (29), was introduced into the otherwise normal splicing [Delta]89 intron (DM[Delta]89; Fig. 2, right) and incorporated into the LCR/[beta]-globin gene expression system as before in the presence and absence of [beta]IVS-I. The results obtained with the stable transfected MEL cell pools is shown in Figure 2. In the interest of brevity only the data from the construct containing [beta]IVS-I are shown. As observed previously, the same relative pattern of results were obtained in the absence of [beta]IVS-I but with lower overall expression levels (data not shown). Cytoplasmic and nuclear as well as total RNA from cells, both before and after induced erythroid differentiation was analysed by the same S1-nuclease protection assay using the human 3'[beta](RI) and [beta]maj-globin internal reference probes (Fig. 1). Analysis of the total RNA samples showed that the level of mRNA accumulation obtained with the donor mutant construct (Fig. 2, lanes 3 and 4) is markedly reduced (~5%) compared to that seen with the normal splicing [Delta]89 gene (Fig. 2, lanes 1 and 2) which mimics the situation in thalassaemic patients (29). Analysis with the 3'[beta](Bam) probe (Fig. 1D) confirmed that the efficiency of splicing of the transcripts from the DM[Delta]89 gene, was virtually completely inhibited and was only 1% of normal (data not shown). As expected a similar result was obtained with the cytoplasmic RNA samples (Fig. 2, lanes 5-8 and 13-18).
Analysis of transcripts in the nuclear RNA fraction provided insight into the fate of the DM[Delta]89 gene product. Since the [Delta]89 and DM[Delta]89 transgenes extend to only 45 bp past the poly(A)-addition site (see Materials and Methods), the 3'[beta](RI) probe which terminates at a PstI site 513 bp beyond this position (Fig. 1D) is able to simultaneously detect 3' uncleaved (UC) transcripts as 257 nt S1-nuclease protected products [Fig. 2, UC[beta](RI)] as well as all correctly 3' cleaved [beta]-globin RNA molecules [Fig. 2, 3'[beta](RI)]. The ratio of the steady state levels of 3'[beta](RI) and UC[beta](RI) products present in the nuclear RNA fractions (Fig. 2, lanes N), therefore provides a measure of the efficiency of 3'-end formation. Once the results obtained with the human 3'[beta](RI) probe are normalised with respect to variations in the level of endogenous murine [beta]maj-globin mRNA, the steady state nuclear levels of both 3' cleaved [3'[beta](RI)] and uncleaved [UC[beta](RI)] DM[Delta]89 transcripts (Fig. 2, right , lanes 19-24) are essentially the same as those of the normally spliced [Delta]89 RNA (Fig. 2, left, lanes 9-12). This indicates that the rate of transcription and 3'-end formation of DM[Delta]89 mRNA is the same as that for [Delta]89. In marked contrast, although the level of nuclear, 3' uncleaved transcripts from a [beta]-globin gene which completely lacks IVS-II sequences ([Delta]; Fig. 2) is similar to that for the DM[Delta]89 and [Delta]89 genes [Fig. 2, right, lane 28, UC[beta](RI)], only ~10% of the total transcripts produced are correctly 3' cleaved [Fig. 2, right 28, 3'[beta](RI) generated product; 11]. These data clearly demonstrate that efficient 3'-end formation is not only strongly enhanced by the presence of [beta]IVS-II sequences, but takes place independently of the splicing reactions since the mutant DM[Delta]89 transcripts are as efficiently processed as the normal splicing [Delta]89 RNA.
A possible complicating factor in our results is that the normal levels of nuclear 3'-end formation of the splice donor DM[Delta]89 gene, may be arising from skipping of exon II. That is, the splicing of the IVS-I donor onto the IVS-II acceptor site may be promoting 3'-end formation independently of any component present in the second intron. Skipping of the upstream exon is frequently observed with splice donor site mutations (29). We therefore assessed exon skipping in our system using a representative sample from the total, cytoplasmic and nuclear RNA fractions from MEL cell pools transfected with the normal [Delta]89 and mutant DM[Delta]89 transgenes described above (Fig. 2). Analysis was by S1-nuclease protection with a mixture of probes labelled to the same specific activity and which simultaneously detect both the 5'- and 3'-end of human [beta]-globin mRNA molecules (Fig. 3B). As the 5'[beta](Acc) probe extends into exon II (Fig. 3B), it will not detect molecules which have undergone skipping. Therefore, if a significant amount of exon II skipping has occurred, the intensity of the signal obtained with the 3'[beta](RI) probe (which will detect all human [beta]-globin mRNA with or without exon II; Fig. 3B), will exceed that seen with the 5' probe. The mutant DM[Delta]89 transgene (Fig. 3A, lanes 2, 5 and 6) gave the same result with all RNA fractions as the control, normal splicing [Delta]89 (Fig. 3A, lanes 1, 3 and 4) demonstrating that no exon II skipping is taking place.
