IsomiRage: From Functional Classification to Differential Expression of miRNA Isoforms

Area of Science:

• Genomics and Bioinformatics
• Molecular Biology
• Cancer Research

Background:

• MicroRNAs (miRNAs) are known for their sequence and length heterogeneity, leading to various isoforms (isomiRs).
• IsomiRs may possess distinct biological activities compared to canonical miRNAs, but their exploration is limited by data scarcity and analytical tool deficiencies.
• Understanding miRNA heterogeneity is crucial for a comprehensive view of gene regulation and cellular function.

Purpose of the Study:

• To develop and present a bioinformatics workflow for the comprehensive characterization and analysis of miRNA variants (isomiRs) in next-generation sequencing (NGS) datasets.
• To distinguish between canonical miRNAs, templated isomiRs, and non-templated isomiRs.
• To enable the grouping of functionally equivalent isomiRs based on modification types to assess miRNA modification patterns in specific biological contexts.

Main Methods:

• A custom bioinformatics pipeline was developed for analyzing small RNA NGS data.
• The pipeline utilizes alignment to a custom database containing canonical miRNAs and all possible 3', 5', and trimmed variants.
• The workflow categorizes isomiRs into templated (alternative dicing) and non-templated (enzymatic modifications like adenylation, uridylation) forms.

Main Results:

• The methodology increased the detection of miRNA species by 40% and identified over 1000 variants in primary epithelial breast cancer cells.
• Most identified modifications were templated isomiRs, resulting from imprecise Drosha or Dicer cleavage.
• Consistent detection of non-templated variants, particularly 3'-end adenylation and uridylation, in both normal and cancer cells suggests frequent miRNA post-transcriptional modification.

Conclusions:

• The developed analytical tool effectively deconvolutes miRNA heterogeneity, enabling detailed characterization of isomiRs.
• The findings highlight the prevalence of miRNA post-transcriptional modifications, including templated and non-templated forms.
• This workflow provides a valuable resource for exploring the functional significance of miRNA isoforms in various biological and disease contexts.