Effect of MBNL RNA binding mutations on MBNL-regulated splicing events
Based on high resolution structures of the TIS11d[25] and MBNL proteins[12, 13] we designed point mutations in each MBNL zinc finger that would disrupt RNA binding, without severely altering the overall fold and structure of the domain. We targeted conserved aromatic residues F36, Y68, F202 and Y236 in ZF 1–4 respectively, and mutated them to alanine (Figure 1A). The mutations were introduced individually, in combinations in the two di-domains (MBNL-FL-M12 and -M34) and into all four ZF domains simultaneously. Similar mutations have since been reported by others[18, 26]. In order to confirm that the mutations disrupt RNA binding, recombinant MBNL1 aa 2–253 was produced with all four zinc fingers mutated and compared to wildtype protein in UV crosslinking assays. While the wild-type crosslinked to the RNA the mutant did not (Figure 1B, lower panel).
We next tested the effects of the MBNL ZF mutants in assays for splicing repression and splicing activation by MBNL1 in HeLa cells. To test splicing repressor activity we used a Tpm1 minigene with a point mutation of the branch point of exon 3, which increases exon 3 skipping in HeLa cells[22, 27]. This minigene responds modestly to simple overexpression of MBNL1. However, upon knockdown of MBNL1 (Figure 1E) exon skipping is reduced substantially (from 35 to 13%, Figure 1C, lanes 1, 2); complementation with overexpressed MBNL1 restores exon skipping to 53% (Figure 1C, lane 10). As a model MBNL-activated exon we used a minigene construct containing a Vldlr exon flanked by globin exons[10], which responds to MBNL1 overexpression by increasing exon inclusion from 14 to 39% (Figure 1D, lanes 1, 9). Note that in order to facilitate comparison of the repressor and activator activities of MBNL1 mutants, we refer throughout to percentage exon skipping of the repressed Tpm1 exon but percentage exon inclusion of the activated Vldlr exon.
Compared to wild type MBNL1, all of the ZF domain single point-mutants had moderately reduced repressor activity, producing exon skipping levels of 32-44% (Figure 1C, lanes 3–6), as did the combined ZNF 3 and 4 mutant (lane 8). Surprisingly, the mutant with combined mutations in ZF 1 and 2 was as active as wild type MBNL1 (lane 7), despite being expressed at similar levels to the other constructs (Figure 1F). The mutant with all four ZF domains impaired showed no activity (Figure 1C, lanes 2 and 9). However, this mutant was consistently expressed at lower levels than the other constructs (Figure 1F), preventing strong conclusions about its activity. We noted that the MBNL proteins with mutations in ZF1 (MBNL-FL-M1 and MBNL-FL-M12) consistently showed the presence of additional slower migrating bands that were detected with FLAG antibodies (Figure 1F). We do not know the explanation for these additional bands, or whether they represent an active fraction of protein. It is therefore possible that higher total levels of active proteins with the M1 mutation might partially mask loss of activity induced by the mutation.
Mutations in ZF1 or 2 had no significant effect upon the ability of MBNL1 to activate Vldlr splicing (Figure 1D, lanes 2,3,9), while mutations in ZF 3 or 4 individually caused a small but significant increase in activity (lanes 4,5). Double mutations of ZF 3 and 4 or 1 and 2 combined were also without significant effect (lanes 6,7), although the 12 mutant was significantly less active than 34 (P < 0.05). Only the quadruple ZF1-4 mutant showed significantly lower activity than WT MBNL1 (lane 8), but again no firm conclusions could be drawn due to the much lower expression levels of this mutant (Figure 1F).
Taken together, the preceding data indicated that both repressor and activator activities of MBNL1 are remarkably tolerant of mutations that impair RNA binding of individual ZF domains, and even mutations of both ZFs within a didomain have limited effects.
MS2 tethering of MBNL1 activation and repression domains
We next compared the activities of deletion mutants of MBNL1 in simple cotransfection and tethered function assays (Figure 2). Consistent with previous data[22] in the knockdown/complementation assay with the Tpm1 reporter, the N-terminal region of MBNL1 (aa 2–253) had similar repressor activity to the full length protein (Figure 2B, lanes 3,5). In contrast, a C-terminal fragment of MBNL1 (aa 239–382) had no activity (Figure 2B, lane 4). Similar effects were seen with the Vldlr reporter; the N-terminal fragment had indistinguishable activity to full length MBNL1 (Figure 2C, lanes 2,4), while the C-terminal fragment was devoid of activator activity (lane 3), despite being expressed to similar levels (Figure 2A).
