2.2. Cell Culture and Transfections

We cultured monkey kidney (COS) and human epithelioma (HeLa) as previously described (Wang et al., 2010). We prepared plasmid DNA by Qiagen Tip-100s or Promega PureYield midi columns. We grew the cells on 60 mm or 100 mm plates (for RT-PCR and co-IPs, respectively) and transfected them when they reached confluence of 50% (if using LT1 from Mirus) or 80% (if using Lipofectamine 2000 from Invitrogen).

For siRNA work, we transfected the siRNA first, followed ~12 h later by the tau DNA, because the optimal ratios of RNA and DNA differ greatly vis-à-vis lipofectamine. We changed the medium 16 hours after transfection and harvested the cells 48 hours after transfection.

2.3. RNA Preparation Reverse Transcription and PCR

We isolated total RNA from transfected cells by the TRIzol method (Invitrogen). We performed reverse transcription and PCR as previously described (Wang et al., 2010). To analyze the endogenous SRp75 levels, we used the Ambion Quantum kit with a ratio of 3:7 18S primers to 18S competimers as the internal control. For the RNAi experiments, vectors pFIV and SM2c-GFP or RNA sc-37007 (Santa Cruz Biotechnology) acted as the negative controls.

For PCR, we used the following primer pairs (shown in Table 1): SPL-LS/SPL-LN for transfected tau, HT7S3/HT11N for endogenous tau, 75-PS/75RRM-1N for endogenous SRp75. We did 27 PCR cycles if analyzing transfected constructs (denaturation 94 °C/1 min, annealing 62 °C/1 min, extension 72 °C/1 min); 30 cycles if analyzing endogenous SRp75 in cells (denaturation 94 °C/1 min, annealing 58 °C/1 min, extension 72 °C/1 min); and 21 cycles if analyzing endogenous SRp75 in polyA⁺ RNA (fetal brain, adult whole brain, cerebellum, hippocampus, spinal cord, skeletal muscle, heart, and liver; Clontech).

We calculated RNA ratios by scanning and averaging the bands from three independent transfections using the ImageJ program. The exception was endogenous SRp75 in polyA⁺ RNA, which we performed only once.

2.4. Protein Expression in Cells and co-IP Assays

We verified expression of the FLAG, Myc and GFP factor constructs by using antibodies against FLAG (mouse M2, Sigma), GFP (mouse, Invitrogen) or myc (rabbit, GeneScript) on Western blots of protein lysates from cells transfected with the constructs (Fig. 4B, 5A–D).

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Fig. 5

SRp75 interacts with hnRNPE2 and hnRNPG, the former a moderate activator and the latter a strong inhibitor of exon 10 splicing and SRp75 and hnRNPG may act synergistically. (AD) Western blots of protein lysate inputs and co-IPs from 1:1 co-transfections of myc-SRp75 and FLAG- or EGFP-factors in COS cells. Protein marker sizes are indicated on the right of each panel. The positions of factors are indicated on the left. (A) Input and (C) co-IP, factors detected by anti-FLAG mouse antibody M2. (B) Input SRp75 detected by anti-myc rabbit antibody. (D) Input (left) and co-IP (right), hnRNPG detected by anti-GFP mouse antibody. (E) RT-PCR of SP/10L in COS cells co-transfected with various combinations of FLAG-SRp75, FLAG-9G8 and EGFP-hnRNPG constructs. Exon ratio calculations, primers, graph conventions and p-value notations are as in Fig. 1C.

We prepared cell lysates from transfected cells using RIPA lysis buffer (50 mM Tris pH 8.0, 150 mM NaCl, 1% Nonidet P-40 [NP-40], 0.5% deoxycholate, 0.1% SDS) containing 1x Protease Inhibitor Cocktail Tablets (Roche Molecular Biochemicals). Lysates were nutated at 4 °C for 1 h, cleared by centrifugation at 13,200 rpm for 15 min at 4 °C and boiled for 10 min prior to running on SDS-PAGE gels.

For protein co-IPs, we prepared protein lysates from cells co-transfected with Myc-SRp75 plus EGFP-hnRNPG or FLAG-9G8, -Nova1 and -hnRNPE2. EGFP-C2 and FLAG-Prdx6 (peroxiredoxin 6) acted as the respective negative controls. After the centrifugation clearing step, we added rabbit anti-myc antibody agarose beads (Sigma) to the supernatants, incubated them with nutation at 4 °C overnight and rinsed the beads 4x with wash buffer (50 mM Tris HCl, pH 7.4, 150 mM NaCl).

