We have established a minigene system to dissect the molecular mechanism underlying alternative splicing of human tau pre-mRNA exon 10, an event important for pathogenesis of neurodegenerative disorder FTDP-17. Our systematic biochemical analyses of wild-type and intronic mutant tau pre-mRNAs demonstrated that single-nucleotide mutations in an evolutionarily nonconserved intronic region enhance splicing between exon 10 and exon 11. Experiments using RNase H and an oligonucleotide complementary to positions −6 to +12 at the splice junction suggest that the intronic mutant tau transcripts harbor a more “open” RNA structure in this region than wild-type tau in the presence or absence of HeLa nuclear extracts. This is consistent with the presence of an RNA stem-loop structure forming around the exon 10 5′ splice site in wild-type tau pre-mRNA. Comparison of the splicing efficiency of wild-type and mutant tau transcripts when various spliceosomal U snRNPs were made limiting by specific 2′-O-methyl oligonucleotides demonstrated that wild-type tau splicing was most sensitive to a reduction in the level of functional U1 snRNP. Finally, the U1 snRNP protection assay and the gel mobility shift experiment with purified U1 snRNP revealed an increase in the binding of U1 snRNP to the 5′ splice site of exon 10 in the DDPAC+14 mutant, correlating well with the increase in splicing efficiency observed in our in vitro splicing assay. These results strongly support the model depicted in Fig. B, where the stem-loop structure in the wild-type tau pre-mRNA is destabilized by FTDP-17 intronic mutations, leading to enhanced recognition of this 5′ splice site by the U1 snRNP-containing early splicing complex and increased formation of Tau4R transcripts.
This stem-loop structure model could also explain the behavior of several other mutations found in a number of patients with tau exon 10 aberrant splicing, including +3 and +13 mutations (6
) in addition to the DDPAC+14 and AusI+16 mutations (Fig. A). It is also consistent with the observation that in the rat (or mouse), the predominant isoform of tau is the exon 10-containing isoform. Exon 10 splicing may be enhanced in these species because of the destabilization of the stem structure caused by the naturally occurring G at position +13 in the rat tau gene (G at both +13 and +16 positions in mouse; see Fig. ); (23
; Jiang and Wu, unpublished data). Thus, a single-nucleotide change in this nonconserved intronic region can have a significant impact on alternative splicing of exon 10.
While this paper was in preparation, two studies were published in which tau exon 10 alternative splicing was examined more extensively using exon-trapping assays (9
). The study by Grover and colleagues lends further support for the stem-loop model. On the other hand, D'Souza and colleagues suggested that the stem-loop structure was not supported by a mutation at +12 that should restore base-pairing in the stem structure (9
). However, results from compensatory-mutation analyses have to be carefully interpreted. First, A-U base pairing (in the +12 compensatory mutant) is expected to be weaker than G-C base-pairing (in the wild-type tau). Second, multiple RNA-RNA and RNA-protein interactions are known to influence splice site selection and splicing efficiency. The stem-loop structure has to be viewed in the context of these multiple interactions. It is likely that events other than the U1 snRNA-pre-mRNA interaction also play important roles in regulating tau exon 10 splicing. In fact, we have found that certain non-U1 snRNP splicing regulators affect tau exon 10 splicing, and we are currently characterizing the differential recognition of the wild-type versus intronic mutant pre-mRNAs by these splicing regulators (Jiang and Wu, unpublished). The single-nucleotide changes around the 5′ splice site may have multiple effects on the RNA-RNA interactions (intramolecular and intermolecular) as well as on RNA-protein interactions. These effects may not be necessarily in the same direction. For example, “compensatory mutations” for the DDPAC+14 and AusI+16 mutants (at positions +1 and −2, respectively) that “restore” the base-pairing interactions in the stem will also affect U1 snRNP binding at the same time. Therefore, the compensatory mutation strategy that we and other groups used may not be optimal for testing the stem-loop model in the presence of the spliceosome.