The preceding data establish the role for [beta]IVS-II sequences in promoting efficient 3'-end formation and transport of mature mRNA to the cytoplasm. The next question that arises is whether the requirement is for an intron per se at that position of the gene. Alternatively, it is possible that some, as yet, uncharacterised element(s) within [beta]IVS-II are contributing to the recruitment of the cleavage and polyadenylation machinery to the primary transcript. In order to test which of these two possibilities is correct, the normal [beta]IVS-II was replaced with [beta]IVS-I or human [alpha]-globin IVS-II ([alpha]IVS-II; Fig. 4A, [beta]I/I and [beta]I/II[alpha]). In addition, we assessed two further constructs in which the fully functional [Delta]89 intron mutant was inserted in place of [beta]IVS-I both with ([beta][Delta]89/I) and without ([beta][Delta]89/-) [beta]IVS-I in the IVS-II position (Fig. 4A). The [beta]-globin genes harbouring these heterologous first or second introns were again incorporated into the micro-locus LCR expression vector as before. Cytoplasmic and nuclear RNA fractions from the two stable transfected, independent MEL cell pools generated with each construct and which had been induced to undergo erythroid differentiation, were again analysed by an S1-nuclease protection assay using the 3'[beta](RI) probe (Fig. 4D) and quantified by PhosphorImager.
The detection of all correctly 3' cleaved transcripts with the 3'(RI) probe shows that the level of cytoplasmic mRNA accumulation (Fig. 4B) with the [beta]IVS-I (lanes 1 and 2) and [alpha]IVS-II (lanes 7 and 8) exchanges, are comparable to each other but only 7-9% of that seen with the normal functioning 256 bp [Delta]RS IVS-II control gene construct (lanes 13 and 14 and Fig. 1). This is despite the finding that analysis with the 3'[beta](Bam) probe (Fig. 1D) which assesses splicing at the [beta]IVS-II position, shows that all detectable [beta]I/I and [beta]I/II[alpha] mRNA is correctly spliced as is that for the positive control gene [Delta]RS (data not shown). The placement of the [Delta]89 intron in the IVS-I position (construct [beta][Delta]89/-) gave full levels of mRNA accumulation even though no intron was present in the IVS-II position (Fig. 4B, lanes 3 and 4). Interestingly, the inclusion of [beta]IVS-I downstream of [Delta]89 in the IVS-II position (construct [beta][Delta]89/I), reduced the level of expression to ~24% of that obtained with the [beta][Delta]RS control (Fig. 4B, lanes 5 and 6).
As before (Fig. 2), the ratio of the signals corresponding to the 3' cleaved [3'[beta](RI)] and uncleaved [UC[beta](RI)] transcripts in the corresponding nuclear RNA fractions, gave a measure of the efficiency of 3'-end formation for these various mutants (Fig. 4C). In the case of the control, normal processed [Delta]RS construct, steady state levels of 3' cleaved transcripts is seven times greater than uncleaved RNA (Fig. 4C, lanes 13 and 14). All the mutant constructs gave smaller ratios corresponding to efficiencies of 3' cleavage of 15.5-16.5% for [beta]I/I and [beta][Delta]89/I (Fig. 4C, lanes 1-2 and 5-6 respectively), 28.5% for [beta]I/II[alpha] (lanes 7 and 8) and 76% for [beta][Delta]89/- (lanes 3 and 4) compared to [Delta]RS. Perhaps the most striking observation is the >4-fold decrease in the efficiency of 3'-end formation between the [beta][Delta]89/- and [beta][Delta]89/I constructs which results from inclusion in the latter of [beta]IVS-I in the IVS-II position. The efficiency of 3' cleavage of [beta][Delta]89/I is similar to [beta]I/I which has IVS-I in both the first and second intron positions. Therefore, the reduction in the levels of cytoplasmic mRNA shown by the majority of these mutants can be accounted for in terms of a decrease in the efficiency of 3'-end formation.
These data demonstrate that [beta]IVS-II exerts the greatest influence on the efficiency of 3'-end formation but that it's distance from the poly(A)-addition site is not crucial since transposition of the IVS-II [Delta]89 intron mutant to the IVS-I position had no pronounced effect on this process. Some particular feature of [beta]IVS-II would appear to be vital as it's role in 3'-end formation could only partially be replaced by heterologous intervening sequences ([beta]IVS-I or [alpha]IVS-II). Therefore, it is not merely the presence of an intron at the IVS-II position of the [beta]-globin gene that is the crucial requirement for efficient 3'-end formation, but that additional properties unique to [beta]IVS-II are also major contributing factors.
