In this study, we report characterization of an exonic splicing enhancer located in exon 10 of the human tau gene with AATAAGAAG as the wild-type sequence. This AG-rich region contains the sequence in which mutations associated with FTDP-17 have been identified. Specifically, two mutations have been found inside this enhancer element, N279K (AAGAAGAAG) and Del280K (AATAAG). We made an additional mutant, dEn, lacking the entire AG-rich element. By systematically comparing the splicing efficiency, protein-RNA interaction profiles, and the splicing complex formation between the wild type and mutant transcripts, we conclude that these mutations alter the activity of this splicing enhancer by increasing (for N279K) or decreasing (for Del280K) interaction with protein factors interacting with this AG-rich element. Among the trans-acting factors interacting with this AG-rich element, we have identified human Tra2β protein. N279K mutation stimulates, whereas Del280K or dEn mutations reduce, the binding of Tra2β to the AG-rich exonic enhancer. The interaction of Tra2β protein with this exonic element correlates well with the copy number of AAG repeats inside the exonic enhancer. Furthermore, down-regulation of endogenous Tra2β expression using RNA interference led to a reduction in tau exon 10 splicing. Our study suggests that Tra2β or Tra2β-like protein(s) may play an important role in affecting the delicate balance of tau isoforms and in pathogenesis of tauopathies involving aberrant tau exon 10 alternative splicing. Further investigation is necessary to determine the spectrum and specificity of individual splicing activators such as Tra2β. Nonetheless, the down-regulation of endogenous Tra2β reduced the tau exon 10 inclusion in a relatively specific manner, and such a treatment partially reverses the exon 10 splicing effect caused by N279K mutation. Our results suggest a new direction for designing therapy for certain FTDP patients (such as those carrying the N279K mutation) based on down-regulation of certain specific splicing activators for tau exon 10 splicing.
The importance of tau exon 10 alternative splicing has been demonstrated by a number of genetic studies that link the mutations affecting exon 10 splicing in the human tau gene to the development of FTDP-17 (15
). These mutations are located either in the intron downstream of exon 10 or inside exon 10. Our previous study has demonstrated that some intronic mutations act at the level of U1 snRNP binding to the 5′ splice site of exon 10 during the earliest step of spliceosome assembly (17
). Exonic mutations inside exon 10 have been studied by several groups (15
). Some of the exonic mutations have been shown to affect the microtubule binding properties of Tau protein(s) (14
), whereas other exon 10 mutations alter the Tau4R/Tau3R ratio presumably by influencing exon 10 splicing (26
). However, the molecular mechanisms underlying the splicing mutations inside exon 10 were not understood.
Exonic splicing enhancers (ESEs) are sequence elements within exons that promote pre-mRNA splicing. A number of ESEs have been identified in tissue-specific or developmentally regulated exons, which typically have weak splice sites and require the ESE for exon inclusion. As shown by our study, the upstream 3′ splice site of exon 10 is weak. Utilization of this suboptimal 3′ splice site was only detectable in the in vitro splicing assay when additional U2AF65 protein was provided in the splicing reactions. The presence of an ESE in exon 10, together with a suboptimal 3′ splice site, provides a fine-tuning mechanism for the regulation of exon 10 inclusion in certain tissues or at certain developmental stages. It is conceivable that during fetal stages of human development, tau exon 10 inclusion is repressed because of either insufficient levels of splicing activator(s) or the presence of trans-acting factors that counteract the activity of embryonically expressed splicing activators. It is equally possible that the fetal pattern of tau exon 10 splicing (almost exclusively exon 10 skipping) is a result of the combination of the above mentioned mechanisms. We are actively investigating these possibilities using both molecular and biochemical approaches.