We analyzed the complexes by Western blotting using rabbit anti-myc (input) and mouse anti-FLAG (input and IP) antibodies (Fig. 5A-D) as detailed in Wang et al. (2010). For detection we used Opti-4CN (Roche). We used primary and secondary antibodies at 1:5,000 dilution.

3.2. Both the RRM and RS domains of SRp75 are required to inhibit exon 10 splicing

To establish which domains of SRp75 are required for regulation of exon 10, we created three SRp75 single-domain constructs as FLAG fusions: Two contain the RNA recognition motif (RRM) domains, and one the serine/arginine-rich (RS) domain (Fig. 4A, 4B). None of them inhibits splicing of exon 10 (Fig. 4C). Also, the RS variant of SRp75 does not boost inclusion of exon 10 (Fig. 4C, lane 5), as is the case with equivalent constructs of other splicing factors (Wang et al., 2004, 2005).

This strongly implies that SR75 exerts its influence through direct binding to the exon 10 RNA but that it also requires interactions with additional factors to discharge its function.

3.3. SRp75 interacts with hnRNPG, another inhibitor of exon 10 splicing, and the two inhibitors appear to act synergistically

Since the behavior of the SRp75 variants indicates that it needs partners, we tested the ability of SRp75 to interact with factors known (9G8, hnRNPG, hnRNPE2, hnRNPE3) or suspected (Nova1) to influence exon 10 splicing via its proximal downstream intron (Fig. 5A–D). Of these, hnRNPE2 and hnRNPE3, members of the hnNRP family, modestly activate splicing of exon 10 (Broderick et al., 2004; Wang et al., 2010), whereas the other three factors inhibit splicing of exon 10 strongly (9G8, hnRNPG) or moderately (Nova1; Gao et al., 2007; Wang et al., 2004).

We tested interactions of SRp75 with other splicing factors with co-IPs. These show that SR75 does not interact with 9G8 (Fig. 5C, lane 2) or hnRNPE3 (Wang et al., 2010). However, it co-precipitates strongly with hnRNPG (Fig. 5D, lane 4) and hnRNPE2 (Fig. 5C, lane 4) and very weakly with Nova1 (Fig. 5C, lane 3).

To test whether the inhibitors that seem to interact by co-IP also enhance each other’s inhibition of exon 10 splicing, we co-transfected SP/10L with FLAG-SRp75, FLAG-9G8 and EGFP-hnRNPG combinations (Fig. 5E). These are triple transfections, so the inhibition effect decreases compared to the double transfections of the other experiments. As a result, addition of FLAG-SRp75 or FLAG-9G8 plus EGFP just misses attaining statistical significance (Fig. 5E, lanes 2 and 3). Addition of FLAG-SRp75 plus FLAG-9G8 shows no inhibition (Fig. 5E, lane 5), suggesting that the two factors may compete for an RNA region – a possibility, given that they apparently interact with residues 12/13 and 14, respectively. However, addition of FLAG-SRp75 plus EGFP-hnRNPG attains high statistical significance (Fig. 5E, lane 6), suggesting that the two factors may act synergistically, a finding congruent with the co-IP results.

The co-IP and co-transfection results suggest the possibility that SRp75 and hnRNPG may act in concert to inhibit exon 10 splicing and also that hnRNPE2 may antagonize the action of SRp75 by either titration or steric interference.

3.4. SRp75 knockdown reverses the inhibition of exon 10 splicing

To investigate if SRp75 is a native inhibitor of tau exon 10 splicing, we tested three siRNA plasmids, three shRNA plasmids and a mixture of siRNAs against it (75i-1, -2 and -3; SM75-1, -2 and -3; and si75, sc-38344 from Santa Cruz; Table 1). We chose HeLa because it expresses SRp75 as well as tau mRNA with 40% exon 10 inclusion.

We had great difficulty suppressing SRp75 in HeLa cells, perhaps because it is abundantly expressed in them (Zahler et al., 1993). Neither the siRNA nor the shRNA plasmids decreased endogenous SRp75 reproducibly in HeLa cells. Si75, a mixture of three SRp75 siRNAs, has little effect on endogenous exon 10; however, it decreases the expression levels of endogenous SRp75 and concommitantly increases inclusion of exon 10 in co-transfected SP/10L, although both changes just miss attaining statistical significance, possibly because the effect is variable (Fig. 6A, 6B, lanes 1 versus 2 and 3 versus 4).

