Recognition of Pre-formed and Flexible Elements of an RNA Stem-Loop by Nucleolin

Bouvet, Philippe; Allain, Frédéric H.‐T.; Finger, L. David; Dieckmann, Thorsten; Feigon, Juli

doi:10.1006/jmbi.2001.4691

Cited by 58 publications

(73 citation statements)

References 57 publications

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“…2). This structural feature, namely, a helical stacking at the 5Ј end of the RNA loop was already observed in several other RNA stem-loop structures (Bouvet et al 2001).…”

Section: Structure Of the Editing Site Before (C66) And After Editingsupporting

confidence: 69%

“…The other nucleotides with no COSY and TOCSY cross-peaks between the H1Ј-H2Ј protons (J H1Ј-H2Ј < 3 Hz) were constrained C3Јendo (␦ = 85 ± 20°). For both RNAs, two sets of structure calculation were run, "G72 anti" and "G72 syn" because not all distance constraints involving G72 could be fulfilled with one single calculation, similar to the Nucleolin binding RNA (Bouvet et al 2001). Two intra-and internucleotide constraints compatible with the G72 anti position were introduced in the first structure calculation set: intra G72 H8-H1Ј, G72 H8-H3Ј constraint distances of 3.7 Å and 3.0 Å and sequential G72 H8-U71 H2Ј, G72 H8-U71 H3Ј constraint distances of 3.7 Å and 4.5 Å.…”

Section: Structure Calculationsmentioning

confidence: 99%

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NMR structure of the apoB mRNA stem–loop and its interaction with the C to U editing APOBEC1 complementary factor

Maris¹,

Masse²,

Chester³

et al. 2005

RNA

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We have solved the NMR structure of the 31-nucleotide (nt) apoB mRNA stem-loop, a substrate of the cytidine deaminase APOBEC1. We found that the edited base located at the 5 end of the octa-loop is stacked between two adenosines in both the unedited (cytidine 6666) and the edited (uridine 6666) forms and that the rest of the loop is unstructured. The 11-nt "mooring" sequence essential for editing is partially flexible although it is mostly in the stem of the RNA. The octa-loop and the internal loop in the middle of the stem confer this flexibility. These findings shed light on why APOBEC1 alone cannot edit efficiently the cytidine 6666 under physiological conditions, the editing base being buried in the loop and not directly accessible. We also show that APOBEC1 does not specifically bind apoB mRNA and requires the auxiliary factor, APOBEC1 complementary factor (ACF), to edit specifically cytidine 6666. The binding of ACF to both the mooring sequence and APOBEC1 explains the specificity of the reaction. Our NMR study lead us to propose a mechanism in which ACF recognizes first the flexible nucleotides of the mooring sequence (the internal loop and the 3 end octa-loop) and subsequently melts the stem-loop, exposing the amino group of the cytidine 6666 to APOBEC1. Thus, the flexibility of the mooring sequence plays a central role in the RNA recognition by ACF.

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“…2). This structural feature, namely, a helical stacking at the 5Ј end of the RNA loop was already observed in several other RNA stem-loop structures (Bouvet et al 2001).…”

Section: Structure Of the Editing Site Before (C66) And After Editingsupporting

confidence: 69%

Section: Structure Calculationsmentioning

confidence: 99%

NMR structure of the apoB mRNA stem–loop and its interaction with the C to U editing APOBEC1 complementary factor

Maris¹,

Masse²,

Chester³

et al. 2005

RNA

Self Cite

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“…The other complexes that have been deposited in the Protein Data Bank (PDB) to date are nucleolin (Allain et al 2000;Bouvet et al 2001), NF-κB (Huang et al 2003), bacteriophage MS2 (Convery et al 1998;Horn et al 2004), thrombin (Long et al 2008), human IgG (Nomura et al 2010), and the G protein-coupled receptor kinase GRK2 (Tesmer et al 2012). A feature common to these specific aptamer-protein interactions is the positive potential of protein surfaces to which each aptamer binds (Fig.…”

Section: Discussionmentioning

confidence: 99%

“…Reduction of conformational heterogeneity is typically observed when aptamers bind to their targets. For example, the loop regions of the 22-nucleotide nucleolin aptamer and the 29-nucleotide NFκB RNA aptamer in the unbound state exchange between different conformations (Bouvet et al 2001;Reiter et al 2008). It is unsurprising that aptamer-protein recognition is mediated in part by flexible regions in the unbound aptamer that provide adaptability in binding.…”

Section: Discussionmentioning

confidence: 99%

An RNA aptamer possessing a novel monovalent cation-mediated fold inhibits lysozyme catalysis by inhibiting the binding of long natural substrates

Padlan

Malashkevich

Almo

et al. 2014

RNA

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RNA aptamers are being developed as inhibitors of macromolecular and cellular function, diagnostic tools, and potential therapeutics. Our understanding of the physical nature of this emerging class of nucleic acid-protein complexes is limited; few atomic resolution structures have been reported for aptamers bound to their protein target. Guided by chemical mapping, we systematically minimized an RNA aptamer (Lys1) selected against hen egg white lysozyme. The resultant 59-nucleotide compact aptamer (Lys1.2minE) retains nanomolar binding affinity and the ability to inhibit lysozyme's catalytic activity. Our 2.0-Å crystal structure of the aptamer-protein complex reveals a helical stem stabilizing two loops to form a protein binding platform that binds lysozyme distal to the catalytic cleft. This structure along with complementary solution analyses illuminate a novel protein-nucleic acid interface; (1) only 410 Å 2 of solvent accessible surface are buried by aptamer binding; (2) an unusually small fraction (∼18%) of the RNA-protein interaction is electrostatic, consistent with the limited protein phosphate backbone contacts observed in the structure; (3) a single Na + stabilizes the loops that constitute the protein-binding platform, and consistent with this observation, Lys1.2minE-lysozyme complex formation takes up rather than displaces cations at low ionic strength; (4) Lys1.2minE inhibits catalysis of large cell wall substrates but not catalysis of small model substrates; and (5) the helical stem of Lys1.2minE can be shortened to four base pairs (Lys1.2minF) without compromising binding affinity, yielding a 45-nucleotide aptamer whose structure may be an adaptable protein binding platform.

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“…Three main techniques have been used to quantify the effect of a mutation on the affinity of a protein-RNA complex, namely, the EMSA [1,20,26,35,352], the SPR [5], and the ITC [34,37]. For a description of these techniques, we refer the reader to specialized reviews [353][354][355][356].…”

Section: Confirmation and Quantification Of Intermolecular Contactsmentioning

confidence: 99%

Structure determination and dynamics of protein–RNA complexes by NMR spectroscopy

Dominguez

Schubert

Duss

et al. 2011

Progress in Nuclear Magnetic Resonance Spectroscopy

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Recognition of Pre-formed and Flexible Elements of an RNA Stem-Loop by Nucleolin

Cited by 58 publications

References 57 publications

NMR structure of the apoB mRNA stem–loop and its interaction with the C to U editing APOBEC1 complementary factor

NMR structure of the apoB mRNA stem–loop and its interaction with the C to U editing APOBEC1 complementary factor

An RNA aptamer possessing a novel monovalent cation-mediated fold inhibits lysozyme catalysis by inhibiting the binding of long natural substrates

Structure determination and dynamics of protein–RNA complexes by NMR spectroscopy

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