Pseudouridine Modification Inhibits Muscleblind-like 1 (MBNL1) Binding to CCUG Repeats and Minimally Structured RNA through Reduced RNA Flexibility.

Myotonic dystrophy type 2 is a genetic neuromuscular disease caused by the expression of expanded CCUG repeat RNAs from the non-coding region of the CCHC-type zinc finger nucleic acid-binding protein (CNBP) gene. These CCUG repeats bind and sequester a family of RNA-binding proteins known as Muscleblind-like 1, 2, and 3 (MBNL1, MBNL2, and MBNL3), and sequestration plays a significant role in pathogenicity. MBNL proteins are alternative splicing regulators that bind to the consensus RNA sequence YGCY (Y = pyrimidine). This consensus sequence is found in the toxic RNAs (CCUG repeats) and in cellular RNA substrates that MBNL proteins have been shown to bind. Replacing the uridine in CCUG repeats with pseudouridine (Ψ) resulted in a modest reduction of MBNL1 binding. Interestingly, Ψ modification of a minimally structured RNA containing YGCY motifs resulted in more robust inhibition of MBNL1 binding. The different levels of inhibition between CCUG repeat and minimally structured RNA binding appear to be due to the ability to modify both pyrimidines in the YGCY motif, which is not possible in the CCUG repeats. Molecular dynamic studies of unmodified and pseudouridylated minimally structured RNAs suggest that reducing the flexibility of the minimally structured RNA leads to reduced binding by MBNL1.

Modifications to toxic CUG RNAs induce structural stability, rescue mis-splicing in a myotonic dystrophy cell model and reduce toxicity in a myotonic dystrophy zebrafish model.

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.

Using affinity chromatography to engineer and characterize pH-dependent protein switches.

Conformational changes play important roles in the regulation of many enzymatic reactions. Specific motions of side chains, secondary structures, or entire protein domains facilitate the precise control of substrate selection, binding, and catalysis. Likewise, the engineering of allostery into proteins is envisioned to enable unprecedented control of chemical reactions and molecular assembly processes. We here study the structural effects of engineered ionizable residues in the core of the glutathione-S-transferase to convert this protein into a pH-dependent allosteric protein. The underlying rational of these substitutions is that in the neutral state, an uncharged residue is compatible with the hydrophobic environment. In the charged state, however, the residue will invoke unfavorable interactions, which are likely to induce conformational changes that will affect the function of the enzyme. To test this hypothesis, we have engineered a single aspartate, cysteine, or histidine residue at a distance from the active site into the protein. All of the mutations exhibit a dramatic effect on the protein's affinity to bind glutathione. Whereas the aspartate or histidine mutations result in permanently nonbinding or binding versions of the protein, respectively, mutant GST50C exhibits distinct pH-dependent GSH-binding affinity. The crystal structures of the mutant protein GST50C under ionizing and nonionizing conditions reveal the recruitment of water molecules into the hydrophobic core to produce conformational changes that influence the protein's active site. The methodology described here to create and characterize engineered allosteric proteins through affinity chromatography may lead to a general approach to engineer effector-specific allostery into a protein structure.