The sequence motif for ESEs can be divergent, although purine-rich motifs represent one of the best characterized elements. Often ESEs function by interacting with SR domain-containing splicing regulators (reviewed in Refs. 53
). ESEs were initially identified by their ability to activate upstream 3′ splice sites (58
). Recent studies also identified splicing enhancers that stimulate downstream 5′ splice site utilization (61
). There have also been reports of bidirectional enhancer elements (62
), and these elements contain multiple domains with distinct regions functioning to facilitate splicing of either upstream or downstream exons. In our study, we have used biochemical approaches to identify a simple AG-rich element that acts bidirectionally to promote exon 10 splicing with both exon 9 and exon 11. We show that one of the important trans-acting factors involved in the function of this exonic splicing enhancer is human Tra2β
protein that acts as an alternative splicing activator. Using a transient transfection assay, we were able to express Tra2β
in a functionally active form at a level comparable with that detected in the rat brain tissue by Western blotting (data not shown). Similar to previous studies, our results show that Tra2β
by itself is not sufficient to complement S100 cytoplasmic extracts for the splicing activity in the manner that certain essential splicing factors of the SR family do (46
). It is likely that Tra2β
protein acts in concert with certain basic essential splicing factors to activate tau exon 10 splicing. Furthermore, Tra2β
protein interacts with tau pre-mRNA by binding to the AG-rich exonic splicing enhancer, as shown by site-specific UV cross-linking experiments. The addition of the transiently expressed Tra2β
protein into the in vitro
splicing reaction further stimulates exon 10 splicing, and such stimulatory effect is more pronounced in the presence of the N279K mutation. Thus, Tra2β
acts as a splicing activator in tau exon 10 splicing. The bidirectional activity of tau gene exonic splicing enhancers and involvement of alternative splicing regulators, in addition to essential splicing factors, make the similarity more obvious between splicing enhancers and transcription enhancers.
SR domain-containing splicing factors play important roles in mammalian pre-mRNA splicing and alternative splicing regulation (for recent reviews, see Refs. 54
, and 65
). In addition to interacting with pre-mRNAs via their RNA recognition motifs, these splicing factors have been proposed to mediate interactions both across introns and across exons (59
). More recently, SR proteins have been reported to support basal AT-AC splicing and to stimulate exonic splicing enhancer important for AT-AC intron splicing (66
is an SR domain-containing protein initially identified as one of the human homologues of Drosophila
SR protein transformer 2 (Tra2) (67
). Systematic biochemical experiments demonstrated that human Tra2β
is a splicing activator interacting with exonic enhancer sequences rich in A and G or GAA repeats rather than a constitutive splicing factor (46
has been reported to activate the splicing of exon 7 of survival of motor neuron genes when overexpressed by transfection (68
). The mechanism by which Tra2β
activates splicing was not clear. Our UV cross-linking and RNase H cleavage/U1 snRNP protection experiments show that increased exon 10 splicing in the tau N279K mutant is associated with increased Tra2β
binding to the exonic splicing enhancer and that the addition of Tra2β
promotes the formation of U1 snRNP-dependent complex at the 5′ splice site of exon 10. Protein-protein interaction between Tra2β
and a U1 snRNP protein U1 70K has been observed,2
and the functional significance of such interaction in promoting U1 binding will be further investigated. These results suggest that one mechanism by which Tra2β
acts to stimulate exon 10 splicing may be by facilitating U1 snRNP binding to the 5′ splice site of exon 10.
A previous study reported that overexpression of human Tra2β
in a chimeric tau minigene system had no effect on human tau gene alternative splicing (69
). It is possible that in this previous study, the failure to detect the effect of Tra2β
was a result of using the artificial chimeric tau minigene rather than the native tau minigene (69
). Alternatively, the presence of endogenous Tra2β
was high enough to mask the effect of overexpression. Our systematic biochemical analyses of the interaction between Tra2β
and the AG-rich splicing enhancer in exon 10 and results from Tra2β
down-regulation experiments strongly suggest an important role of Tra2β
in tau exon 10 alternative splicing regulation. A definitive in vivo
role of Tra2β
in this alternative splicing event awaits future exploration using strategies such as targeted gene deletion.