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The Journal of Immunology, 2006, 176: 2381-2388.
Copyright © 2006 by The American Association of Immunologists

Position-Dependent Repression and Promotion of DQB1 Intron 3 Splicing by GGGG Motifs1

Jana Královicová and Igor Vorechovsky2

Division of Human Genetics, University of Southampton, School of Medicine, Southampton, United Kingdom


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Alternative splicing of HLA-DQB1 exon 4 is allele-dependent and results in variable expression of soluble DQbeta. We have recently shown that differential inclusion of this exon in mature transcripts is largely due to intron 3 variants in the branch point sequence (BPS) and polypyrimidine tract. To identify additional regulatory cis-elements that contribute to haplotype-specific splicing of DQB1, we systematically examined the effect of guanosine (G) repeats on intron 3 removal. We found that the GGG or GGGG repeats generally improved splicing of DQB1 intron 3, except for those that were adjacent to the 5' splice site where they had the opposite effect. The most prominent splicing enhancement was conferred by GGGG motifs arranged in tandem upstream of the BPS. Replacement of a G-rich segment just 5' of the BPS with a series of random sequences markedly repressed splicing, whereas substitutions of a segment further upstream that lacked the G-rich elements and had the same size did not result in comparable splicing inhibition. Systematic mutagenesis of both suprabranch guanosine quadruplets (G4) revealed a key role of central G residues in splicing enhancement, whereas cytosines in these positions had the most prominent repressive effects. Together, these results show a significant role of tandem G4NG4 structures in splicing of both complete and truncated DQB1 intron 3, support position dependency of G repeats in splicing promotion and inhibition, and identify positively and negatively acting sequences that contribute to the haplotype-specific DQB1 expression.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Eukaryotic mRNAs are transcribed as precursors containing intervening sequences or introns that are subsequently removed by splicing (1). This process is conducted by the spliceosome, a macromolecular complex assembled by the stepwise association of the pre-mRNA substrate with small nuclear ribonucleoprotein particles (snRNPs;3 U1, U2, U4/U6, and U5) and a large number of non-snRNP proteins (1). In addition to these trans-acting factors, spliceosome assembly requires the presence of cis-elements in the precursor (pre-)mRNAs: the 5' splice sites (5'ss), 3'ss, the polypyrimidine tract (PPT), and the branchpoint sequence (BPS). In higher eukaryotes, these signal recognition sequences are degenerate and often insufficient to define exon-intron boundaries. Auxiliary elements that activate or repress splicing, known as exonic and intronic splicing enhancers (ISEs) or silencers, allow the authentic splice sites to be correctly recognized among pseudosites that have similar signal sequences but outnumber genuine splice sites by an order of magnitude (2, 3). Splicing silencers and enhancers have been identified through mutations or naturally occurring variants, selection experiments, and bioinformatics approaches (4, 5, 6, 7, 8), and some of them have been shown to interact with RNA-binding factors that positively or negatively regulate splicing, including serine/arginine-rich (SR) proteins and heterogenous nuclear RNPs. SR proteins typically mediate splicing enhancement by binding to exon splicing enhancers (reviewed in Refs.4 and 9), although they may also repress splicing (10, 11). The auxiliary elements are important not only in constitutive, but also in alternative splicing, which generates distinct mRNAs from a single gene and plays a major role in regulating gene expression and enhancing proteomic diversity (reviewed in Ref.12).

A growing number of studies have shown that pre-mRNA splicing can be influenced by cis-acting elements that contain repetitive guanosines (Gs), largely acting as ISEs (13, 14, 15, 16, 17, 18, 19, 20, 21, 22). The GGG motifs (G3) are abundant near the 5' ends of human introns and to a lesser extent at the 3' ends (15, 23, 24, 25), suggesting that they play a role in exon definition. The G3 runs were found to be underrepresented in ~100 nt intronic segments downstream of brain-specific cassette exons as compared with controls (26), pointing to their potential general importance in alternative splicing. In addition, G3 are common in relative enhancer and silencer classification by unanimous enrichment-predicted ISE hexamers downstream of the 5'ss and upstream of the 3'ss and exhibit significant species-specific differences in the prevalence (7). Apart from the enhancing effect of G repeats on splicing, the guanosine quadruplet (G4) runs located close to the 5'ss have been recently shown to mediate silencing of a brain-region-specific exon of the GRIN1 gene (27). G-rich repeats have been proposed to function cooperatively in combination with exonic UAGG elements (27), but the ambiguous effects of G repeats on splicing are poorly understood.

