Tertiary Structure Mapping of the Pri-miRNA miR-17~92

Abstract

The understanding of RNA in regulating gene expression has exploded over the past 15 years. MicroRNAs (miRNAs) have vastly expanded the role of RNA in gene regulation beyond spliceosomal, ribosomal, and messenger RNAs. Approximately one half of miRNAs are polycistronic, where two or more miRNAs are encoded on a single pri-miRNA transcript, termed a miRNA cluster. The six miRNAs of the miR-17~92 cluster are contained within a ~800 nucleotide region within intron 3 of the cl13orf25 ~7 kb pri-miRNA transcript. We recently reported on the tertiary structured domain of miR-17~92 and its role in modulating miRNA biogenesis. The key finding was that the cluster structure explained the differential processing of the miRNA hairpins by Drosha. This work demonstrated the need to consider pri-miRNA tertiary structure in miRNA biogenesis. Since biochemical structure probing is typically performed on relatively short RNAs (≤200 nucleotides), we had to adapt these methodologies for application on large RNAs (~800 nucleotide miR-17~92 pri-miRNA). We present here our adaptation of a protection footprinting method using ribonucleases to probe the structure of the ~800 nucleotide miR-17~92 pri-miRNA. We outline the technical difficulties involved in probing large RNAs and data visualization using denaturing polyacrylamide gel electrophoresis and how we adapted the existing approaches to probe large RNAs. The methodology outlined here is generally applicable to large RNAs including long noncoding RNAs (lncRNA).