RNA-DNA triplex-forming miRNAs define an evolutionarily recent chromatin regulatory mechanism

Martin, M.; Jalife, C.; Mendez, C.; Montecinos, C.; Brown-Brown, D.; Diaz-Tejeda, F.; Caffi, V.; Zavala, K.; Kugler, F.; Molina-Gonzalez, L.; Valenzuela-Nieto, G.; Laengst, G.; Mardones, G. A.; Munita, R.; Opazo, J. C.; Maldonado, R.

doi:10.64898/2026.02.25.707792

bioRxiv · 10.64898/2026.02.25.707792

RNA-DNA triplex-forming miRNAs define an evolutionarily recent chromatin regulatory mechanism

Martin, M.·Jalife, C.·Mendez, C.·Montecinos, C.·Brown-Brown, D.·Diaz-Tejeda, F.·Caffi, V.·Zavala, K.·Kugler, F.·Molina-Gonzalez, L.·Valenzuela-Nieto, G.·Laengst, G.·Mardones, G. A.·Munita, R.·Opazo, J. C.·Maldonado, R.

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Abstract

MicroRNAs (miRNAs) are best known for their role in post-transcriptional gene regulation in the cytoplasm. However, a subset of miRNAs has been detected in the nucleus, suggesting additional regulatory functions. Here, we systematically characterize chromatin-associated small non-coding RNAs in the human pancreatic cancer cell line PANC-1. Using chromatin RNA immunoprecipitation coupled with small RNA sequencing, we show that the chromatin-associated small RNA population differs markedly from the bulk nuclear RNA pool and is strongly enriched in miRNAs. Among these, miR-21 represents the most abundant chromatin-associated species. Sequence analyses revealed that a subset of these miRNAs fulfills the requirements for RNA-DNA triplex formation at genomic regulatory regions. Gel-shift assays further demonstrate that Argonaute2 (Ago2) directly interacts with triple-helical nucleic acid structures in vitro, suggesting a potential mechanistic link between triplexes and Ago2-chromatin engagement. Evolutionary analyses indicate that these triplex-forming chromatin-associated miRNAs are largely restricted to anthropoid primates, in contrast to broadly conserved non-triplex-forming miRNAs. Together, our results identify a population of chromatin-associated miRNAs and provide evidence for a potential structural mechanism linking miRNAs, Ago2, and chromatin.

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Martin, M., Jalife, C., Mendez, C., Montecinos, C., Brown-Brown, D., Diaz-Tejeda, F., Caffi, V., Zavala, K., Kugler, F., Molina-Gonzalez, L., Valenzuela-Nieto, G., Laengst, G., Mardones, G. A., Munita, R., Opazo, J. C., Maldonado, R.. 2026-02-26. RNA-DNA triplex-forming miRNAs define an evolutionarily recent chromatin regulatory mechanism. https://doi.org/10.64898/2026.02.25.707792

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