DQB1 is a highly polymorphic MHC class II gene encoding the beta-chains of membrane heterodimers DQ that are critical for the development of adaptive immune responses and tolerance. The gene has >60 alleles that are classified into five lineages designated DQB1*02 through *06 (28). DQB1 exon 4, which encodes the transmembrane domain of DQbeta, is fully included only in DQB1*02 and *05 mRNAs, whereas the DQB1*03, *04, and *06 alleles show partial exon exclusion. Proteins immunoprecipitated with a mAb against DQ were found in supernatants of cultured cells that produce mRNAs lacking this exon, but not in those that do not generate these transcripts (29). Differential skipping of exon 4 results from poor recognition of the 3'ss of intron 3, which is largely due to single nucleotide polymorphisms (SNPs) that weaken the BPS and PPT (30). DQB1 intron 3 contains several polymorphic G repeats, but their significance in the allele-specific expression of this gene is unclear.

In this study, we have used splicing reporter assays to systematically examine the effects of G repeats on intron 3 splicing. We show that two G4 motifs arranged in tandem upstream of the exon 4 BPS are key G-rich ISEs, whereas the same motifs located just proximal to the 5'ss inhibit intron 3 splicing. Our results support position dependency of these elements in splicing enhancement and repression and identify important intronic signatures that influence allele-dependent expression of this disease gene.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Splicing reporter constructs

The development of allele-specific DQB1 splicing reporters was described previously (30). For mutagenesis, we used a reporter construct that contained exons 3 through 6 of the DQB1*0602 allele (Fig. 1A). This minigene was cloned into the mammalian expression vector pCR3.1 (Invitrogen Life Technologies) as described elsewhere (30). Mutated minigenes were prepared by overlap-extension PCR (31) and validated by sequencing as described previously (32). Truncations in intron 3 were introduced as previously described (30). The mutated constructs contained the following replacements of the suprabranch region (Fig. 1A): Ran1, random sequence generated by an online random sequence tool, available at http://tandem.bu.edu/rsg.html; ran2, a randomly mutated suprabranch sequence of adenovirus major late as described elsewhere (33); and IIIa, sequence upstream of an adenovirus BPS that was previously implicated in a repressive effect of ASF/SF2 on splicing (10). DQB1 exon 5, which encodes a cytoplasmic portion of the beta-chain, is not included on the HLA-DQB1*0602 allele due to an inactivating 3'ss SNP of intron 4 (34, 35). Full sequences of intron 3 and 4/5 are shown as a multiple alignment of DQB1 alleles.4 Exon 4 sequences are available from the HLA/IMGT database (28). The nucleotide sequence of the wild-type DQB1 segment 2 (Fig. 1A) was TGG ACT TCA ACT CCT CAG CAG G.


Figure 1
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  		FIGURE 1. DQB1 sequences upstream of the BPS enhance intron 3 splicing. A, Schematic representation of splicing reporter constructs. Open boxes in the upper panel represent exons (E; drawn to scale); lines represent introns (IVS; not to scale). Scale units are base pairs (bp). Slash indicates intron truncations as described previously (30 ). Segments 1 and 2 are shown as small open rectangles; the exon 4 BPS is shown as a small closed rectangle. Correctly spliced and intron 3-retaining isoforms are shown schematically above and below the splicing reporter construct, respectively. The lower panel shows nucleotide sequences of wild-type and mutated segment 1. G4 in position IVS3–33-36 and IVS3–40-43 of the wild-type sequence are boxed. Cytosine substitutions introduced in G4 repeats are underlined. The relative distance (bp) from the 3'ss of intron 3 is indicated on a scale at the bottom. SNPs IVS3–36A/G and IVS3–38A/G are denoted by stars. B, Sequence-specific enhancement of intron 3 splicing. RNA products are schematically shown on the right. White, gray, and black boxes represent exons 3, 4, and 6 as in panel A; retained intron 3 is shown as a line. ‘No transfection’ and ‘no template’ controls are not shown. The type of DQB1 construct (complete or truncated intron 3) used for mutagenesis is shown at the bottom.

