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Combinatorial mutagenesis of MBNL1 zinc fingers elucidates distinct classes of regulatory events.

ABSTRACT: The RNA binding protein and alternative splicing factor Muscleblind-like 1 (MBNL1) has been a topic of intense study due to its role in myotonic dystrophy (DM) pathogenesis. MBNL1 contains four zinc finger (ZF) RNA binding domains arranged in two pairs. Through combinatorial mutagenesis of the ZF domains, we demonstrate that the pairs of ZFs have differential affinity for RNA and subsequently differential splicing activities. We evaluated splicing and binding activity for six MBNL1-mediated splicing events and found that the splicing activity profiles for the ZF mutants vary among transcripts. Clustering analysis of splicing profiles revealed that two distinct classes of MBNL1 pre-mRNA substrates exist. For some of the RNA transcripts tested, binding and splicing activity of the ZF mutants correlated. However, for some transcripts it appears that MBNL1 exerts robust splicing activity in the absence of RNA binding. We demonstrate that functionally distinct classes of MBNL1-mediated splicing events exist as defined by requirements for ZF-RNA interactions.

SUBMITTER: Purcell J

PROVIDER: S-EPMC3457334 | biostudies-literature | 2012 Oct

REPOSITORIES: biostudies-literature

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Combinatorial mutagenesis of MBNL1 zinc fingers elucidates distinct classes of regulatory events.

Purcell Jamie J Oddo Julia C JC Wang Eric T ET Berglund J Andrew JA

Molecular and cellular biology 20120813 20

The RNA binding protein and alternative splicing factor Muscleblind-like 1 (MBNL1) has been a topic of intense study due to its role in myotonic dystrophy (DM) pathogenesis. MBNL1 contains four zinc finger (ZF) RNA binding domains arranged in two pairs. Through combinatorial mutagenesis of the ZF domains, we demonstrate that the pairs of ZFs have differential affinity for RNA and subsequently differential splicing activities. We evaluated splicing and binding activity for six MBNL1-mediated spli ...[more]

PMID: 22890842

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Project description:Protein engineering by combinatorial site-directed mutagenesis evaluates a portion of the sequence space near a target protein, seeking variants with improved properties (e.g., stability, activity, immunogenicity). In order to improve the hit-rate of beneficial variants in such mutagenesis libraries, we develop methods to select optimal positions and corresponding sets of the mutations that will be used, in all combinations, in constructing a library for experimental evaluation. Our approach, OCoM (Optimization of Combinatorial Mutagenesis), encompasses both degenerate oligonucleotides and specified point mutations, and can be directed accordingly by requirements of experimental cost and library size. It evaluates the quality of the resulting library by one- and two-body sequence potentials, averaged over the variants. To ensure that it is not simply recapitulating extant sequences, it balances the quality of a library with an explicit evaluation of the novelty of its members. We show that, despite dealing with a combinatorial set of variants, in our approach the resulting library optimization problem is actually isomorphic to single-variant optimization. By the same token, this means that the two-body sequence potential results in an NP-hard optimization problem. We present an efficient dynamic programming algorithm for the one-body case and a practically-efficient integer programming approach for the general two-body case. We demonstrate the effectiveness of our approach in designing libraries for three different case study proteins targeted by previous combinatorial libraries--a green fluorescent protein, a cytochrome P450, and a beta lactamase. We found that OCoM worked quite efficiently in practice, requiring only 1 hour even for the massive design problem of selecting 18 mutations to generate 10? variants of a 443-residue P450. We demonstrate the general ability of OCoM in enabling the protein engineer to explore and evaluate trade-offs between quality and novelty as well as library construction technique, and identify optimal libraries for experimental evaluation.

Dataset Information

Combinatorial mutagenesis of MBNL1 zinc fingers elucidates distinct classes of regulatory events.

Publications

Combinatorial mutagenesis of MBNL1 zinc fingers elucidates distinct classes of regulatory events.

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