As reported previously[22] replacement of the downstream MBNL1 binding element in Tpm1 with a binding site for MS2 coat protein led to a ~3-fold decrease in exon skipping (Figure 2E, lanes 1,2). Addition of MS2 coat protein had little effect (lane 6), while fusion proteins of MS2 with full length MBNL1 or just the N-terminal region led to high levels of exon skipping (lanes 3,4). In contrast, the C-terminal region of MBNL1 fused to MS2 had a significant, but much more modest effect than full length MBNL1-MS2 (lane 5). Replacement of the reported MBNL1 binding site containing two GC motifs downstream of the Vldlr exon with a single MS2 site reduced exon inclusion from 14% to 2% (Figure 2F, lanes 1,2), consistent with the activity of this element as an MBNL-dependent splicing enhancer in mouse embryonic fibroblasts[10]. Co-transfection with MS2 protein had no effect (lane 6), while full length MBNL1-MS2 restored exon inclusion levels (lane 3). As in the repression assay, the N-terminal of MBNL1-MS2 had full activity, while the C-terminal region had partial activity (lanes 4,5). These data indicate that the N-terminal region of MBNL1 has full activity in simple co-transfection and artificial tethering repression and enhancing assays, while the C-terminal region was inactive in simple cotransfections and had partial activity in tethered assays.
In the artificial tethering assay, the MS2 domain serves to recruit the fusion protein to the regulated RNA, presumably bypassing the RNA-binding function of at least some of the ZF domains. To explore this issue we introduced the RNA binding mutations into the ZF domains of the MBNL-N-MS2 construct (Figure 3). Tethering of the WT MBNL-N-MS2 downstream of Tpm1 exon 3 increased exon skipping from 20% (lanes 1,10) to 71% (lane 9). Individual mutations in ZF1-4 or combined mutations in ZF3 and 4 had no effect on activity (lanes 2–5, 7). However, combined mutations in ZF1 and 2 drastically reduced activity (lane 6), even though the protein was expressed (Figure 3B). Indeed, exon skipping levels in the presence of the ZF12 mutant were not significantly different from MS2 alone or no cotransfection (lane 6, compared to lanes 1 or 10). The quadruple mutant in ZF1-4 was also inactive, but again the protein was expressed at very low levels (lane 8 and Figure 3B). The complete loss of activity upon ZF12 mutation in the tethered repressor assay is in stark contrast to the more modest effects in the simple cotransfection assay (Figures 1C and3A). In the tethered activation assay the single mutations in ZF1, 3 and 4, and the combined mutation of ZF3 and 4 led to a modest but significant increase in activity while the ZF2 mutation was without effect (Figure 3C, lanes 2–5,7 compared to 9). Only the dual ZF12 mutant showed decreased activity (lane 6) but the effect was modest compared to the loss of repressor activity.
MBNL1 is thought to dimerize through its C terminus[16, 28]. However, the crystal structure of ZF34 revealed a dimerization contact involving the RNA binding face of ZF4 in one subunit, with the reverse face of ZF4 in the other subunit[13]. We tested the effects of individual or combined mutations in Tyrosine 224 (Y224S) and Glutamine 244 (Q244N), which are predicted to impair the potential dimerization contact, but not RNA binding (Figure 4A). These mutations had no effect upon the tethered repressor (4C) or activator (Figure 4D) activities of MBNL-N-MS2, or on the direct activation of Vldlr by full length MBNL1 (Figure 4B). These results suggest that the observed crystal contact between MBNL1 subunits is not important for MBNL1 function.
MBNL1 binding to RNA species from MBNL-regulated exons
Having investigated the role of the MBNL1 ZF domains in splicing repression and activation, we next tested the binding of MBNL1-N to RNAs containing the MBNL binding elements of Vldlr and Tpm1 by electrophoretic mobility shift assay (Figure 5). We compared binding of WT MBNL1-N with the mutants in ZF12 (M12) and ZF34 (M34). WT MBNL1 bound to the Vldlr and upstream Tpm1 elements, Tpm1 URE, with Kd in the 0.5 – 1 nM range, while binding to the downstream Tpm1 element Tpm1 Dugc, was approximately 10-fold lower affinity (Figure 5A, Kd 25–50 nM). With the Vldlr RNA a second binding event was also observed with a much lower affinity; we observed no additional binding events to either of the Tpm1 elements, even though their length is sufficient to accommodate multiple binding sites[15]. Mutation of ZF34 reduced the affinity of binding to all three RNAs by about ~20-fold (Figure 5B). In contrast, the effects of mutations in ZF12 were far more drastic; no stable complexes were observed on any of the RNAs, even when up to 2 μM MBNL protein was used (Figure 5C). Thus, the N-terminal ZF12 domains are more important for both binding to Tpm1 and Vldlr RNAs, as well as for tethered activity.