 
Cell cultures and transfections

Highly transfectable human embryonic kidney 293T cells were grown under standard conditions in RPMI 1640 supplemented with 10% (v/v) FCS (Invitrogen Life Technologies). Transient transfections were performed in 6- or 12-well plates using FuGENE 6 (Roche) as described previously (30, 36), with a plating density ~2 and ~1 x 105 cells/well, respectively. The medium was changed when reaching ~50% confluency 2 h before transfection. Plasmids were purified with the Wizard Plus SV Minipreps (Promega). Cells were harvested for RNA extraction 48 h posttransfection.

Detection of RNA products

Total RNA was isolated using Tri-Reagent (Sigma-Aldrich) and treated with DNase I (Ambion) according to the manufacturers’ recommendations. First-strand cDNA was reverse transcribed using oligo(dT)15 primers and Moloney murine virus reverse transcriptase (Promega). PCR products were amplified with primers directed to vector sequences and with a combination of cDNA and vector primers as described (30) to validate the ratios of RNA products. Exon inclusion levels were measured with FluorImager 595 using FluorQuant and Phoretix software (Nonlinear Dynamics) as described previously (30, 36).

UV cross-linking

A 115-nt PCR product was amplified with primers A (5'-TAA TAC GAC TCA CTA TAG GGT GGA CTT CAA CTC CTC AGC AGG GAT; T7 promoter sequence is in italics) and B (5'-CTG GGC AGA TTC AGA CT; Fig. 1A) and plasmid reporter DNA as a template. PCR products were gel purified using the MinElute kit (Qiagen). Riboprobes were transcribed using the MAXIScript kit (Ambion) in the presence of [{alpha}-32P]GTP (Amersham). RNAs were incubated in 10-µl reaction mixtures containing HeLa nuclear and S100 extracts (4C Biotech), 2 mM MgCl2, 0.5 mM ATP, 20 mM creatine phosphate, RNasin, and 1 µg/µl tRNA at 30°C for 20 min. The samples were irradiated on ice with a 254-nm UV cross-linker (Ultralum) at 1.9 J/cm2 and digested with RNase T1 (0.8 U/µl) and RNase A (0.4 U/µl) (Ambion) at 37°C for 30 min. Cross-linked proteins were resolved by SDS-PAGE followed by autoradiography at –70°C.

Exon 4 inclusion of murine H2-IA genes

Mouse cDNA samples were prepared from total RNA isolated from freshly prepared thymus, spleen, and PBMC of strains C3H (haplotype H-2k), C57BL (H-2b), and DBA (H-2d) using oligo(dT)15 primers. Amplification primers were 523 (AGK AAT GGG GAC TGG ACC TTC; K is G and T), 939 (AGA CAG AGA CTG GGG GAC TCC), 68 (CAG GGA CTG AGG GCG GAA ACT), and 907 (GCT GAG GTG GTG GAT ACA ATA). Mouse strains were purchased from Harlan U.K.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
A G-rich segment upstream of the BPS modulates splicing of DQB1 intron 3

Intron 3 is weakly spliced due to poor recognition of the 3'ss on the DQB1*03, DQB1*04, and DQB1*06 alleles, resulting in exon 4 skipping (30). In contrast, the remaining two DQB1 lineages show constitutional inclusion of this exon in mRNAs (30). Allele-dependent intron 3 retention and exon 4 skipping are largely influenced by the IVS3–24A/C/T and IVS3–6C/T SNPs in the BPS and PPT, respectively, with a subset of exon 4 variants contributing to differential exon skipping only to a small extent (30).

