35 research outputs found
Effect of the frequency of intramolecular motions on the NMR relaxation in liquid state temperature regime
Metal cation regulation of enzyme dynamics is a key factor influencing the activity of S adenosyl L homocysteine hydrolase from Pseudomonas aeruginosa
CRYSTAL STRUCTURE OF THE ISOMORPHOUS COMPLEXES TETRAAQUABIS(2,6-DIHYDROXY-BENZOATO-O)(2,6-DIHYDROXY-BENZOATO-O,O)TERBIUM(III) AND HOLMIUM(III) DIHYDRATE
Mature MiRNAs Form Secondary Structure, which Suggests Their Function beyond RISC
<div><p>The generally accepted model of the miRNA-guided RNA down-regulation suggests that mature miRNA targets mRNA in a nucleotide sequence-specific manner. However, we have shown that the nucleotide sequence of miRNA is not the only determinant of miRNA specificity. Using specific nucleases, T1, V1 and S1 as well as NMR, UV/Vis and CD spectroscopies, we found that miR-21, miR-93 and miR-296 can adopt hairpin and/or homoduplex structures. The secondary structure of those miRNAs in solution is a function of RNA concentration and ionic conditions. Additionally, we have shown that a formation of miRNA hairpin is facilitated by cellular environment.Looking for functional consequences of this observation, we have perceived that structure of these miRNAs resemble RNA aptamers, short oligonucleotides forming a stable 3D structures with a high affinity and specificity for their targets. We compared structures of anti-tenascin C (anti-Tn-C) aptamers, which inhibit brain tumor glioblastoma multiforme (GBM, WHO IV) and selected miRNA. A strong overexpression of miR-21, miR-93 as well Tn-C in GBM may imply some connections between them. The structural similarity of these miRNA hairpins and anti-Tn-C aptamers indicates that miRNAs may function also beyond RISC and are even more sophisticated regulators, that it was previously expected. We think that the knowledge of the miRNA structure may give a new insight into miRNA-dependent gene regulation mechanism and be a step forward in the understanding their function and involvement in cancerogenesis. This may improve design process of anti-miRNA therapeutics.</p></div
Enzymatic probing (A, B, E, F) and NMR analysis (C, D, G, H, I) of miR-21 (A-D), miR-93 (E-H) and miR-296 (I).
<p><b>A, B, E, F</b>. Cleavage patterns obtained for limited hydrolysis of 5'-end labeled miR-21 (<b>A, B</b>) and miR-93 (<b>E, F</b>) with RNase V1, nuclease S1 and RNase T1 in native conditions. Lanes: C - reaction control; L – OH ladder; T1 - limited hydrolysis by RNase T1 (0.025u/µl) in denaturing condition. <b>A</b>. Lines V1 - limited hydrolysis with RNase V1 (0, 0.03125, 0.0625 or 0.125 u/µl). <b>B</b>. Lanes T1(N) - limited hydrolysis with RNase T1 (0, 0.04, 0.02 or 0.01 u/µl) in native conditions. <b>E</b>. Line V1 - limited hydrolysis with RNase V1 (0.125 u/µl). <b>F</b>. Lines S1 - limited hydrolysis with nuclease S1 (0.00475 u/µl). The increasing concentrations of RNase V1 and RNase T1 are indicated by arrows. Cleavage sites are indicated in autoradiogram. <b>C, D, G, H, I</b>. NMR analysis of miR-21 (<b>C, D</b>) miR-93 (<b>G, H</b>) and miR-296 (<b>I</b>) structures. <b>C</b>. The imino region of <sup>1</sup>H NMR spectrum of miR-21 (0.7 mM) recorded at 7°C in H<sub>2</sub>O:D<sub>2</sub>O (90%:10%) with 150 mM sodium chloride, 10 mM phosphate buffer and 0.1 mM EDTA. Resonances arising from the hairpin form are indicated with *. <b>D</b>. Imino region of the <sup>1</sup>H-<sup>1</sup>H 2D NOESY spectrum of miR-21 at 15°C in H<sub>2</sub>O:D<sub>2</sub>O (90%:10%) with 150 mM sodium chloride, 10 mM phosphate buffer and 0.1 mM EDTA. The lines indicate the imino proton connectivity. <b>G</b>. The imino region of <sup>1</sup>H NMR spectrum of miR-93 (0.75 mM) recorded at 25°C in H<sub>2</sub>O:D<sub>2</sub>O (90%:10%) with 150 mM sodium chloride (top) or 50 mM sodium chloride (bottom), 10 mM phosphate buffer and 0.1 mM EDTA. Resonances arising from the hairpin form are indicated with *. <b>H</b>. Imino region of the <sup>1</sup>H-<sup>1</sup>H 2D NOESY spectrum of miR-93 at 15°C in H<sub>2</sub>O:D<sub>2</sub>O (90%:10%) with 150 mM sodium chloride, 10 mM phosphate buffer and 0.1 mM EDTA. The lines indicate the imino proton connectivity. <b>I</b>. The imino region of <sup>1</sup>H NMR spectrum of miR-296 (0.3 mM) recorded at 25°C in H<sub>2</sub>O:D<sub>2</sub>O (90%:10%) with 150 mM sodium chloride, 10 mM phosphate buffer and 0.1 mM EDTA.</p
Distribution of linear and nonlinear human miRNAs and of hairpin stem location in predicted human secondary structure (A); and folding distribution of human mature miRNAs strands (B).
<p>Distribution of linear and nonlinear human miRNAs and of hairpin stem location in predicted human secondary structure (A); and folding distribution of human mature miRNAs strands (B).</p
miR-21, miR-93 and miR-296 stability in GBM lysate.
<p><b>A, B</b>. Hydrolysis of 5′-end labeled miR-21, miR-93 and miR-296 in 0.01 mg/ml GBM lysate. <b>C</b>. Half-lives of miR-21, miR-93 and miR-296.</p