BIOPHYSICAL AND BIOCHEMICAL CHARACTERIZATION OF NtRH35: A SPLICING-RELATED Nicotiana tabacum HELICASE

RNA helicase 35 (NtRH35) is a DEAD-box protein recently identified in Nicotiana tabacum through its interaction with SCI1 (Stigma/Style Cell-cycle Inhibitor 1), a regulator of cell proliferation in the floral meristem of various plant species. Previous studies have shown that NtRH35 binds RNA and associates with components of the splicing machinery, similar to its well-characterized human homolog, the DDX41 helicase. However, the molecular mechanisms underlying its function remain poorly understood, and plant homologs have not yet been thoroughly studied. In this work, we confirmed the NtRH35–SCI1 interaction in vitro and found it to be ligand-independent — it occurs even in the absence of nucleic acids, nucleotides, and under high-salt conditions. Preliminary isothermal titration calorimetry (ITC) experiments showed a strong interaction, with dissociation constants around 100 nM. We also confirmed the ATPase activity of NtRH35, while its helicase activity is currently under investigation. Additionally, truncated versions of both proteins were produced, allowing us to identify the specific regions involved in their interaction.

This work was supported by Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES 88887.172952/2025-00) and by Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP 19/24774-1).