Integration of Expressed Sequence Tag Data Flanking Predicted RNA Secondary Structures Facilitates Novel Non-Coding RNA Discovery

Paul M Krzyzanowski,Miguel A Andrade-Navarro,Michael A Rudnicki,Feodor D Price,Enrique M Muro,Matthew E Hudson

doi:10.1371/journal.pone.0020561

Paul M Krzyzanowski, Miguel A Andrade-Navarro + Show 4 more

Open Access

https://doi.org/10.1371/journal.pone.0020561

Copy DOI

Abstract

Many computational methods have been used to predict novel non-coding RNAs (ncRNAs), but none, to our knowledge, have explicitly investigated the impact of integrating existing cDNA-based Expressed Sequence Tag (EST) data that flank structural RNA predictions. To determine whether flanking EST data can assist in microRNA (miRNA) prediction, we identified genomic sites encoding putative miRNAs by combining functional RNA predictions with flanking ESTs data in a model consistent with miRNAs undergoing cleavage during maturation. In both human and mouse genomes, we observed that the inclusion of flanking ESTs adjacent to and not overlapping predicted miRNAs significantly improved the performance of various methods of miRNA prediction, including direct high-throughput sequencing of small RNA libraries. We analyzed the expression of hundreds of miRNAs predicted to be expressed during myogenic differentiation using a customized microarray and identified several known and predicted myogenic miRNA hairpins. Our results indicate that integrating ESTs flanking structural RNA predictions improves the quality of cleaved miRNA predictions and suggest that this strategy can be used to predict other non-coding RNAs undergoing cleavage during maturation.

Highlights

MicroRNAs have emerged to be important and powerful factors in most biological processes. miRNAs are small (,21 nt) species of non-coding RNA molecules that regulate cognate mRNAs exhibiting sequence complementarity. miRNAs are first transcribed as long primary transcripts containing hairpin-like structures which are excised by DGCR8/Drosha dependent endonuclease cleavage to form pre-miRNAs [1,2,3], which are exported into the cytoplasm
To determine whether Expressed Sequence Tag (EST) could be used as a general data source in novel miRNA prediction, we examined whether a trend of association exists between the EST termini around known miRNAs in human and mouse
We inferred that miRNA predictions might be generated by identifying genomic regions potentially generating structured RNA with corresponding EST evidence in proximity of their 39 terminus

Summary

Introduction

MicroRNAs (miRNAs) have emerged to be important and powerful factors in most biological processes. miRNAs are small (,21 nt) species of non-coding RNA (ncRNA) molecules that regulate cognate mRNAs exhibiting sequence complementarity. miRNAs are first transcribed as long primary transcripts (pri-miRNAs) containing hairpin-like structures which are excised by DGCR8/Drosha dependent endonuclease cleavage to form pre-miRNAs [1,2,3], which are exported into the cytoplasm. Pre-miRNAs exported to the cytoplasm are processed by the Dicer ribonuclease to form short dsRNA duplexes, which supply the RISC complex with mature miRNA strands [4,5,6]. Mature miRNAs are thought to coordinate rapid transcriptional transitions by repressing large numbers of mRNA transcripts in parallel [7]. During cell growth and differentiation, miRNAs have been shown to function during key transitions between cell states where they facilitate transcriptional reprogramming. Due to their ability to control numerous target transcripts simultaneously, miRNAs have emerged as regulators of gene expression in an increasing number of biological processes involving development and tissue regeneration Due to their ability to control numerous target transcripts simultaneously, miRNAs have emerged as regulators of gene expression in an increasing number of biological processes involving development and tissue regeneration (e.g. [11,12,13,14])

Methods

Results

Discussion

Conclusion