A 20-nt region upstream of the BPS has been shown to bind components of splicing factor SF3a/SF3b in a sequence-independent manner and to facilitate U2 snRNP anchoring (33, 37). To study the significance of polymorphic suprabranch sequences in DQB1 splicing, we examined the splicing pattern of the DQB1*0602 minigene with truncated intron 3. This reporter pre-mRNA generates a mixture of intron 3-retaining and correctly spliced transcripts 48 h posttransfection (30), thus providing a sensitive assay for testing auxiliary cis-elements. We replaced two adjacent 22-nt segments (designated 1 and 2; Fig. 1A; see intron 3 alignments4) upstream of exon 4 BPS with random sequences (designated ran1 and ran2) and with a sequence upstream of adenovirus BPS (termed IIIa). The latter sequence was previously associated with a repressive effect of the SR protein ASF/SF2 on splicing (10). Segment 1 was separated from the BPS by two adenosines, while segment 2 was immediately adjacent to segment 1 further upstream (Fig. 1A and footnote 4). We transiently transfected wild-type and mutated reporter constructs into 293T cells and examined their splicing products. Intron 3 splicing was impaired in all constructs in which segment 1 was replaced, whereas identical substitutions of segment 2 did not reduce splicing, except for a small inhibitory effect of the randomly generated sequence ran1 (Fig. 1B).

Because segment 1 had tandem arranged G4 runs that were absent in segment 2 (Fig. 1A), we next tested whether its stimulating effect on splicing is influenced by these elements. We introduced G to C transversions in both G4 and examined RNA products after transfection of wild-type and mutated constructs. All transversions dramatically increased intron 3 retention (Fig. 1B). Together, these results suggested that segment 1 contains G-rich ISE(s) and that the G repeats may be critical for the enhancing effect mediated by segment 1.

G4 upstream of the BPS are key G-rich ISEs of DQB1 intron 3

Next, we examined the influence of G repeats on DQB1 splicing systematically by mutating each G3 and G4 in exon 4 and in flanking intronic sequences (Fig. 2A and footnote 4). The wild-type DQB1*0602 construct carrying truncated intron 3 has two runs of G3 and three runs of G4. The latter motifs are close to the 5'ss and, in a tandem arrangement, just upstream of exon 4 BPS. This BPS, which is located in intron positions –21 to –27 relative to the 3'ss, is the best match to a mammalian consensus within 40 nt upstream of authentic 3'ss (30), was predicted through comparison of human and mouse introns (38) and confirmed by mutagenesis and branchpoint mapping (30). The G4 run adjacent to the BPS is invariable, whereas the G4 motif further upstream and the intervening sequence between the two G4 contain G/A SNPs IVS3–38 and IVS3–36, respectively (Fig. 1A).


Figure 2
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  		FIGURE 2. G4 upstream of the BPS are the most effective G-rich ISEs. A, The upper panel shows a schematic representation of G3 and G4 repeats (open boxes) in exon 4 (gray box) and flanking intronic sequences (lines). Arrows indicate the G4 motifs upstream of the BPS (closed rectangle). The lower panel shows RNA products of splicing reporters mutated systematically in each G run in exon 4 and flanking intronic sequences. % spl., percentage of correctly spliced products relative to the sum of natural transcripts, transcripts with intron 3 retention and exon skipping. Means and SD (SD) were computed from two duplicate transfection experiments. B, Effect of mutations in intron 3 and exon 4 G repeats on splicing of the DQB1 minigenes containing complete intron 3. % ES, percentage of exon 4 skipping; % IR, percentage of intron retention; hd, heteroduplexes. Reporter constructs are schematically shown below each panel.

