Telomeric RNA and HP1α form interfacial clusters that stabilize HP1α-DNA condensates.

Phase separation of biomolecules into distinct material states is expanding our understanding of cellular processes, particularly nuclear chromatin organization linked to gene regulation. Heterochromatin, containing DNA, repressive histone marks, and HP1α protein, functions in gene silencing and telomeric regulation. While HP1α and DNA phase separation contributes to heterochromatin formation, underlying mechanisms remain unclear. Given telomeric RNAs' proposed role in heterochromatin formation, we investigated interactions between telomeric RNA, DNA, and HP1α. We demonstrate that HP1α phase separates with both Telomeric Repeat containing RNA (TERRA) and DNA. When both nucleic acids are present, HP1α forms multiphase condensates where HP1α-TERRA clusters localize at the HP1α-DNA phase interface. This multiphase architecture results from sequence-specific TERRA-HP1α interactions. TERRA RNA clusters stabilize DNA condensates and modulate their interfacial properties. This work reveals a previously uncharacterized role for TERRA transcripts in regulating HP1α condensates and provides insights into how structured non-coding RNAs influence protein condensate material properties and organization. These findings advance understanding of complex biomolecular interactions governing nuclear compartmentalization through cooperative phase separation mechanisms, highlighting the sophisticated regulatory roles of non-coding RNAs in chromatin organization.