Cellular conditions of weakly chelated magnesium ions strongly promote RNA stability and catalysis

Abstract

Most RNA folding studies have been performed under non-physiological conditions of high concentrations (≥10 mM) of Mg²⁺_free, while actual cellular concentrations of Mg²⁺_free are only ~1 mM in a background of greater than 50 mM Mg²⁺_total. To uncover cellular behavior of RNA, we devised cytoplasm mimic systems that include biological concentrations of amino acids, which weakly chelate Mg²⁺. Amino acid-chelated Mg²⁺ (aaCM) of ~15 mM dramatically increases RNA folding and prevents RNA degradation. Furthermore, aaCM enhance self-cleavage of several different ribozymes, up to 100,000-fold at Mg²⁺_free of just 0.5 mM, indirectly through RNA compaction. Other metabolites that weakly chelate magnesium offer similar beneficial effects, which implies chelated magnesium may enhance RNA function in the cell in the same way. Overall, these results indicate that the states of Mg²⁺ should not be limited to free and bound only, as weakly bound Mg²⁺ strongly promotes RNA function under cellular conditions.

Fig. 1

Amino acid-chelated magnesium promotes RNA thermostability. a Melting curves of drz-spur-3 ribozyme in bacterial conditions; 13.3 mM Mg²⁺_total (orange: Mg²⁺ high), 96 mM glutamate and 13.3 mM Mg²⁺_total (red: GluCM), 106 mM amino acids and 16.0 mM Mg²⁺_total (cyan: Aa₄CM), 11.3 mM EDTA and 13.3 mM Mg²⁺_total (green: EDTACM), and 2 mM Mg²⁺_total (blue: Mg²⁺ low). b, c Melting curves in b eukaryotic conditions and c 0.01 mM Mg²⁺_free condition, chosen to test Mg²⁺ sensitivity. The same colors as a indicate the same concentrations of the binding donors with different total magnesium concentrations (3.6 mM, 4.6 mM, or 0.5 mM, and 0.074 mM, 0.093 mM, or 0.01 mM, respectively)

Fig. 2

Amino acid-chelated magnesium reduces RNA degradation according to thermal denaturation experiment. Successive thermal denaturation experiments are conducted using cleaved drz-spur-3 ribozyme. a Closed and open symbols indicate first and second successive thermal denaturation curves, respectively. Arrows indicate decrease of the maximum dA_260nm·dT⁻¹ from the first melting curves to the second melting curves. Free Mg²⁺ is 2 mM, 0.5 mM, or 0.01 mM, while chelated Mg²⁺ was also present and achieved in different chelators as provided in the figure. b Summary of RNA stabilization in each condition. RNA stabilization at a given condition was estimated by dividing the maximum dA_{260 nm}·dT⁻¹ in the second melting curves by that in the first melting curves

Fig. 3

Amino acid-chelated magnesium reduces RNA degradation according to ILP reactivities. a ILP on the drz-spur-3 ribozyme. Lane 1–2 and 20–21 are RNase T1 ladders (G). Lane 3 and 19 are alkaline hydrolysis ladders (OH^–). Lane 4–6 are time-dependent ILPs in the presence of 13.3 mM Mg²⁺_total. Lane 7–9 are that in the presence of 96 mM glutamate and 13.3 mM Mg²⁺_total (2 mM Mg²⁺_free). Lane 10–12 are that in the presence of 106 mM amino acids and 16 mM Mg²⁺_total (2 mM Mg²⁺_free). Lane 13–15 are that in the presence of 11.3 mM EDTA and 13.3 mM Mg²⁺_total (2 mM Mg²⁺_free). Lane 16–18 are that in the presence of 2 mM Mg²⁺_total for a control. Concentrations of binding donors are the same as in Fig. 1. b Relative ILP reactivity at 24 h at each nucleotide position is shown. The relative reactivity in high Mg²⁺ condition was set at 1.0. The error bars show average errors (n = 2). c ILP reactivity in the presence of 13.3 mM Mg²⁺ referenced to U49. Nucleotides colored in blue show the positions where ILP reactivity is greater than 7.5% of U49. Red arrows denote positions of ILP reactivity where gluCM offers protection

Fig. 4

Amino acid-chelated magnesium stimulates self-cleavage of the CPEB3 ribozyme. a Self-cleaving reaction at 0.1 mM Mg²⁺_free condition. Fraction (f_cleaved) vs time plot is shown. Black open squares show 0.1 mM Mg²⁺_total as a control, red open squares show 0.66 mM EDTA, red filled squares show 96 mM glutamate, and black squares show 0.76 mM Mg²⁺_total. b Self-cleaving reaction at 0.5 mM Mg²⁺_free condition. c Self-cleaving reaction at 2 mM Mg²⁺_free condition. Concentrations of binding donors for b and c are provided in Fig. 1; for a, binding donor concentrations are 96 mM and 0.66 mM for glutamate and EDTA. d k_obs values for the cleaving reaction at the various magnesium conditions. The error bars mean S.D. (n = 4). The symbols and colors in b, c, and d are same with a

Fig. 5

Coordinated water molecules are critical for RNA stabilization. a Melting curves of drz-spur-3 ribozyme in the background of metabolites. 3 mM Mg²⁺_total (orange) and 28 mM of glutamate (red), malate (green), citrate (cyan), or EDTA (black) conditions were tested. As a control, 1.1 mM Mg²⁺_total (blue) condition was performed. The estimated concentrations of Mg²⁺_free are provided. K_D’s are given in the main text. b Chemical structure of glutamate and magnesium. Glutamate interacts with magnesium through N, O-chelation. c Chemical structure of EDTA and magnesium. EDTA interacts with magnesium through O, O, O, O, N, N-chelation

Fig. 6

Proposed effects of weakly chelated magnesium on RNA. Illustration of the effects of amino acids and aaCM for RNA. Amino acids alone increase the folding free energy of RNA, either having no effect or destabilizing the RNA. At the same time, aaCM decreases the folding free energy of RNA, stabilizing the RNA and promoting high catalytic activity