 
Examination of RNA products after transfection of mutated minigenes showed that intron 3 splicing was markedly repressed by mutations that disrupt tandem G runs located upstream of the BPS (Fig. 2A). Single or double mutations of G3 further upstream reduced splicing less (IVS3–51-52), while most mutations of G3 in exon 4 had no or only minor effects (mutations 60/61, 75, 76/77, 81/82). By contrast, mutations in the G repeats that were located close to the splice sites of intron 3 (IVS3+7 and E4+2/3) improved intron 3 splicing.

To confirm the importance of suprabranch G4 runs in the context of complete intron 3, we mutated a subset of G repeats in constructs carrying the wild-type, 509-nt intron with a total of 11 G3. As expected, mutations of the suprabranch G4 showed the largest reduction of splicing, whereas mutations in the remaining G repeats had only minor or no effects, including tandem arranged G3 in the middle of intron 3 (nucleotide position 265 and 274, Fig. 2B and footnote 4). Interestingly, G3 mutations in positions 60–61 of exon 4, which were in the vicinity of a previously identified exon splicing enhancer 5D (30), markedly decreased exon skipping.

Together, these results showed that G4 located just upstream of BPS are key G-rich ISEs that promote splicing of both full-length and truncated DQB1 intron 3, whereas a G4 motif located 5'ss proximal acts as an intronic splicing silencer.

A critical role of central G4 residues in splicing enhancement

To define a role of individual nucleotides in both suprabranch G4 ISEs, we mutated systematically G in each position into A, T, and C and transfected mutated reporters into 293T cells (Fig. 3, A and B). Mutations of the second and third Gs were most effective in repressing intron 3 splicing. G to C substitutions resulted in the strongest inhibition of intron 3 splicing in both G4 runs, with uridines and adenosines exhibiting smaller effects.


Figure 3
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  		FIGURE 3. G to C substitutions in central positions of suprabranch G4 are the most potent repressor mutations. A, Systematic mutagenesis of proximal G4. Mutations are shown at the indicated intronic positions above the panel. Means of two transfection experiments in triplicate (SD) are shown below. RNA products are schematically shown on the right. % spl., percentage of correctly spliced transcripts. B, Systematic mutagenesis of distal G4. C, Influence of the IVS3–38G/A SNP and the AC box on intron 3 splicing. The AC dinucleotide is located just downstream of the proximal G4 in positions –31 to –32 (Fig. 1A). Mutated construct are shown above, D, The length of inter-G4 sequence and intron 3 splicing. Sequences of tri- and hexanucleotides inserted between the tandem G4 are shown above.

 
A central nucleotide in the 3-nt intervening sequence between the two G4 runs (Fig. 1A) is polymorphic, with the A allele associated with the DQB1*02 lineage and the G allele with the remaining lineages (28, 30). Assuming previously determined allelic frequencies at DQB1 (39) and absolute linkage disequilibrium between exon 2 specificities and this variant, the frequency of the A allele is ~25% and ~22% in white and black populations, respectively. The first nucleotide of the proximal G4 motif is also polymorphic, with the invariant G allele present in the DQB1*02, *05, and *06 lineages (28, 30). To test the influence of these variants on splicing, we prepared reporter constructs containing mutations to the other allele in these positions. Both the IVS3–38G->A and IVS3–36G->A mutations reduced intron 3 splicing to a small extent (Fig. 3, A and C), consistent with an increased exon skipping of the latter SNP, which was previously observed for constructs with complete intron 3 (30).

The structure of the suprabranch ISE (G4N3G4; Fig. 1) was remarkably similar to G3N3G3 elements identified previously in the human {alpha}-globin gene (22) and a G4N3G4 motif found in the gene for human growth hormone (20). The activity of the latter ISE was modified by an adjacent AC element (20). Because the 3' G4 run just upstream of the DQB1 BPS was also followed by this dinucleotide (Fig. 1A), we systematically substituted each A–32C–31 position with the remaining nucleotides (Fig. 3C). Although the ratio of correctly spliced to intron 3-retaining transcripts was similar, all substitutions of C–31 increased exon skipping, suggesting that this nucleotide plays an important role in exon definition.