Since [beta]IVS-I failed to replace [beta]IVS-II, hybrids between these two introns were constructed in order to map the region(s) within IVS-II which are essential for enhancing the efficiency of 3'-end formation. The first hybrid intron consisted of the 5'-half of [beta]IVS-I (75 bp) linked to the last 60 bp of [beta]IVS-II (construct 5'I/3'II; Fig. 4A). The second hybrid intron had the reverse configuration, namely the first 70 bp of [beta]IVS-II linked to the 3'-half (75 bp) of [beta]IVS-I (construct 5'II/3'I; Fig. 4A). Human [beta]-globin genes harbouring these hybrid introns were incorporated within the [beta]LCR expression system as usual and used to generate stable transfected pools of MEL cells. Cytoplasmic and nuclear RNA from cells induced to undergo erythroid differentiation was analysed as before using the 3'[beta](RI) and 3'[beta](Bam) probes in an S1-nuclease protection assay (Fig. 4). The results with the 3'(RI) probe show that only the 5'I/3'II intron construct (Fig. 4B, lanes 11 and 12) gives levels of cytoplasmic mRNA that are comparable (45%) to those seen with the control [Delta]RS gene (Fig. 4B, lanes 13 and 14). The 5'II/3'I intron containing gene gave levels of mRNA accumulation that were only 1-2% of those seen with the control (Fig. 4B, lanes 9 and 10). Analysis with the 3'[beta](Bam) probe did however show, that all the 5'II/3'I mRNA that was detected, was correctly spliced as was that for the 5'I/3'II gene (data not shown). The analysis of the corresponding nuclear RNA fractions (Fig. 4C), showed that the efficiency of 3'-end formation [3'[beta](RI):UC[beta](RI) ratio] obtained with the 5'I/3'II gene was 85% (lanes 11 and 12) of the control [Delta]RS construct whereas that for 5'II/3'I was only 10% of normal (lanes 9 and 10). These data therefore indicate, that the terminal 60 nt of [beta]IVS-II are sufficient to promote efficient 3'-end formation and mRNA accumulation.
This paper presents a detailed analysis in vivo of the role of introns in the production of stable mRNA at physiological levels of expression from within a natural chromatin context. We have previously shown that the accumulation of cytoplasmic human [beta]-globin mRNA in stable transfected MEL cells is dependent upon the presence of the second intron (11) and that this is not an erythroid-specific requirement but equally applies in non-erythroid cells (data not shown). We have exploited the properties of the human [beta]LCR to drive quantitative, physiological levels of expression of mutant [beta]-globin genes to further map and characterise the essential role of [beta]IVS-II in the production of cytoplasmic mRNA.
Our early studies indicated, but did not prove, that [beta]IVS-II played a crucial role in promoting efficient 3'-end formation (11). The analysis of two [beta]IVS-II mutants defective in splicing, one due to size ([Delta]HH; Fig. 1) and another possessing a GT -> AC 5' splice donor mutation (DM[Delta]89; Fig. 2), clearly showed that efficient 3'-end formation of the transcripts from these genes, was still taking place despite the virtual complete abolition of splicing. This was particularly evident when steady state levels of nuclear transcripts from the normal splicing ([Delta]89) and splice donor mutant (DM[Delta]89) genes was compared (Fig. 2). Here, the levels of correctly 3' cleaved DM[Delta]89 RNA were the same as that of [Delta]89 (Fig. 2, compare lanes 10-12 and 20, 22 and 24) suggesting an accumulation of unprocessed transcripts within the nucleus. The placement of normal splicing heterologous ([beta]IVS-I and [alpha]IVS-II) introns in the [beta]-globin second intron position, could only partially (7-9%) replace the function of [beta]IVS-II (Fig. 4B, lanes 1 and 2 and 7 and 8) resulting primarily from a reduction in the efficiency of 3'-end formation (Fig. 4 C, lanes 1 and 2 and 7 and 8). These observations indicate that some, as yet uncharacterised unique feature(s) of [beta]IVS-II are contributing to efficient 3'-end formation. The transposition of the normal splicing [Delta]89 variant of [beta]IVS-II to the IVS-I position (Fig. 4A, construct [beta][Delta]89/-), gave full levels of cytoplasmic mRNA accumulation (Fig. 4B, lanes 3 and 4) and retained essentially normal levels in the efficiency of 3'-end formation (Fig. 4C, lanes 3 and 4). However, the inclusion of [beta]IVS-I downstream of [Delta]89 in the IVS-II position, markedly reduced the efficiency of 3' cleavage to only 16.5% of normal (Fig. 4C, lanes 5 and 6) as was observed for the [beta]I/I gene which possesses [beta]IVS-I at both the intron I and II positions of the [beta]-globin gene (Fig. 4C, lanes 1 and 2). Although the contribution of [beta]IVS-II is not dependent on the distance to the poly(A)-addition site, it's strong positive influence on 3'-end formation can be blocked by a heterologous intron placed in closer proximity to the poly(A)-addition elements. The analysis of [beta]IVS-I/[beta]IVS-II hybrid introns (Fig. 4B and C, lanes 9-12) mapped the region within [beta]IVS-II that is crucial for promoting efficient 3' cleavage to the terminal 60 nt of this intron.
