3.1. HnRNPE3 moderately activates splicing of exon 10
Chromosome 21 contains four known or putative splicing factors: U2AF35, RBM11, SRA4 (SFRS15, KIAA 1172) and hnRNPE3 (poly-(rC) binding protein 3, PCBP3). All but RBM11 are located within the Down syndrome critical region between 21q22.1 to 21q22.3 (Gardiner et al., 2002
). RBM11 was already in a eukaryotic expression vector (pCMV-SPORT6), and we placed the other three into N-terminal FLAG vectors. Our Westerns show that all three express in cells ( shows FLAG-hnRNPE3; data are not shown for U2AF35 and SRA4).
Co-transfection of the factors with tau exon 10 showed that U2AF35 and RBM11 do not affect exon 10 splicing, but hnRNPE3 and SRA4 moderately increase exon 10 inclusion (). Previous work from our laboratory had shown that exon 10 splicing is also activated by hnRNPE2, a close relative of hnRNPE3 (Broderick et al., 2004
). Given these results, we decided to concentrate on hnRNPE3.
When hnRNP proteins influence splicing by direct binding to the pre-mRNA, they invariably bind to intronic elements (Martinez-Contreras et al., 2007
). Additionally, hnRNPE proteins bind poly-(rC) sequences, hence their alternative appellation (PCBP; Makayev and Liebhaber, 2002
). There is one C triplet just downstream of exon 10, at positions +19 to +21 (-, shaded). To find out which region hnRNPE3 interacts with, we did co-transfections and RNA-protein pulldowns of deletion and point mutants of the downstream intron of exon 10 with hnRNPE3 (, ). We did not do co-tranfections of hnRNPE3 with point mutants M12, M13, M14 and M16, because they produce almost exclusively 10+
(Gao et al., 2007
; Wang et al., 2003
) and thus would not show additional activation by hnRNPE3.
3.2. HnRNPE3 activates splicing of exon 10 by binding to the C triplet in its downstream proximal intron
As we previously described (Gao et al., 2007
; Wang et al., 2003
) and also show here (, , odd-numbered lanes), the splicing behavior of our mutants defines region 11-18 as an intronic splicing silencer (ISS) and region 19-26 as an intronic splicing enhancer (ISE), in agreement with FTDP pedigrees and results from other laboratories (Andreadis, 2006
; Liu and Gong, 2008
The co-transfections show that hnRNPE3 can no longer activate splicing of exon 10 in mutant Δ19/26, which lacks the C triplet (, lane 7 versus 8). HnRNPE3 is also less effective in activating mutant Δ23/29 (, lane 9 versus 10). Additionally, hnRNPE3 is unable to activate splicing of mutant M19, in which the first C of the triplet has become a G (, lane 5 versus 6). The M19 mutation completely abolishes inclusion of exon 10 in human FTDP pedigrees and transfection assays (Broderick et al., 2004
; Stanford et al., 2003
The pulldowns of the equivalent constructs with FLAG-hnRNPE3 show that hnRNPE3 interacts directly with exon 10 (, lane 1 versus 2). HnRNPE3 binds to the intronic construct, E13+30, nearly as strongly as it binds to E10+30, and much more strongly than it binds to the exonic one, E80 (, lanes 2 to 4).
In the pulldowns of the deletions, hnRNPE3 binds as strongly to deletion Δ11/18 as it does to E13+30 (, lane 6). It binds weakly to deletion Δ19/26 that lacks the C triplet (, lane 7) but also binds weakly to deletions Δ3/10 and Δ23/29 that contain it (, lanes 5 and 8). In the pulldowns of the point mutations, hnRNPE3 binds to M13 as strongly as it does to E13+30 (, lane 5). It binds very weakly to M19 which no longer has a C triplet (, lane 8), but binds equally weakly to M11, 12, 14 and 16 (, lanes 3, 4, 6 and 7).
These results indicate that hnRNPE3 regulates splicing of exon 10 by binding to the C triplet, but that its binding is influenced by the details and configuration of the local sequence.
3.3. The N-terminal KH domain of hnRNPE3 is essential for activation of exon 10 splicing
To establish which domains of hnRNPE3 are required for regulation of exon 10, we created two hnRNPE3 deletion variants as FLAG fusions: hnRNPE3N and hnRNPE3C contain the first and last two KH domains of hnRNPE3, respectively (). In co-transfections, hnRNPE3N is as effective as full-length hnRNPE3 in increasing exon 10 splicing (, lane 3), whereas hnRNPE3C has essentially no effect (, lane 4). The results strongly suggest that the N-terminal KH domain of hnRNPE3 is required for activation of exon 10 splicing.
