Abstract
BackgroundAlternative splicing contributes to the diversity of the proteome, and provides the cell with an important additional layer of regulation of gene expression. Among the many RNA binding proteins that regulate alternative splicing pathways are the Muscleblind-like (MBNL) proteins. MBNL proteins bind YGCY motifs in RNA via four CCCH zinc fingers arranged in two tandem arrays, and play a crucial role in the transition from embryonic to adult muscle splicing patterns, deregulation of which leads to Myotonic Dystrophy. Like many other RNA binding proteins, MBNL proteins can act as both activators or repressors of different splicing events.ResultsWe used targeted point mutations to interfere with the RNA binding of MBNL1 zinc fingers individually and in combination. The effects of the mutations were tested in assays for splicing repression and activation, including overexpression, complementation of siRNA-mediated knockdown, and artificial tethering using MS2 coat protein. Mutations were tested in the context of both full length MBNL1 as well as a series of truncation mutants. Individual mutations within full length MBNL1 had little effect, but mutations in ZF1 and 2 combined were more detrimental than those in ZF 3 and 4, upon splicing activation, repression and RNA binding. Activation and repression both required linker sequences between ZF2 and 3, but activation was more sensitive to loss of linker sequences.ConclusionsOur results highlight the importance of RNA binding by MBNL ZF domains 1 and 2 for splicing regulatory activity, even when the protein is artificially recruited to its regulatory location on target RNAs. However, RNA binding is not sufficient for activity; additional regions between ZF 2 and 3 are also essential. Activation and repression show differential sensitivity to truncation of this linker region, suggesting interactions with different sets of cofactors for the two types of activity.
Highlights
Alternative splicing contributes to the diversity of the proteome, and provides the cell with an important additional layer of regulation of gene expression
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
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
Summary
Alternative splicing contributes to the diversity of the proteome, and provides the cell with an important additional layer of regulation of gene expression. Among the many RNA binding proteins that regulate alternative splicing pathways are the Muscleblind-like (MBNL) proteins. Like many other RNA binding proteins, MBNL proteins can act as both activators or repressors of different splicing events. Pre-mRNA splicing is a critical part of mRNA maturation, and alternative splicing is a well established method of generating diversity and exerting control over the proteome. Investigations suggested that proteins of the SR family generally act as splicing activators, while proteins of the hnRNP family typically act as repressors. More recent global analyses of the activities of RNA binding proteins has indicated that many of them show both activator or repressor activity, depending on the site at which they bind to the target pre-mRNA [4]
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