MiRScore: A Rapid and Precise MicroRNA Validation Tool
Allison Vanek, Sam Griffiths-Jones, Blake C. Meyers, Saima Shahid, Michael J. Axtell
Abstract
MicroRNAs (miRNAs) are small non-protein-coding RNAs that regulate gene expression in many eukaryotes. Next-generation sequencing of small RNAs (small RNA-seq) is central to the discovery and annotation of miRNAs. Newly annotated miRNAs and their longer precursors encoded by MIRNA loci are typically submitted to databases such as the miRBase microRNA registry following the publication of a peer-reviewed study. However, genome-wide scans using small RNA-seq data often yield high rates of false-positive MIRNA annotations, highlighting the need for more robust validation methods. miRScore was developed as an independent and efficient tool for evaluating new MIRNA annotations using sRNA-seq data.
Introduction
MicroRNAs (miRNAs) are a class of small, non-coding RNAs that regulate gene expression within eukaryotes. This regulation typically occurs when a miRNA, which is loaded into an RNA-induced silencing complex (RISC), imperfectly base pairs to a target messenger RNA (mRNA). The RISC then frequently acts as an endonuclease to cleave the mRNA or to otherwise inhibit its translation. miRNA-directed regulation of mRNAs is crucial in various biological processes such as developmental timing, metabolism, and defensive pathways in both plants and animals. Although miRNA biogenesis varies somewhat between animals and plants, the fundamental aspects of miRNA structure and function are conserved.
Materials and Methods:
Annotated MIRNAs from Oryza sativa, Arabidopsis thaliana, Zea mays, Mus musculus, and Homo sapiens were tested using miRScore version 0.3.2. Mature miRNA and hairpins sequences for each species were downloaded from either miRBase version 22.1 or MirGeneDB version 3.0. The miRNA file submitted to miRScore for each species contained sequences for both the annotated miRNA and miRNA* sequence where applicable. Several miRNAs from miRBase in some species began at position one of their respective hairpins and did not allow for evaluation of the miRNA duplex, particularly the two-nucleotide 3’ overhang structure.
Discussion
miRScore is primarily designed as a quick secondary filter to analyze new MIRNA annotations prior to submission or acceptance into a permanent repository. Because it assesses the validity of an annotation with respect to specific sRNA-seq datasets, it is not appropriate to conclude that a miRNA whose annotation is not supported by specific datasets is not a bona fide miRNA.
Acknowledgments
We thank the Penn State Genomics Core Facility (RRID: SCR_023645) for sRNA-seq services. We thank Steven Runo and Claude dePamphilis for the gift of Striga hermonthica seed.
Citation: Vanek A, Griffiths-Jones S, Meyers BC, Shahid S, Axtell MJ (2025) miRScore: A rapid and precise microRNA validation tool. PLoS Comput Biol 21(11): e1013663. https://doi.org/10.1371/journal.pcbi.1013663
Editor: Mark Ziemann, Burnet Institute, AUSTRALIA
Received: December 16, 2024; Accepted: October 25, 2025; Published: November 3, 2025.
Copyright: © 2025 Vanek et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: The miRScore software is an open-source python project available under the permissive MIT license. Documentation, source code, and test examples are freely available on the github page at https://github.com/Aez35/miRScore. Newly generated small RNA-seq data are available from NCBI GEO under accession GSE282265.
Funding: This work was supported by the National Science Foundation (2130884 to MJA and BCM; 2450802 to BCM), the Biotechnology and Biological Sciences Research Council (BB/W018438/1 to SGJ), and a seed grant from The Huck Institutes of the Life Sciences at Penn State (un-numbered award to MJA). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
