Many novel mammalian microRNA candidates identified by extensive cloning and RAKE analysis

Abstract

MicroRNAs are 20- to 23-nucleotide RNA molecules that can regulate gene expression. Currently > 400 microRNAs have been experimentally identified in mammalian genomes, whereas estimates go up to 1000 and beyond. Here we show that many more mammalian microRNAs exist. We discovered novel microRNA candidates using two approaches: testing of computationally predicted microRNAs by a modified microarray-based detection system, and cloning and sequencing of large numbers of small RNAs from different human and mouse tissues. Together these efforts experimentally identified 348 novel mouse and 81 novel human microRNA candidate genes. Most novel microRNAs candidates are not conserved beyond mammals, and ~10% are taxon-specific. Our analyses indicate that the entire microRNA repertoire is not remotely exhausted.

Figure 1.

Schematic representation of the modified RAKE assay (A) and experimental results obtained for known (B) and novel (C) candidate miRNAs. (A) A 44K microarray with a tiling path of 60-mer probes that are attached with their 3′ end to the glass surface was designed for the Agilent platform. Each DNA probe consists of a universal 3′ spacer sequence (black) followed by 22 nt of sequence complementary to the microRNA candidate (red), three thymidine nucleotides (green), and a short universal spacer (black). Unlabeled small RNA is hybridized to these arrays, followed by a Klenow extension reaction in the presence of biotinylated dATP. As miRNAs function as a primer for extension and the thymidines are the only template for extension, a complete 3′-end match is required for biotin incorporation and streptavidin fluorophore-mediated detection in the final step. (B) Schematic representation of the miR-293 pre-miRNA with predicted secondary structure and RAKE results. The mature miRNA (red) and numbers above the sequence indicate the 3′ end that fully matches the respective tiling path probe on the array. The strongest signal in the RAKE assay is obtained for probe 14, confirming the known 3′ end of miR-293. A weaker signal is obtained for probe 8, consistent with sensitive detection of the star sequence, which is produced as a side product from the hairpin structure by Drosha and Dicer nucleases that cut double-stranded RNA with a 2-nt 3′ overhang. The star sequence cannot be detected for all positive miRNAs. (C) RAKE results for three candidate miRNAs confirm the existence of novel miRNAs and identify the 3′ end of the mature miRNAs. For cand208 the star sequence can be detected (probe 7), whereas for cand973 multiple probes are positive with a rapidly decreasing intensity around the predominant probe, most likely representing imprecise 3′ end processing.

Figure 2.

The cloning frequency for known and novel mouse and human miRNAs is significantly different. Although many known miRNAs were not picked up in our cloning experiments and about half of the novel miRNAs were identified multiple times, a relatively high fraction of novel miRNA candidates was identified only once, indicating that small RNA sequencing efforts have not been exhausted.

Figure 3.

Overlap of cloned small miRNAs and RAKE results for known (A) and novel (B) mouse miRNA candidates. Known microRNAs were found to varying degrees in the different experiments, but most are detected in limited numbers of samples in both human and mouse. Most novel microRNA candidates were identified by the combination of computational prediction and RAKE confirmation approach.

Figure 4.

Chromosomal location of human (A) and (B) miRNAs. (Red) Noval miRNA candidates, (green) known miRNAs. Clusterd miRNAs are stacked