Abstract
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.
Highlights
MicroRNAs are short, non-coding RNAs that regulate gene expression by diminishing translation of their target messenger RNAs [1,2]
We report here characterization by NMR spectroscopy and in-line probing of an oligonucleotide model of this RNA
Exchangeable Protons and Secondary Structure We initially investigated the NMR spectrum of RNA 1 (Figure 2), which directly models the pre-element of miR-21
Summary
MicroRNAs (miRNAs) are short, non-coding RNAs that regulate gene expression by diminishing translation of their target messenger RNAs [1,2] Whereas their normal function is regulation of development and cellular responses to stress [3], the aberrant expression of specific miRNAs is associated with a wide range of diseases, including cancer [4] and heart disease [5,6]. MiR-21 is a miRNA that is elevated in both cancer and heart disease It is highly expressed in a variety of tumors [7], contributing to the cancer phenotype by diminishing translation of tumor suppressor genes [8,9,10,11,12]. An understanding of the factors that regulate miRNA expression is essential to efforts to therapeutically target specific disease-related miRNAs [14,15] and to gaining a basic understanding of the roles of miRNAs in biology
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