3.4. HnRNPE3 interacts with hnRNPE2, another modest activator of exon 10 splicing and the two activators act additively
We tested the interaction of hnRNPE3 with SRA4 and with factors known (9G8, hnRNPE2) or suspected (SRp75, Nova1) to bind to the proximal downstream intron of exon 10. Of these, SRA4 and hnRNPE2, a member of the hnNRPE family, also modestly activate splicing of exon 10 (Broderick et al., 2004
; ), whereas the other three factors inhibit splicing of exon 10 strongly (9G8) or moderately (SRp75, Nova1; Gao et al., 2007
; Wang et al., 2003
The co-IPs show that hnRNPE3 does not interact with 9G8, Nova1 or SRp75 (, lanes 2-4). However, it co-precipitates with hnRNPE2 (, lane 5). HnRNPE proteins are known to interact (Kim et al., 2000
). Our result suggests that the two proteins could potentially act as a heterodimer. HnRNPE3 also interacts weakly with SRA4 (, lane 6).
To test whether the three factors that seem to interact by co-IP also act additively as exon 10 splicing activators, we co-transfected SP/10L with hnRNPE3, SRA4 and hnRNPE2 combinations (). The results indicate that the three factors act additively (, lanes 4-6). The three together (, lane 6) are as active as hnRNPE3+hnRNPE2 (, lane 5) and slightly less active than hnRNPE3+SRA4 (, lane 4). This may reflect either a problem with co-expressing so many plasmids or steric interference between the three factors.
The co-IP and co-transfection results suggest the possibility that the three activators might act as a heterotrimer, though definitive proof will require additional interaction experiments.
3.5. HnRPNE3 shRNA reverses the activation of exon 10 splicing
To establish that hnRNPE3 is a native activator of tau exon 10 splicing, we tested three siRNA and three shRNA constructs against it (E3i-1, -2 and -3 and SME3-1, -2 and -3; shows the sequences and their location within hnRNPE3). We chose HeLa because it expresses hnRNPE3 and tau and its tau is 40% 10+. Neuroblastoma cells are useless for testing RNAi against splicing activators, because their tau almost entirely lacks exon 10.
Three of the siRNAs and two of the shRNAs showed no influence on endogenous hnRNPE3 in HeLa cells. The exception was SME3-1, which decreased the expression level of endogenous hnRNPE3 significantly (, lane 3 versus 4). SME3-1 had an extremely slight effect on endogenous tau exon 10. In contrast, it strongly decreased inclusion of exon 10 in SP/10L co-transfected in a 1:1 ratio with the SME3-1 plasmid (, lane 1 versus 2).
Fig. 6 Suppression of hnRNPE3 by shRNA decreases inclusion of exon 10. 1 ug of SP/10L was transfected into HeLa cells together with 1 ug of shRNA construct SME3-1 or control vector SM2c-GFP. (A) A representative result of the effect of SME3-1 on transfected (more ...)
Tau mRNA is known to have a long half-life (Aronov et al., 1999
). This, plus the fact that hnRNPE3 is a modest activator, may explain the inability of SME3-1 to affect endogenous tau. Nevertheless, decrease of endogenous hnRNPE3 results in decrease of inclusion of exon 10 in a transfected construct which undergoes active transcription concommitant with the shRNA. The result strengthens the conclusion that hnRNPE3 is an endogenous activator of tau exon 10 splicing, though it falls just short of providing definitive proof on its own.
3.6. HnRNPE3 is preferentially expressed in neuronal tissues
Given the effect of hnRNPE3 on exon 10 splicing, we wanted to determine its expression profile. Quantitative PCR with competing 18S primers shows that hnRNPE3 is exclusively expressed in neuronal tissues and is also adult-specific (). It is entirely absent in skeletal muscle, heart and liver (, lanes 6-8) and barely present in fetal brain (, lane 1). However, it is present in all the compartments of adult brain. Its relative expression levels are cortex>>cerebellum≈spinal cord>hippocampus (, lanes 2, 3, 5 and 4, respectively).
Fig. 7 HnRNPE3 is restricted to the central nervous system and is adult-specific. (A) Quantitative RT-PCR reactions were done on polyA+ RNA. Primer pair: E3-KH2NewS/E3-KH2NewN. 18S:competimer ratio was 2:8. (B) Expression of hnRNPE2 relative to 18S. (C) Expression (more ...)