Because the tandem G4 runs upstream of the BPS are the only G repeats that are very close to one another in intron 3, it is possible that the GxNyGx structures have a stronger effect on splicing if the y values are small. To test whether the length of the gap between G runs is important for DQB1 splicing, we inserted an additional 3 and 6 bp after the first nucleotide of the intervening sequence (Fig. 3D). However, transfection of the mutated constructs into 293T cells and examination of their splicing pattern did not reveal significant alterations of intron removal, suggesting that the distance between the two runs in this range is not critical.

Finally, because G4-rich sequences have been shown to act as binding sites for the member(s) of the H/F family heterogeneous nuclear RNPs (hnRNPs) (13, 18, 19, 40, 41, 42, 43, 44, 45, 46, 47), we attempted to identify proteins that bind to suprabranch G runs. Both wild-type and mutated GTP-labeled pre-mRNA substrates were subjected to UV cross-linking in HeLa nuclear and S100 cytoplasmic extracts (Fig. 4). Both substrates cross-linked to several proteins of ~45–50 kDa (Fig. 4), a size range corresponding to hnRNPs H/H'/F observed previously for G4-containing pre-mRNAs (27). This pattern was similar for the wild-type and mutated reporters, presumably due to the presence of other G4 repeats in the substrate, although the C–42 pre-mRNA gave a somewhat weaker signal.


Figure 4
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  		FIGURE 4. Detection of protein binding to a G4-containing DQB1 pre-mRNAs. UV cross-linking of uniformly [{alpha}-32P] GTP-labeled wild-type (wt) and mutated RNA probes to ~45–50 kDa proteins in HeLa nuclear (NE) or S100 (lane 1) extracts. A NE-specific ~68 kDa protein was present in both UV-treated and untreated samples (not shown). The pattern of ~45–50 kDa proteins corresponds to that observed for the hnRNP H/H'/F subfamily (27 ).

 
Together, these results showed that central G4 guanosines were the most important nucleotides for the G4N3G4-mediated splicing enhancement, whereas the influence of polymorphic variants or inter-G4 distance on splicing was minor or insignificant. They also showed an important role of cytosine –31 in exon 4 inclusion and confirmed a previously observed cross-linking pattern, consistent with binding the hnRNP H/H'/F family of proteins.

Exon 4 of the mouse H2-IAbeta is constitutively included in mRNA

Alternative splicing of conserved exons is frequently species specific and these events modify conserved domains in proteins more frequently than other classes of alternative splicing (48). Human and mouse exons encoding the transmembrane domain of DQbeta are highly conserved, with a nucleotide identity of ~90% (28, 49, 50). We, therefore, examined the inclusion of mouse exon 4 in the H2-I-Abeta mRNAs of three inbred mouse strains carrying haplotypes that were previously fully sequenced (Fig. 5A and footnote 4). However, RT-PCR with two independent primer pairs revealed no detectable amounts of transcripts lacking the transmembrane exons (Fig. 5B). This suggests that, in contrast to the weakly spliced human exon 4 (30), the mouse homolog is constitutively included in mature transcripts on the available haplotypes. Interestingly, examination of sequence alignments of the mouse (49, 50) and the rat (51) introns showed a complete absence of tandem arranged G runs upstream of the predicted BPS.4 This finding raises the hypothesis that the suprabranch G-rich ISEs identified in this study have been selected in humans to promote splicing of poorly recognized intron 3 and to maintain sufficient expression of natural DQB1 transcripts that encode membrane-bound molecules.