Several reports in recent years have indicated a close link between splicing and polyadenylation (30). The final intron of a given gene both in vitro (14) and in vivo (11,13) has been particularly implicated in this process. The occurrence of sequences upstream of the AAUAAA signal which affects the efficiency of 3'-end formation and polyadenylation is not without precedent. A number of U-rich USEs for various viral encoded genes have been described which stimulate polyadenylation (16,31,32). The first USE to be described for a cellular gene but which lacks a U-rich structure, is that for the human C2 complement gene (17). Furthermore, pre-mRNA processing efficiency elements may be present within the coding regions of intronless genes (33).
In the case of the SV40 late polyadenylation signal, in vitro experiments indicate that the effect of the USE (AUUUGURA) appears to be mediated by U1A protein as part of U1 snRNP binding to this site (34,35) interacting with the CPSF polyadenylation factor (36). In contrast, the binding of the human U1A snRNP protein alone to a region within the 3'UTR of its own pre-mRNA, results in an inhibition of polyadenylation (37) mediated by direct interaction between U1A and the poly(A)-polymerase (38). Therefore, the same U1A protein but in a different context can act both as a stimulator or inhibitor of polyadenylation. The binding of U1 snRNP via the U1A protein to the SV40 late USE, provides a mechanism by which the splicing and polyadenylation processes can be linked. A similar mechanism may be operating in regulating alternative processing of pre-mRNA encoding calcitonin/calcitonin gene-related peptide (CGRP). The inclusion of exon 4 requires sequences present within the intron downstream of the polyadenylation site for exon 4 (39). This `polyadenylation enhancer' is contained within a region of 127 nt and consists of a 5' splice donor site and an upstream polypyrimidine tract which have been found to bind U1snRNA and polypyrimidine tract binding (PTB) protein respectively (40). Binding studies in vitro indicate that U1snRNA and PTB stimulate the association of the polyadenylation factor CstF to the exon 4 poly(A)-addition site.
We thank Mitchell Finer and Michel Sadelain respectively for the [Delta]HH and [alpha]IVS-II mutant [beta]-globin gene constructs. The authors also wish to thank Angus Lamond, Ernesto Yagüe and Desmond Chow for constructive criticism of this manuscript. The support of the Medical Research Council, UK, Zeneca (formerly ICI) Pharmaceuticals, Therexsys UK, the European Union Human Capital and Mobility and Biomed 2 programmes is gratefully acknowledged.
Nucleic Acids Research
Pages
Introduction
Materials And Methods
Tissue culture
Plasmid DNA constructs and generation of [beta]IVS-II mutants
Extraction of total, nuclear and cytoplasmic RNA
Analysis of RNA by S1-nuclease protection
Results
Minimising the size of a fully functional [beta]IVS-II
Efficient 3'-end formation requires [beta]IVS-II sequences but occurs independently of the splicing reaction
The splice donor DM[Delta]89 RNA does not undergo exon II skipping
[beta]IVS-II cannot be efficiently exchanged for a heterologous intron
The essential function of [beta]IVS-II localises to the 3'-end of the intron
Discussion
Acknowledgements
References
REFERENCES
This page is run by Oxford University Press, Great Clarendon Street, Oxford OX2 6DP, as part of the OUP Journals
Comments and feedback: www-admin{at}oup.co.uk
Last modification: 23 Jan 1998
Copyright© Oxford University Press, 1998.
