Computational Prediction of DNA-RNA Triplex Formation Sites Reveals Novel Regulatory Links Between lncRNAs and Key Fertility Genes in Retinta Cattle

Background: Long non-coding RNAs (lncRNAs) are increasingly recognized as key regulators of gene expression, playing pivotal roles in diverse biological processes, including reproduction. This study identified and characterized lncRNAs located near fertility-associated genes in Retinta beef cattle, exploring their potential regulatory roles via DNA–RNA triplex formation using in silico approaches. Methods: We applied an integrative bioinformatics pipeline to identify potential triplex interactions, predicting structurally accessible regions within the lncRNAs and demonstrating the statistical enrichment of binding sites across known regulatory genomic elements. Results: Twelve protein-coding genes previously linked to female fertility or male scrotal circumference were analyzed, revealing 16 unique lncRNAs within ±50 kb windows, predominantly on BTA5. We predicted high-confidence triplex-forming oligonucleotides (TFOs) for most gene-lncRNA pairs. Our results suggest robustness and sequence specificity, as interactions were disrupted by sequence permutation or when a control background sequence was used. RNA secondary-structure analysis revealed that TFOs generally lie in exposed regions, supporting their accessibility for triplex formation. Furthermore, promoter and regulatory regions of fertility-associated genes were enriched in predicted triplex target sites (TTSs), with some overlapping CpG islands and enhancer regions, leading to the hypothesis that these lncRNAs might play a role in epigenetic regulation. Conclusions: Overall, these findings establish computationally derived hypotheses regarding the potential molecular mechanisms by which lncRNAs may modulate reproductive efficiency in cattle and highlight specific lncRNAs as promising targets for functional studies and marker-assisted breeding.