NMR characterization of an oligonucleotide model of the miR-21 pre-element.

We have used NMR spectroscopy to characterize an oligonucleotide stem loop structure based on the pre-element of an oncogenic microRNA, miR-21. This predicted stem-loop structure is cleaved from the precursor of miR-21 (pre-miR-21) by the nuclease Dicer. It is also a critical feature recognized by the protein complex that converts the primary transcript (pri-miR-21) into the pre-miRNA. The secondary structure of the native sequence is poorly defined by NMR due to rapid exchange of imino protons with solvent; however, replacement of two adjacent putative G•U base pairs with G•C base pairs retains the conformation of the hairpin observed by chemical probing and stabilizes it sufficiently to observe most of the imino proton resonances of the molecule. The observed resonances are consistent with the predicted secondary structure. In addition, a peak due to a loop uridine suggests an interaction between it and a bulged uridine in the stem. Assignment of non-exchangeable proton resonances and characterization of NOEs and coupling constants allows inference of the following features of the structure: extrahelicity of a bulged adenosine, deviation from A-form geometry in a base-paired stem, and consecutive stacking of the adenosines in the 5' side of the loop, the guanosine of the closing base pair, and a cross-strand adenosine. Modeling of the structure by restrained molecular dynamics suggests a basis for the interaction between the loop uridine, the bulged uridine in the stem, and an A•U base pair in the stem

Mg²⁺-stimulated (in-line) cleavage of RNAs 1 and 2.

<p>A. Electrophoretic analysis of cleavage. Mg²⁺: RNA treated with 5 mM MgCl₂. OH: hydrolysis ladder. T1: Ribonuclease T1 digestion. Untreated RNA in lanes labeled “-“. Bands due to guanosines identified from RNAse T1 digestion are indicated. B. Mapping of cleavage onto predicted secondary structures of <b>1</b> and <b>2</b>. Cleavage after each nucleotide is indicated by a line with length proportional to band intensity.</p

Stereoviews of structural features apparent in the NMR-based model of 2.

<p>A. Stacking of consecutive purines G13–A15 and cross-strand stacking of A20. B. Orientation of U12 with respect to U18. The fourteen lowest energy structures were aligned on U18 and the pyrimidine rings of U18 and U12 are shown.</p

Portion of the NOESY spectrum showing NOEs between H8/H6/H2 (7.4–8.0 ppm) and H1′/H5 (5.2–6.0 ppm) protons.

<p>Mixing time was 400 ms and temperature was 25°C. Cross-peaks due to NOEs from a nucleotide aromatic proton to the H1′ proton of its own sugar are labeled. A. Sequential NOEs from C9 to U12. B. Sequential NOEs from G13 to U16. Crosspeaks due to H2 of A14 and A15 and the U16 H5 to U16 H6 are labeled. C. Sequential NOEs from U16 to C21.</p

Structural features of pri-miRNAs.

<p>The stem and apical loop sequences of pri-miR-21 are shown. Arrows indicate sites of cleavage by Drosha and Dicer, as indicated. The sequence of nucleotides shown in large font is investigated in this work.</p