Figure 5
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  		FIGURE 5. Exon 4 of murine H2-IAbeta is constitutively included in mRNA. A, RT-PCR with two pairs of PCR primers (left) using cDNA preparations from three H2-IAbeta-expressing tissues (top) of three mouse strains (bottom) that carry haplotypes d (49 ), b, and k (50 ). RNA products are shown schematically on the right side. B, multiple alignment of the available H2-IAbeta alleles at the 3'ss of intron 3. SNPs are shown as stars. Putative branch point is shown as å. Full intron 3 sequences are available as a multiple alignment.4

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
Membrane proteins are often regulated by mechanisms that release their soluble forms from its membrane anchorage (52). More than 40% of alternatively spliced genes that encode single-pass transmembrane proteins produce splice variants lacking exons coding for the transmembrane domains, potentially generating soluble protein isoforms (53). Alternatively spliced HLA transcripts that lack transmembrane exons enter the endocytic compartment, are released by cells, and can be detected as soluble molecules (sHLA) in body fluids (29, 54). sHLAs may induce apoptosis of alloreactive T cells in vitro, modulate immune responses, and play a role in the induction and maintenance of peripheral tolerance (55, 56). In addition, serum levels of sHLA are increased in inflammatory and autoimmune diseases and correlate positively with disease activity and autoantibody titers (54, 57), further supporting a physiological role for sHLA.

In healthy individuals, serum sHLA levels vary on different HLA haplotypes (58) and in vitro, the amount of soluble DQ molecules in culture supernatants has been linked to the haplotype-specific expression of transcripts lacking DQB1 exon 4 (29). In this, and in our recent study (30), we have begun to dissect molecular mechanisms underlying differential expression of mRNA isoforms lacking exon 4, the most prominent example of allele-dependent alternative splicing in the MHC class II region. We have identified a suprabranch segment that promoted splicing of both full-length and truncated DQB1 intron 3 and found that G repeats in this segment were critical enhancer elements (Fig. 1). Because mutations of these motifs markedly diminished natural transcripts in our minigene splicing assays, germline mutations of these intronic sequences are likely to alter the expression of DQB1. Likewise, a subset of polymorphisms in these elements such as IVS3–31G/A (Fig. 3C) is likely to contribute to the allele-specific splicing pattern of DQB1. The importance of intronic mutations or variants in gene expression is supported by recent estimates exploiting the complete set of disease genes with a newly developed probabilistic model, suggesting that ~62% of human disease gene mutations alter splicing and that splicing mutations are the most common cause of hereditary disorders (59). Indeed, deficient pre-mRNA splicing has been shown to result in HLA null alleles (60, 61, 62). Although these cases are uncommon, they are likely to be underrepresented because intronic regions of the MHC class II and other disease genes have been analyzed for mutations only exceptionally. Similarly, clonal somatic mutations in these intronic elements in tumor cells may contribute to a failure to express mature HLA Ags.

In addition to DQB1, the {alpha}-chain-encoding DQA1 gene also undergoes allele-dependant alternative splicing of exon 4. Apart from the recently described diversity of DQA1 mRNAs generated by alternative polyadenylation (63, 64), this gene produces exon 4-lacking transcripts spliced to downstream splice sites in the 3' untranslated region. Our analysis of expressed sequence tag libraries showed that DQA1 sequences lacking exon 4 were derived from alleles in linkage disequilibrium with exon-skipping transcripts DQB1*03/*04 (GenBank accession numbers CA309994.1, BF891581.1, BF891575.1, BF963070.1, BF896819.1, BF891027.1, BF950596.1, BF891573.1, and BF890947.1) and DQB1*06 (BG537384), suggesting that they may participate in the expression of soluble DQ molecules.

