Structural insights into RNA recognition by the alternative-splicing regulator muscleblind-like MBNL1
Structural Biology Program, Memorial Sloan-Kettering Cancer Center, 439 East 67th Street, New York, NY 10021, USA.Nature Structural & Molecular Biology (Impact Factor: 13.31). 12/2008; 15(12):1343-51. DOI: 10.1038/nsmb.1519
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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- "Molecular dynamics simulations suggested that 5 0 CU/3 0 UC internal loops exist in a dynamic equilibrium between two conformations (Childs-Disney et al., 2014). A complex of tetrameric MBNL1 bound to a model RNA recognition site has been characterized by X-ray crystallography (Teplova and Patel, 2008) (Figure 4A); a complex of two MBNL1 zinc fingers with two single-stranded RNAs has also been refined from X-ray crystallographic data (Teplova and Patel, 2008). The specificity of MBNL1 binding to imperfect (with multiple U 3 U or CU 3 UC internal loops) rather than to fully base-paired stem-loop was demonstrated experimentally (Kino et al., 2004; Warf and Berglund, 2007). "
ABSTRACT: RNAs adopt diverse folded structures that are essential for function and thus play critical roles in cellular biology. A striking example of this is the ribosome, a complex, three-dimensionally folded macromolecular machine that orchestrates protein synthesis. Advances in RNA biochemistry, structural and molecular biology, and bioinformatics have revealed other non-coding RNAs whose functions are dictated by their structure. It is not surprising that aberrantly folded RNA structures contribute to disease. In this Review, we provide a brief introduction into RNA structural biology and then describe how RNA structures function in cells and cause or contribute to neurological disease. Finally, we highlight successful applications of rational design principles to provide chemical probes and lead compounds targeting structured RNAs. Based on several examples of well-characterized RNA-driven neurological disorders, we demonstrate how designed small molecules can facilitate the study of RNA dysfunction, elucidating previously unknown roles for RNA in disease, and provide lead therapeutics. Copyright © 2015 Elsevier Inc. All rights reserved.
- "This question is addressed in a paper reporting the crystallographic complex of zinc finger domains from the alternative splicing regulator protein MBNL1 and CGCUGU (80). The model shows the protein interacting with a short single-stranded RNA, in particular with the GC step in its sequence. "
Article: Structural studies of CNG repeats[Show abstract] [Hide abstract]
ABSTRACT: CNG repeats (where N denotes one of the four natural nucleotides) are abundant in the human genome. Their tendency to undergo expansion can lead to hereditary diseases known as TREDs (trinucleotide repeat expansion disorders). The toxic factor can be protein, if the abnormal gene is expressed, or the gene transcript, or both. The gene transcripts have attracted much attention in the biomedical community, but their molecular structures have only recently been investigated. Model RNA molecules comprising CNG repeats fold into long hairpins whose stems generally conform to an A-type helix, in which the non-canonical N-N pairs are flanked by C-G and G-C pairs. Each homobasic pair is accommodated in the helical context in a unique manner, with consequences for the local helical parameters, solvent structure, electrostatic potential and potential to interact with ligands. The detailed three-dimensional profiles of RNA CNG repeats can be used in screening of compound libraries for potential therapeutics and in structure-based drug design. Here is a brief survey of the CNG structures published to date.
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- "First , the N - terminal zinc finger of Pan3 conveys sequence specificity by binding to polyA RNA prefer - entially over other polyribonucleotides ( Supplementary Fig S4 ) . The NMR solution structure of the scPan3 zinc finger ( Fig 2 ) revealed a similarity to other zinc fingers that bind RNA ( Hudson et al , 2004 ; Teplova & Patel , 2008 ; Kuhlmann et al , 2014 ) . Each of these other proteins contains tandem zinc fingers within the same polypeptide chain ( four in MBNL1 , two in TIS11d , and seven in Nab2 ) , probably because the affinity of a single finger is insufficient to provide binding specificity . "
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.
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