MS2 tethering of MBNL1 truncations
To analyse further the roles of the pairs of tandem zinc fingers we tested a series of deletion mutations based on MBNL-N-MS2. These included a C-terminal deletion series (previously tested on Tpm1[22]), a natural deletion variant lacking the C-terminal half of the linker (Δ116-183), an N-terminal deletion series, and the linker alone. The linker sequence is predicted to be unstructured, but parts of it are highly conserved and have been shown previously to have a role in MBNL activities[17, 18, 22]. We expressed these proteins as MS2-fusions (Figure 6B) and analysed their activity when recruited to either the downstream Tpm1 (repressed, Figure 6C) or Vldlr (activated, Figure 6D) sites.
When recruited downstream of the MBNL-repressed Tpm1 exon 3 (the Tpm1-ΔbpDMS2 minigene) zinc fingers 3 and 4 and the C-terminal part of the linker could be removed individually or in combination with no effect (Figure 6C, lanes 4–6). C-terminal truncations beyond amino acid 116 led to diminished activity (lanes 6–9). Complete removal of the linker sequence and an alpha-helix of zinc finger 2 leaving only the first two zinc fingers results in an inactive protein (lane 9). Despite the importance of the linker region, when recruited alone it had no activity above MS2 alone (lane 10, 17). Zinc fingers 3 and 4 along with the complete preceding linker region, or with just the C-terminal part of the linker, were partially active (lanes 11,12). However, this activity required the linker sequence as ZF34 alone were inactive (lane 13). These data show that the N-terminal part of the protein comprising amino acids 2–102, encompassing the first two zinc fingers plus a third of the linker sequence, constitutes a minimal repressor domain. Introduction of RNA binding mutations into ZF1 or 2 individually drastically reduced activity of the 2–116 repressor domain, while combined mutation of ZF 1 and 2 abolished activity (compare lanes 14–16 with lane 6).
A similar, but not identical, response to the mutations was seen in the Vldlr context (Figure 6D). In this case, more of the linker sequence was required for full activity with progressively diminishing activity upon C-terminal deletion into the linker (Figure 6D, lanes 5–9, Figure 6E). Deletion of the second pair of zinc fingers (lane 5) or internal deletion of the C-terminal part of the linker (lane 4) had no effect on activity. As for repressor activity, the linker sequence alone was inactive (lane 10), but in combination with the second pair of zinc fingers the fusion protein retained substantial activity (lane 11), albeit less than the first pair (lane 5). This activity was reduced further by removal of the N-terminal part of the linker (lane 12) and abolished when only ZF34 remained (lane 13). Thus splicing activation is more dependent than repression upon the full linker sequence (Figure 6E). Although the activity of the 2-116 construct was already diminished, we tested the importance of RNA binding. Abrogating the RNA binding capacity of the 116 construct led to a severe, albeit not total reduction in activity (Figure 6D, lanes 14–16 compared to 6).
We analysed RNA binding of some of the C-terminal deletion fragments (Figure 5D-F). The miminal repressor domain MBNL1-2-116 bound to the Vldlr and Tpm1 RNAs with affinity reduced compared to the complete N-terminus but actually higher than the N-terminus with point mutations in ZF34 (Figure 5D compared to 5A,B). In addition, the 2–116 protein showed additional subsequent binding events on all three RNAs, consistent with the fact that it has only 2 ZFs that can contact the RNA. The 2–91 protein, which showed almost complete loss of tethered activation activity (Figure 5D lanes 6–8) bound to each RNA with affinity similar to 2–116 (Figure 5E), emphasizing that RNA binding is necessary but not sufficient for activity. Finally, the 2–72 protein, which lacks an experimentally observed C-terminal extension to the ZF2 domain[12, 13] failed to bind RNA at any concentration, confirming the importance of the additional α-helix (Figure 5F).
PTB associates with Vldlr RNA but does not regulate its splicing
MBNL and PTB act as co-repressors of Tpm1 splicing[22]. Pull-downs with biotinylated Vldlr RNA indicated that PTB was one of the major binding proteins in HeLa nuclear extract (data not shown). We therefore asked whether PTB acted synergistically or antagonistically with MBNL1 in the regulation of Vldlr splicing. As shown earlier, overexpression of MBNL1 promoted skipping of Tpm1 exon 3 but inclusion of the Vldlr exon (Figure 7B, C lanes 1,2). Overexpression of PTB had little effect on Tpm1 splicing (Figure 7C lane 3), as PTB is not limiting in HeLa cells[29]. However, PTB/nPTB knockdown led to decreased exon skipping (lanes 1 and 4). In contrast, Vldlr splicing was unresponsive to either overexpression or knockdown of PTB (Figure 7C lanes 1,3,4), suggesting that binding of PTB to Vldlr is non-functional. Furthermore, the activating effect of MBNL was not reduced upon PTB knockdown, and actually appeared to be slightly increased (Figure 7C lane 5). Therefore, while MBNL1 and PTB cooperate to repress Tpm1 splicing, MBNL1 acts independently to activate Vldlr splicing, and PTB binding to Vldlr appears to be non-functional.