Structural insights into RNA recognition by the alternative-splicing regulator muscleblind-like MBNL1.
ABSTRACT Muscleblind-like (MBNL) proteins, regulators of developmentally programmed alternative splicing, harbor tandem CCCH zinc-finger (ZnF) domains that target pre-mRNAs containing YGCU(U/G)Y sequence elements (where Y is a pyrimidine). In myotonic dystrophy, reduced levels of MBNL proteins lead to aberrant alternative splicing of a subset of pre-mRNAs. The crystal structure of MBNL1 ZnF3/4 bound to r(CGCUGU) establishes that both ZnF3 and ZnF4 target GC steps, with site-specific recognition mediated by a network of hydrogen bonds formed primarily with main chain groups of the protein. The relative alignment of ZnF3 and ZnF4 domains is dictated by the topology of the interdomain linker, with a resulting antiparallel orientation of bound GC elements, supportive of a chain-reversal loop trajectory for MBNL1-bound pre-mRNA targets. We anticipate that MBNL1-mediated targeting of looped RNA segments proximal to splice-site junctions could contribute to pre-mRNA alternative-splicing regulation.
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ABSTRACT: CUG repeat expansions in the 3' UTR of dystrophia myotonica protein kinase (DMPK) cause myotonic dystrophy type 1 (DM1). As RNA, these repeats elicit toxicity by sequestering splicing proteins, such as MBNL1, into protein-RNA aggregates. Structural studies demonstrate that CUG repeats can form A-form helices, suggesting that repeat secondary structure could be important in pathogenicity. To evaluate this hypothesis, we utilized structure-stabilizing RNA modifications pseudouridine (Ψ) and 2'-O-methylation to determine if stabilization of CUG helical conformations affected toxicity. CUG repeats modified with Ψ or 2'-O-methyl groups exhibited enhanced structural stability and reduced affinity for MBNL1. Molecular dynamics and X-ray crystallography suggest a potential water-bridging mechanism for Ψ-mediated CUG repeat stabilization. Ψ modification of CUG repeats rescued mis-splicing in a DM1 cell model and prevented CUG repeat toxicity in zebrafish embryos. This study indicates that the structure of toxic RNAs has a significant role in controlling the onset of neuromuscular diseases.Nucleic Acids Research 10/2014; · 8.81 Impact Factor
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ABSTRACT: The conserved eukaryotic Pan2-Pan3 deadenylation complex shortens cytoplasmic mRNA 3' polyA tails to regulate mRNA stability. Although the exonuclease activity resides in Pan2, efficient deadenylation requires Pan3. The mechanistic role of Pan3 is unclear. Here, we show that Pan3 binds RNA directly both through its pseudokinase/C-terminal domain and via an N-terminal zinc finger that binds polyA RNA specifically. In contrast, isolated Pan2 is unable to bind RNA. Pan3 binds to the region of Pan2 that links its N-terminal WD40 domain to the C-terminal part that contains the exonuclease, with a 2:1 stoichiometry. The crystal structure of the Pan2 linker region bound to a Pan3 homodimer shows how the unusual structural asymmetry of the Pan3 dimer is used to form an extensive high-affinity interaction. This binding allows Pan3 to supply Pan2 with substrate polyA RNA, facilitating efficient mRNA deadenylation by the intact Pan2-Pan3 complex.The EMBO Journal 05/2014; · 10.75 Impact Factor
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ABSTRACT: Expanded non-coding RNA repeats of CUG and CCUG are the underlying genetic causes for myotonic dystrophy type 1 (DM1) and type 2 (DM2), respectively. A gain-of-function of these pathogenic repeat expansions is mediated at least in part by their abnormal interactions with RNA binding proteins such as MBNL1 and resultant loss of activity of these proteins. To study pathogenic mechanisms of CCUG repeat expansions in an animal model, we created a fly model of DM2 that expresses pure, uninterrupted CCUG repeat expansions ranging from 16 to 720 repeats in length. We show that this fly model for DM2 recapitulates key features of human DM2 including RNA repeat-induced toxicity, ribonuclear foci formation and changes in alternative splicing. Interestingly, expression of two isoforms of MBNL1, MBNL135 and MBNL140, leads to cleavage and concurrent up-regulation of the levels of the RNA repeat transcripts, with MBNL140 having more significant effects than MBNL135. This property is shared with a fly CUG repeat expansion model. Our results suggest a novel mechanism for interaction between the pathogenic RNA repeat expansions of myotonic dystrophy and MBNL1.Human Molecular Genetics 10/2014; · 6.68 Impact Factor