In this study, we investigated several aspects of alternative splicing regulation by MBNL proteins. We identified a 273-nt-long segment in the IR pre-mRNA containing alternatively spliced exon 11 that is necessary and sufficient for regulation of exon 11 by MBNL1 and MBNL3. We demonstrated that MBNL1 directly interacts with a 30-nt region downstream of exon 11 that contains three separate consensus-binding sequences. Similar clusters of binding sites have been identified in other MBNL targets (14
). Binding of MBNL1 to each of the motifs contributes to binding of MBNL1 to the downstream intron. These results support the possibility of cooperative binding correlating with previous data demonstrating potential dimerization of MBNL1 (36
). Importantly, the 30-nt region was required for responsiveness of exon 11 splicing to MBNL1 or MBNL3 expression demonstrating that the 30-nt segment mediates splicing regulation via direct interactions with MBNL1. These results are consistent with recently published results (37
). While there was no direct interaction of MBNL1 on the upstream intronic sequence, the response to MBNL expression was higher than the background levels (construct F, ). This implies that MBNL proteins can still respond to splicing through an indirect mechanism potentially through protein–protein interactions of another RNA-binding factor. However, results from construct H () are inconsistent with a response element located within intron 10. Therefore, the nature of an MBNL response element in intron 10 remains to be delineated.
The consensus motif that we used in this study was YGCY(U/G)Y (10
). It was recently found that the simplified form of YGCY motif is enough for MBNL1 binding (13
). The binding sites that were identified in the present study contained one YGCY motif and several YGC or GCY motifs. Secondary structure of RNA can also play a role in the interaction of RNA-binding proteins (38
) and MBNL1 binds to GC base-pairs interrupted by pyrimidine mismatches (36
). We were unable to identify a secondary structure within the functionally defined 30
nt MBNL response element in IR (data not shown), suggesting that the probes form single-stranded RNA structures as previously described (14
A second goal of this investigation was to identify regions of MBNL1 and MBNL3 that are required for splicing activation or repression and are separate from the RNA-binding domains. We performed parallel analyses on MBNL1 and MBNL3 because both strongly induce IR exon 11 inclusion and cTNT exon 5 skipping and yet differ outside of the RNA-binding domains. To test the ability of MBNL deletion mutants to bind RNA in vivo
, we took advantage of the ability of MBNL proteins to bind and colocalize with CUG-repeat RNA nuclear foci (41
The analyses of MBNL1 and MBNL3 protein domains provided several conclusions. First, for both MBNL1 and MBNL3, regions required for positive splicing activity were localized in regions that were separate from the RNA-binding domains (A). Most deletions that lost the majority of splicing activity retained the ability to bind RNA (MBNL1 mutations 3, 4, G and MBNL3 mutations 3, G), indicating that a domain required for intrinsic splicing activity rather than RNA binding was affected by the deletion. We cannot rule out that deletions resulted in general disruption of protein structure resulting in a loss of activity, although each truncated protein was expressed at similar levels as the FL proteins. N-terminal deletions were essentially replaced by the N-terminal fusion with GFP rather than being simply truncated and for one deletion, MBNL3 mutation G, replacing the deleted region by a luciferase segment did not restore activity. Our results also indicate that efficient splicing repression of cTNT exon 5 requires the second pair of zinc fingers. In addition, while MBNL1 mutation 4 was inactive on cTNT exon 5, it retained nearly half of its activity on IR exon 11, indicating that the effect on cTNT splicing regulation was likely to be specific to repressor activity and not a general loss of activity. MBNL3 mutant 4 lost RNA-binding activity even though the deletion was 56 residues from the zinc-finger domains. It is unclear whether this deletion disrupted general protein structure having a secondary effect on RNA binding or this region of the protein could provide a more specific role in promoting or stabilizing RNA binding.
Second, for both MBNL1 and MBNL3, either the N-terminal or central pair of zinc-finger RNA-binding domains was sufficient to function as splicing activators of IR. MBNL1 and MBNL3 mutants 2 and E contain either the N-terminal or central zinc-finger domains, respectively, and retain substantial activation and repression activity. The observation that different RNA-binding domains within the same protein target the protein appropriately has also been observed for the CELF proteins which contain RRM-type RNA-binding domains (35
Third, MBNL1 and MBNL3 contain more than one activation domain. For MBNL1, this is based on results from mutants 4 and E in which N- and C-terminal regions of the protein each have nearly 50% activity of full-length and overlap by only 21 residues. As mutant F retains more than a quarter of full-length activity with no residues overlapping with mutant 4, separate halves of MBNL1 can activate IR exon 11. The MBNL1 domains required for repression are between residues 80 and 160 (we consider the 11% activity of MBNL1 mutant F for cTNT repression to be negligible) and between residues 222 and 302. For MBNL3, the majority of the activity for both repression and activation is located between residues 81 and 176 based on results from mutants 2 and E. The most telling is deletion of 73 residues from mutant E to mutant G, which completely eliminates activity. Importantly, the inactive deletion mutant (G) retains RNA-binding activity, indicating that this region is required for an activity other than RNA binding.
Interestingly, the regions within MBNL1 and MBNL3 that are required for splicing regulation have sequence similarities and even identity with regions within CELF proteins that are required for splicing regulation (B). Regions rich in alanine, glutamine and methionine as well as spaced leucines or isoleucines are suggestive of protein–protein interaction domains. Overall, our results support a model in which separate domains of MBNL proteins function to bind specific motifs within the RNA and interact directly with components of the spliceosome or with co-regulators to mediate splicing regulation. It will be of particular interest to identify these interacting proteins and to compare the proteins that are required for activation and repression.
Splicing repression of cTNT exon 5 by MBNL1 results from its antagonism of U2AF65 binding upstream of the regulated exon (39
). The mechanism by which MBNL activates splicing remains elusive. However, the results from this study have identified the sites on IR pre-mRNA where MBNL binds and the regions of the MBNL1 and MBNL3 proteins that are required for splicing regulation, which likely contribute to its mechanism of splicing activation.