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Fig. 6

Suppression of SRp75 slightly increases inclusion of exon 10, although the changes in both SRp75 and exon 10 just miss attaining statistical significance. 1 ug of SP/10L was transfected into HeLa cells following transfection of control or Si75 RNA (Santa Cruz). (A) A representative result of the effect of si75 on transfected tau (left) and endogenous SRp75 (right). DNA marker sizes are indicated between the panels. The exon 10, 18S and SRp75 PCR products are indicated on the left and right of the panels, respectively. (B, left panel) Quantitative RT-PCR of tau exon 10. (B, right panel) Quantitative RT-PCR of endogenous SRp75. The ratio of SRp75 to 18S is indicated. Primer pair: 75-PS/75RRRM-1N. The 18S:competimer ratio was 3:7. Exon ratio calculations, primers, graph conventions and p-value notations are as in Fig. 1C.

The result prompts the conclusion that SRp75 is an endogenous activator of tau exon 10 splicing, though it falls short of providing definitive proof.

4.2. SRp75 requires hnRNPG to exert its full regulatory effect on tau exon 10 and its action may be antagonized by hnRNPE2 and its interacting factors

The SR protein 9G8, which inhibits splicing of exon 10 more strongly than SRp75, binds to the same region as SRp75 and requires a wild-type residue at position 14 (Gao et al., 2007; Wang et al., 2004). However, SRp75 and 9G8 do not interact (Fig. 5C) nor act additively to enhance suppression of exon 10 (Fig. 5E). Instead, both inhibitors interact with hnRNPE2, a modest activator of exon 10 splicing (Broderick et al., 2004), as evidenced by co-IPs for SRp75 (Fig. 5C) and yeast two-hybrid assays for 9G8 (Funke et al., 1996).

These interactions imply that hnRNPE2 can antagonize the action of both SRp75 and 9G8. Our previous work indicates that it may do so by interacting with hnRNPE3, another modest activator of exon 10 splicing which binds to the C triplet in position 19–21 and also interacts with SR factor SRA4 (Fig. 8; Wang et al., 2010). The hnRNPE2/hnRNPE3 complex may prevent binding of SRp75 and 9G8 either sterically (by “covering” a region of the exon 10 RNA which would otherwise be available to either inhibitor) or kinetically (by titrating out the inhibitors).

If 9G8 and SRp75 act independently, their concentrations within a cell may determine which one becomes the crucial factor for exon 10 exclusion. This scenario may also explain the binding of SRp75 to deletion I10-Δ11/18 in vitro (Fig. 2). This deletion brings a CA motif, originally at position 21–22, to position 13–14 of the intron. The newly proximal CA motif may be sufficient to bind SRp75 (but not to suppress exon 10 splicing, which requires SRp75 cofactors), whereas 9G8 can no longer bind to this deletion (Gao et al., 2007).

Whereas 9G8 seems not to need partners to fully inhibit tau exon 10 splicing, SRp75 may need hnRNPG or other co-factors to exert its full effect. Co-IPs and co-transfections show that SRp75 and hnRNPG interact (Fig. 5D) and may act synergistically (Fig. 5E). Previous work (Wang et al., 2004) has shown that, unlike SRp75, the N-terminal region of hnRNPG (which contains both its RRM and a recently identified novel RNA binding domain; Heinrich et al., 2009) is not required to inhibit splicing of exon 10, suggesting it acts via protein interactions.

HnRNPG also interacts with htra2-beta1, antagonizing the action of the latter (Heinrich et al., 2009; Hoffman and Wirth, 2002; Nasim et al., 2003). Htra2-beta1 modestly activates exon 10 splicing by interacting with a purine-rich enhancer within the exon (defined by FTDP-17 mutations Δ280K and N279K). However, the inhibitory effect of hnRNPG persists when the purine-rich region is destroyed (Wang et al., 2005). The interaction with SRp75 pinpoint the additional entry point of the hnRNPG influence on exon 10.

Fig. 8 shows a speculative model that summarizes our cumulative knowledge of the splicing regulation at the proximal downstream intron of tau exon 10. The binding sites of 9G8, SRp75 and hnRNPE3 and the interactions between hnRNPE3, hnRNPE2, 9G8, SRp75 and hnRNPG have been defined (Broderick et al., 2004; Gao et al., 2007; Wang et al., 2004, 2010; this work). Future work will allow us to determine if additional elements and factors play a role in regulating exon 10 splicing via this region.

This work was supported by NIH grants R01 AG18486 to A. A. and R21 HD056195 to A. A. and S. S. We want to thank Dr. Alonso Ross for his generous subsidy of the shRNA clones via the UMMS SiRNA Core.