The role of U1 snRNP in mammalian pre-mRNA splicing has been well established (for reviews, see references 2
, and 52
). It has been demonstrated that differential binding of U1 snRNP could affect 5′ splice site recognition in both yeast and mammalian cells (10
). It is worth noting that these previous studies have all focused on either the upstream exonic region or the intronic region at the U1 snRNP binding site less than 10 nucleotides from the splice junction. Sequences further downstream in the intron have been found to be less conserved (47
). Our results demonstrate that a nonconserved intronic region outside the U1 snRNP binding site can also influence U1 snRNP binding via formation of a secondary structure(s) that masks the U1 snRNP binding site. The formation of secondary structures that potentially sequester 5′ splice sites has been proposed as a mechanism to regulate alternative 5′ splice site selection (12
). However, there has been little direct biochemical evidence demonstrating differential U1 snRNP binding in the pre-mRNA with proposed secondary structures (4
). In yeast cells, a systematic analysis to examine the effects of secondary structures on U1 snRNP binding using artificial hairpins to sequester the 5′ splice site of the yeast RP51A intron has been carried out (22
). Pre-mRNAs containing hairpin structures with longer than 9 consecutive base pairs began to show a reduction in splicing in vivo, whereas structures with up to 6 consecutive base pairs had little effect on splicing efficiency (22
). Our study demonstrates that even a single-nucleotide change at position +14 or +16, which potentially disrupts one base-pairing in a 6-bp stem involving the 5′ splice site, leads to a significant increase in splicing of tau exon 10 both in vitro and in vivo. In addition, these mutations are located in a nonconserved region of the intron. This is significant and prompts revised strategies for identifying potentially important genes for human diseases, since many have focused only on evolutionarily conserved regions.
It should be pointed out that this secondary-structure model is not inconsistent with the potential involvement of factors other than U1 snRNP. It is possible that the intronic mutations also disrupt the interaction of certain splicing-repressing factors (either protein or RNA). The potential involvement of splicing factors other than U1 snRNP in differentiating wild-type and intronic mutant tau pre-mRNAs was suggested by several observations. In human brain tissues and in cells transfected with tau minigenes, the increase in exon 10 splicing in both AusI+16 and DDPAC+14 mutant tau pre-mRNAs, compared with wild-type tau, appeared more dramatic than the difference observed in the RNase H cleavage assay in the absence of nuclear extract. In this study (Fig. ) and in a previous study (30
), the ratio of Tau4R to Tau3R was increased from 1 in wild-type tau to 3 or higher in AusI+16 tau and 4 or higher in DDPAC+14 tau. SR proteins have been shown to bind directly to 5′ splice sites (69
) and recruit and/or stabilize the binding of the U1 snRNP to pre-mRNA (11
). In our previous studies, we have demonstrated interaction between SR proteins and U1 70K, a U1 snRNP protein (67
), and distinct functional activities of SR proteins in alternative 5′ splice site selection (68
) as well as in alternative exon inclusion (33
). Recently, a yeast U1 snRNP protein, Nam8p, was shown to interact with nonconserved intronic sequences and affect 5′ splice site selection (51
). U5 PRP8 yeast and human homologs have been shown to interact with 5′ and 3′ splice sites (53
), although their role in regulating alternative splicing is not yet clear. It is possible that some of these proteins, or other novel or known alternative splicing regulators, also play a role in tau alternative splicing.
Abnormal pre-mRNA splicing has been implicated in the pathogenesis of a large number of human diseases, including neurodegenerative disorders such as amyotrophic lateral sclerosis (42
). Almost all splicing mutations reported in human diseases either weaken recognition by spliceosomal snRNPs or cause activation of cryptic splice sites, leading to exon skipping, intron retention, or usage of cryptic splice sites (28
). The FTDP-17-associated intronic mutations analyzed in this study represent the first case in which single-nucleotide mutations cause increased rather than decreased splicing of an alternatively spliced exon, thereby altering the balance between different isoforms of normal gene products and leading to neurodegeneration. Our study provides strong evidence that enhanced U1 snRNP binding to a normal alternative splice site, as a result of single-nucleotide mutations in the nonconserved intronic region outside of the U1 snRNP binding site, can be a pathogenic mechanism. Such aberrant splicing can cause alteration in the delicate balance of different alternative splicing products. Considering the size of introns compared with exons and the complexity of alternative splicing regulation in mammalian genes, it is likely that simple alterations in the balance of different isoforms of critical genes as a result of aberrant splicing could be a more important mechanism for pathogenesis of human diseases than previously appreciated.