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.
Edited by Ronald C. WekMyotonic 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 (⌿) resulted in a modest reduction of MBNL1 binding. Interestingly, ⌿ 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. Myotonic dystrophy type 1 (DM1)3 is a genetic neuromuscular disease caused by expression of expanded CUG repeats in the 3Ј UTR of the dystrophia myotonica protein kinase (DMPK) gene. Similar to DM1, myotonic dystrophy type 2 (DM2) is caused by expression of expanded CCUG repeats in an intron of the CCHC-type zinc finger nucleic acid-binding protein (CNBP) gene. DM1 and DM2 occur when the CUG/CCUG repeats are expanded beyond 100 repeats, and patients can have up to thousands of CUG/CCUG repeats (1, 2). A primary component of the currently accepted DM1 and DM2 disease mechanism is that expanded CUG/CCUG repeats sequester RNAbinding proteins (primarily the Muscleblind-like family), which prevents these proteins from performing their functions in cells (3, 4).The members of the Muscleblind-like family of proteins (MBNL1, MBNL2, and MBNL3) bind RNA and regulate several RNA processing pathways, including alternative splicing, premiRNA biogenesis, mRNA localization, alternative polyadenylation, and circular RNA generation (5-9). MBNL proteins bind to the consensus YGCY RNA sequence (6, 10). CUG and CCUG repeats are composed of YGCY motifs creating hundreds or thousands of perfect MBNL-binding sites resulting in large numbers of MBNL proteins binding to the repeats and forming nuclear foci (11). When MBNL proteins are sequestered, they are unable to regulate RNA processing events, and consequently, many DM1 and DM2 symptoms are caused by misregulated alternative splicing and potentially the loss of other MBNL activities (12). It is therefore important to understand how MBNL proteins bind to their toxic and cellular RNA substrates to develop mechanisms to alleviate MBNL sequestration in DM1 and DM2.MBNL pro...
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 pHdependent 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.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.