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Water-mediated recognition of t1-adenosine anchors Argonaute2 to microRNA targets.

ABSTRACT: MicroRNAs (miRNAs) direct post-transcriptional regulation of human genes by guiding Argonaute proteins to complementary sites in messenger RNAs (mRNAs) targeted for repression. An enigmatic feature of many conserved mammalian miRNA target sites is that an adenosine (A) nucleotide opposite miRNA nucleotide-1 confers enhanced target repression independently of base pairing potential to the miRNA. In this study, we show that human Argonaute2 (Ago2) possesses a solvated surface pocket that specifically binds adenine nucleobases in the 1 position (t1) of target RNAs. t1A nucleotides are recognized indirectly through a hydrogen-bonding network of water molecules that preferentially interacts with the N6 amine on adenine. t1A nucleotides are not utilized during the initial binding of Ago2 to its target, but instead function by increasing the dwell time on target RNA. We also show that N6 adenosine methylation blocks t1A recognition, revealing a possible mechanism for modulation of miRNA target site potency.

SUBMITTER: Schirle NT

PROVIDER: S-EPMC4606517 | biostudies-literature | 2015 Sep

REPOSITORIES: biostudies-literature

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Water-mediated recognition of t1-adenosine anchors Argonaute2 to microRNA targets.

Schirle Nicole T NT Sheu-Gruttadauria Jessica J Chandradoss Stanley D SD Joo Chirlmin C MacRae Ian J IJ

eLife 20150911

MicroRNAs (miRNAs) direct post-transcriptional regulation of human genes by guiding Argonaute proteins to complementary sites in messenger RNAs (mRNAs) targeted for repression. An enigmatic feature of many conserved mammalian miRNA target sites is that an adenosine (A) nucleotide opposite miRNA nucleotide-1 confers enhanced target repression independently of base pairing potential to the miRNA. In this study, we show that human Argonaute2 (Ago2) possesses a solvated surface pocket that specifica ...[more]

PMID: 26359634

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Project description:BackgroundAdenosine stress T1 mapping is an emerging magnetic resonance imaging method to investigate coronary vascular function and myocardial ischemia without application of a contrast agent. Using gene-modified mice and 2 vasodilators, we elucidated and compared the mechanisms of adenosine myocardial perfusion imaging and adenosine T1 mapping.MethodsWild-type (WT), A2AAR-/- (adenosine A2A receptor knockout), A2BAR-/- (adenosine A2B receptor knockout), A3AR-/- (adenosine A3 receptor knockout), and eNOS-/- (endothelial nitric oxide synthase knockout) mice underwent rest and stress perfusion magnetic resonance imaging (n=8) and T1 mapping (n=10) using either adenosine, regadenoson (a selective A2AAR agonist), or saline. Myocardial blood flow and T1 were computed from perfusion imaging and T1 mapping, respectively, at rest and stress to assess myocardial perfusion reserve and T1 reactivity (ΔT1). Changes in heart rate for each stress agent were also calculated. Two-way ANOVA was used to detect differences in each parameter between the different groups of mice.ResultsMyocardial perfusion reserve was significantly reduced only in A2AAR-/- compared to WT mice using adenosine (1.06±0.16 versus 2.03±0.52, P<0.05) and regadenoson (0.98±026 versus 2.13±0.75, P<0.05). In contrast, adenosine ΔT1 was reduced compared with WT mice (3.88±1.58) in both A2AAR-/- (1.63±1.32, P<0.05) and A2BAR-/- (1.55±1.35, P<0.05). Furthermore, adenosine ΔT1 was halved in eNOS-/- (1.76±1.46, P<0.05) versus WT mice. Regadenoson ΔT1 was approximately half of adenosine ΔT1 in WT mice (1.97±1.50, P<0.05), and additionally, it was significantly reduced in eNOS-/- mice (-0.22±1.46, P<0.05). Lastly, changes in heart rate was 2× greater using regadenoson versus adenosine in all groups except A2AAR-/-, where heart rate remained constant.ConclusionsThe major findings are that (1) although adenosine myocardial perfusion reserve is mediated through the A2A receptor, adenosine ΔT1 is mediated through the A2A and A2B receptors, (2) adenosine myocardial perfusion reserve is endothelial independent while adenosine ΔT1 is partially endothelial dependent, and (3) ΔT1 mediated through the A2A receptor is endothelial dependent while ΔT1 mediated through the A2B receptor is endothelial independent.

Project description:The unique cytokine interleukin-18 (IL-18) acts synergistically with IL-12 to regulate T-helper 1 and 2 lymphocytes and, as such, seems to underlie the pathogenesis of various autoimmune and allergic diseases. Several anti-IL-18 agents are in clinical development, including the recombinant human antibody ABT-325, which is entering trials for autoimmune diseases. Given competing cytokine/receptor and cytokine/receptor decoy interactions, understanding the structural basis for recognition is critical for effective development of anti-cytokine therapies. Here we report three crystal structures: the murine antibody 125-2H Fab fragment bound to human IL-18, at 1.5 A resolution; the 125-2H Fab (2.3 A); and the ABT-325 Fab (1.5 A). These structures, along with human/mouse IL-18 chimera binding data, allow us to make three key observations relevant to the biology and antigenic recognition of IL-18 and related cytokines. First, several IL-18 residues shift dramatically (> 10 A) upon binding 125-2H, compared with unbound IL-18 (Kato, Z., Jee, J., Shikano, H., Mishima, M., Ohki, I., Ohnishi, H., Li, A., Hashimoto, K., Matsukuma, E., Omoya, K., Yamamoto, Y., Yoneda, T., Hara, T., Kondo, N., and Shirakawa, M. (2003) Nat. Struct. Biol. 10, 966-971). IL-18 thus exhibits plasticity that may be common to its interactions with other receptors. Related cytokines may exhibit similar plasticity. Second, ABT-325 and 125-2H differ significantly in combining site character and architecture, thus explaining their ability to bind IL-18 simultaneously at distinct epitopes. These data allow us to define the likely ABT-325 epitope and thereby explain the distinct neutralizing mechanisms of both antibodies. Third, given the high 125-2H potency, 10 well ordered water molecules are trapped upon complex formation in a cavity between two IL-18 loops and all six 125-2H complementarity-determining regions. Thus, counterintuitively, tight and specific antibody binding may in some cases be water-mediated.

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Water-mediated recognition of t1-adenosine anchors Argonaute2 to microRNA targets.

Publications

Water-mediated recognition of t1-adenosine anchors Argonaute2 to microRNA targets.

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