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  A. Miccio, R. Cesari, F. Lotti, C. Rossi, F. Sanvito, M. Ponzoni, S. J. E. Routledge, C.-M. Chow, M. N. Antoniou, and G. Ferrari In vivo selection of genetically modified erythroblastic progenitors leads to long-term correction of {beta}-thalassemia PNAS, July 29, 2008; 105(30): 10547 - 10552. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 F. Rigo and H. G. Martinson Functional Coupling of Last-Intron Splicing and 3'-End Processing to Transcription In Vitro: the Poly(A) Signal Couples to Splicing before Committing to Cleavage Mol. Cell. Biol., January 15, 2008; 28(2): 849 - 862. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P. Malik and P. I. Arumugam Gene Therapy for {beta}-Thalassemia Hematology, January 1, 2005; 2005(1): 45 - 50. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L. M. Salati, W. Szeszel-Fedorowicz, H. Tao, M. A. Gibson, B. Amir-Ahmady, L. P. Stabile, and D. L. Hodge Nutritional Regulation of mRNA Processing J. Nutr., September 1, 2004; 134(9): 2437S - 2443S. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 N. CUSTODIO, C. CARVALHO, I. CONDADO, M. ANTONIOU, B. J. BLENCOWE, and M. CARMO-FONSECA In vivo recruitment of exon junction complex proteins to transcription sites in mammalian cell nuclei RNA, April 1, 2004; 10(4): 622 - 633. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J C Sanchez-Gutierrez, T Benlloch, M A Leal, B Samper, I Garcia-Ripoll, and J E Feliu Molecular analysis of the aldolase B gene in patients with hereditary fructose intolerance from Spain J. Med. Genet., September 1, 2002; 39(9): e56 - 56. [Full Text] [PDF]
|
|
|
|
 |
|
 H. Tao, W. Szeszel-Fedorowicz, B. Amir-Ahmady, M. A. Gibson, L. P. Stabile, and L. M. Salati Inhibition of the Splicing of Glucose-6-phosphate Dehydrogenase Precursor mRNA by Polyunsaturated Fatty Acids J. Biol. Chem., August 16, 2002; 277(34): 31270 - 31278. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. M. Shayakhmetov, C. A. Carlson, H. Stecher, Q. Li, G. Stamatoyannopoulos, and A. Lieber A High-Capacity, Capsid-Modified Hybrid Adenovirus/Adeno-Associated Virus Vector for Stable Transduction of Human Hematopoietic Cells J. Virol., February 1, 2002; 76(3): 1135 - 1143. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 F. Moreau-Gaudry, P. Xia, G. Jiang, N. P. Perelman, G. Bauer, J. Ellis, K. H. Surinya, F. Mavilio, C.-K. Shen, and P. Malik High-level erythroid-specific gene expression in primary human and murine hematopoietic cells with self-inactivating lentiviral vectors Blood, November 1, 2001; 98(9): 2664 - 2672. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Zertal-Zidani, R. Ducrocq, C. Weil-Olivier, J. Elion, and R. Krishnamoorthy A novel {delta}{beta} fusion gene expresses hemoglobin A (HbA) not Hb Lepore: Senegalese {delta}0{beta}+ thalassemia Blood, August 15, 2001; 98(4): 1261 - 1263. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. E. Rubin, P. Pasceri, X. Wu, P. Leboulch, and J. Ellis Locus control region activity by 5'HS3 requires a functional interaction with beta -globin gene regulatory elements: expression of novel beta /gamma -globin hybrid transgenes Blood, May 15, 2000; 95(10): 3242 - 3249. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. Zhao, L. Hyman, and C. Moore Formation of mRNA 3' Ends in Eukaryotes: Mechanism, Regulation, and Interrelationships with Other Steps in mRNA Synthesis Microbiol. Mol. Biol. Rev., June 1, 1999; 63(2): 405 - 445. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. Zufferey, J. E. Donello, D. Trono, and T. J. Hope Woodchuck Hepatitis Virus Posttranscriptional Regulatory Element Enhances Expression of Transgenes Delivered by Retroviral Vectors J. Virol., April 1, 1999; 73(4): 2886 - 2892. [Abstract] [Full Text]
|
|
|
|
|
|
P. R. Provost and Y. Tremblay Length Increase of the Human alpha -Globin 3'-Untranslated Region Disrupts Stability of the Pre-mRNA but Not That of the Mature mRNA J. Biol. Chem., September 22, 2000; 275(39): 30248 - 30255. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
B. Amir-Ahmady and L. M. Salati Regulation of the Processing of Glucose-6-phosphate Dehydrogenase mRNA by Nutritional Status J. Biol. Chem., March 23, 2001; 276(13): 10514 - 10523. [Abstract] [Full Text] [PDF]
|
|
| |||||||||