In contrast to the suprabranch G repeats, G4 motifs located close to the 5'ss inhibited intron 3 splicing (Fig. 2A). Comparison of the previously reported G3 and G4 runs that affected splicing (Table I) suggests that, except for a G repeat bridging the 5'ss (46), their 5'ss proximal location was associated with splicing inhibition, whereas their more distal position with splicing promotion. The effect of G4 motifs is likely to depend on the relative distance from the splice sites that are sequentially recognized by a number of splicing factors. The proximity of these motifs to the splice sites might involve a distinct set of interactions that would account for the observed silencing effects. The comparison in Table I also reveals that G-rich ISEs are often tandem arranged structures that are separated by an intervening sequence of a variable length and composition. The length of the intervening sequence is often very short, up to several nucleotides, with longer sequences often within the 12–18 nt range, raising a speculation that their overall length distribution may not be random. A recent analysis of the intervening loops in G quadruplexes showed that the loop length was significantly deviated from a random distribution, suggesting that the intervening sequences, which play a role in determining the quadruplex stability, are under selective pressure (65). Interestingly, some of the intervening sequences in tandem G repeats (Table I) matched those that are among the most frequent in quadruplex loops, such as CCT, CT, CC, AAA, and AA (66). Although extending the gap between G repeats did not alter splicing (Fig. 3D), we could not exclude a confounding effect of newly introduced nucleotides.


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  		Table I. Comparison of the effects of intronic G3 and G4 repeats on splicing of mammalian pre-mRNA substrates

 
The inhibitory influence of G4 close to the 5'ss and stimulatory effects of identical G runs located further away from the 5'ss strikingly resembled those recently described for sequences flanking the CI cassette exon of the GRIN1 gene (27). The GRIN1 G4 is located in the same intron position relative to the 5'ss (+5 to +8) as the 5' proximal G4 in DQB1 intron 3 (Fig. 2A and footnote 4) and cytosine substitutions in the GRIN1 motif also markedly enhanced exon inclusion (27). The presence of adenosine in position IVS3+7 on the DQB1*05 alleles is likely to weaken this silencer and contribute to efficient splicing of intron 3, which is characteristic of this DQB1 lineage (30). Although 5'ss proximal G4 were found to be associated with exon skipping (27), DQB1 exon 3 is constitutively included in the mRNA, but allele-specific differences in the inclusion of this highly polymorphic, beta2 domain-encoding exon cannot be excluded.

Our systematic mutagenesis of suprabranch G repeats showed that cytosines were the most efficient repressors of intron 3 splicing, with the second and third position being the most effective. This pattern is very similar to that observed for tandem G4 runs in an ISE located upstream of the putative BPS of GH1 exon 4 (20). In addition, G to C substitutions in the 5'ss proximal GRIN1 GGGG motif were also powerful activators of splicing (27). A similar systematic analysis of a splicing silencer element derived from a sense Alu repeat showed that cytosines had the strongest stimulatory effect on exon inclusion (67).

In summary, our results showed that G4N3G4 structures upstream of the branch site promote splicing of DQB1 intron 3 and that the central Gs in both repeats are the most powerful splicing enhancers. A significant influence of G repeats on splicing and exon inclusion suggests that naturally occurring DNA variants that remove or create these elements may alter the amounts of natural transcripts or relative expression of alternatively spliced isoforms, thus contributing to interindividual differences in gene expression and phenotypic variability. In addition to exonic variants that commonly affect exon inclusion (4, 30, 68, 69), the influence of G-rich intronic variants on gene expression, such as a GGG insertion/deletion polymorphism in an OCA1 intron (70), may be underappreciated, and it will be interesting to examine variants in these motifs in future studies.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by the Juvenile Diabetes Research Foundation International, the Wellcome Trust Value in People Award, and the Annual Grant from the University of Southampton. Back

2 Address correspondence and reprint requests to Dr. Igor Vorechovsky, Division of Human Genetics, University of Southampton School of Medicine, MP808, Southampton, U.K. E-mail address: igvo{at}soton.ac.uk Back

3 Abbreviations used in this paper: snRNP, small nuclear ribonucleoprotein particle; BPS, branchpoint sequence; G3, guanosine triplets; G4, guanosine quadruplets; IVS, intervening sequence or intron; ISE, intronic splicing enhancers; SNP, single nucleotide polymorphism; hnRNP, heterogeneous nuclear RNP; sHLA, soluble molecules; ss, splice sites; PPT, polypyrimidine tract. Back

4 The online version of this article contains supplemental material. Back

Received for publication August 31, 2005. Accepted for publication November 16, 2005.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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