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.
"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. "
[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.
Nucleic Acids Research 06/2014; 42(13). DOI:10.1093/nar/gku536 · 9.11 Impact Factor
"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 . "
[Show abstract][Hide abstract] 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; 33(14). DOI:10.15252/embj.201488373 · 10.43 Impact Factor
"CUG repeat RNA crystallizes as an A-form helix (Mooers et al., 2005), with C and G bases paired and Us unpaired, and additional biochemical studies have shown that a mismatched RNA hairpin structure is important for recognition by MBNL1 (Warf and Berglund, 2007). However, structures of MBNL1 zinc fingers cocrystallized with CGCUGU RNA suggested that MBNL1 recognizes singlestranded RNA (Teplova and Patel, 2008). Additionally, the roles of motif spacing and of intervening sequences between tandem motifs remain largely uncharacterized. "
[Show abstract][Hide abstract] ABSTRACT: Specific protein-RNA interactions guide posttranscriptional gene regulation. Here, we describe RNA Bind-n-Seq (RBNS), a method that comprehensively characterizes sequence and structural specificity of RNA binding proteins (RBPs), and its application to the developmental alternative splicing factors RBFOX2, CELF1/CUGBP1, and MBNL1. For each factor, we recovered both canonical motifs and additional near-optimal binding motifs. RNA secondary structure inhibits binding of RBFOX2 and CELF1, while MBNL1 favors unpaired Us but tolerates C/G pairing in motifs containing UGC and/or GCU. Dissociation constants calculated from RBNS data using a novel algorithm correlated highly with values measured by surface plasmon resonance. Motifs identified by RBNS were conserved, were bound and active in vivo, and distinguished the subset of motifs enriched by CLIP-Seq that had regulatory activity. Together, our data demonstrate that RBNS complements crosslinking-based methods and show that in vivo binding and activity of these splicing factors is driven largely by intrinsic RNA affinity.
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