Poster abstracts
Poster number 21 submitted by Koppany Bodor
Magnesium Ions Increase, but Potassium Ions Decrease, the Thermal Stability of the MALAT1 RNA Triple Helix
Koppany T. Bodor (Department of Chemistry and Biochemistry, University of Notre Dame), Mika J. Schievelbein (Department of Chemistry and Biochemistry, University of Notre Dame), Jessica A. Brown (Department of Chemistry and Biochemistry, University of Notre Dame)
Abstract:
Accumulation of human metastasis-associated lung adenocarcinoma transcript 1 (MALAT1), a nuclear-localized long noncoding RNA, is associated with cancer. A unique feature of MALAT1 is a 3′-terminal triple helix, which prevents degradation. The triple helix has one confirmed binding-partner: methyltransferase-like protein 16 (METTL16), an mRNA methyltransferase that binds but does not modify the triple helix. Previous studies have suggested that METTL16 preferentially binds to a less thermostable form of the MALAT1 triple helix and that the thermal stability of the MALAT1 triple helix depends on salt concentrations. Salt concentrations are generally lower in cancer, which may favor a less stable triple helix that stabilizes the MALAT1 triple helix•METTL16 complex. This study tests how salt conditions modulate triple helix thermal stability and the MALAT1 triple helix•METTL16 binding interaction. Here, low (0.1 mM MgCl2 and 0-150 mM KCl), middle-low (0.5 mM MgCl2 and 0-150 mM KCl), middle-high (1 mM MgCl2 and 0-150 mM KCl), and high (5 mM MgCl2 and 0-150 mM KCl) ionic conditions were tested at pH 7 and pH 5. UV thermal denaturation assays showed up to three melting transitions, which represent a structured region of unknown identity (50-60°C), Hoogsteen interactions specific to the triple helix (60-70°C), and Watson-Crick interactions (70-80 °C). For the Hoogsteen melting transition, an increase in MgCl2 concentration correlates with increased stability, while KCl reduces stability. A lack of KCl hyper stabilizes the triple helical Hoogsteen interactions, as Hoogsteen and Watson-Crick denaturation peaks are indistinguishable. This result contrasts with the Watson-Crick melting transition, where the thermal stability positively correlates to increasing salt, whether it be monovalent or divalent. Salt-dependent trends at pH 5 are similar. However, at pH 5, the Hoogsteen melting transition is more stable (60-80 °C) due to increased cytosine protonation at the N3 position (pKa = 4.3). These findings will be correlated with electrophoretic mobility shift assays of MALAT1 triple helix•METTL16 to determine the relationship between triple helix stability and binding affinity. These results will show how cancer cell conditions may affect the MALAT1 triple helix•METTL16 complex.
Keywords: RNA Triple Helix