Finely tuned conformational dynamics regulate the protective function of the lncRNA MALAT1 triple helix

Abstract

Nucleic acid triplexes may regulate many important biological processes. Persistent accumulation of the oncogenic 7-kb long noncoding RNA MALAT1 is dependent on an unusually long intramolecular triple helix. This triplex structure is positioned within a conserved ENE (element for nuclear expression) motif at the lncRNA 3' terminus and protects the entire transcript from degradation in a polyA-independent manner. A requisite 3' maturation step leads to triplex formation though the precise mechanism of triplex folding remains unclear. Furthermore, the contributions of several peripheral structural elements to triplex formation and protective function have not been determined. We evaluated the stability, conformational fluctuations, and function of this MALAT1 ENE triple helix (M1TH) protective element using in vitro mutational analyses coupled with biochemical and biophysical characterizations. Using fluorescence and UV melts, FRET, and an exonucleolytic decay assay we define a concerted mechanism for triplex formation and uncover a metastable, dynamic triplex population under near-physiological conditions. Structural elements surrounding the triplex regulate the dynamic M1TH conformational variability, but increased triplex dynamics lead to M1TH degradation. Taken together, we suggest that finely tuned dynamics may be a general mechanism regulating triplex-mediated functions.

Figure 1.

Peripheral elements regulate thermostability of the MALAT1 ENE triple helix. (A) Secondary structure of the full-length MALAT1 ENE triple helix (M1^TH) including Watson–Crick interactions (—), Hoogsteen interactions (), A-minor interactions (−−), and a wobble base pair (•). A 5′ GTP (grey) is added to the sequence to facilitate transcription. The A-rich tail is colored blue and structural regions are indicated in brackets. (B) The crystal structure of the MALAT1 triple helix core (PDB: 4PLX) (33). The three strands are colored orange, pink, and blue. The two short loops (gray) were engineered to facilitate crystallization. (C) Secondary structure of trimolecular M1^ABsT. M1^Bs is colored orange, M1^A is colored pink, and M1^Tail is colored blue, as in B. (D) Secondary structure of bimolecular M1^ET, which eliminates the basal linker. The ENE motif is colored cyan and the M1^Tail is colored as in C. (E) Secondary structure of bimolecular M1^AB, wherein the basal linker is included and the apical P2 helix is truncated. M1^A is colored as in C and M1^B is colored green. In C–E, a 5′ GTP (gray) is added to the sequence to facilitate transcription. (F) Normalized UV melt profiles for M1^TH (gray), M1^ABsT (orange), M1^ET (blue), M1^AB (green) from 20 to 85°C in 20 mM HEPES•KOH, pH 7.4, 1 mM MgCl₂, 25 mM NaCl and 25 mM KCl. Triplex formation for all constructs is explicitly demonstrated in Supplementary Figure S2. Triplex and duplex melting occur in the same transition for the bimolecular M1^AB. All experiments were performed in duplicate. A single representative melting profile is plotted. (G) Triplex melting temperatures (T_m,1) for the unimolecular, bimolecular, and trimolecular constructs, colored as in F. Large changes in T_m,1 for the bi- and tri-molecular RNA relative to the full-length RNA demonstrate the contributions of peripheral elements to triplex stability.

Figure 2.

Triplex stabilities vary across a matrix of monovalent and divalent concentrations. Melting profiles monitored by DS-FRET for M1^ET (A) and M1^AB (B) under four different magnesium concentrations (∼0.05, 0.2, 0.5 and 1 mM, with increasing concentrations colored from black to purple) in 25 mM total monovalent salt (equimolar NaCl and KCl). See Supplemental Figure S5 for samples of raw Cy3 and Cy5 melting profiles. Inset: Peaks in a plot of the negative derivative of the raw melting profile are used to determine T_m,1 for each experiment. (C, D) Tertiary thermal stability (T_m,1) landscapes of M1^ET (C) and M1^AB (D) monitored using DS-FRET across multiplexed ionic conditions (2.6, 5.7, 8.8, 15.1, 27.6, 52.6, 102.6 and 202.6 mM total monovalent salt (equimolar KCl and NaCl); and ∼0.05, 0.1, 0.2, 0.3, 0.5, 0.6, 0.8 and 1 mM MgCl₂). n = 3 experiments; S.D. A black circle on each stability landscape indicates approximate physiological ionic conditions (∼150 mM monovalent, 1 mM MgCl₂).

Figure 3.

Stability landscape of M1^TH determined by differential scanning fluorimetry. (A) Melting profiles of M1^TH monitored by RiboGreen fluorescence using DSF under four different magnesium concentrations (0.1, 0.2, 0.3 and 0.6 mM MgCl₂, with magnesium concentrations increased from black to purple) in 51 mM total monovalent salt (equimolar NaCl and KCl). Inset: The negative derivative of the raw melting profile is used to determine the melting temperature transitions. Two melting transitions are recorded by DSF, with T_m values consistent with UV melt experiments (Table S2). (B) Tertiary thermal stability landscape of M1^TH monitored by DSF under multiplexed ionic conditions (2.6, 5.7, 8.8, 15.1, 27.6, 52.6, 102.6 and 202.6 mM total monovalent salt; and 0.1, 0.2, 0.3, 0.5, 0.6, 0.8 and 1 mM MgCl₂). n = 3 experiments; S.D. Thermostability of the unimolecular M1^TH is considerably higher than physiological temperatures. A circle on the stability landscape indicates approximate physiological ionic conditions (∼150 mM monovalent, 1 mM MgCl₂).

Figure 4.

Triplex conformational variability. Conformational landscapes of (A) M1^ET and (B) M1^AB monitored by FRET (E_FRET = I_Cy5 / (I_Cy3 + I_Cy5) at room temperature in different ionic conditions (2.6, 5.7, 8.8, 15.1, 27.6, 52.6, 102.6 and 202.6 mM total monovalent salt (equimolar NaCl and KCl); and ∼0.05, 0.1, 0.2, 0.3, 0.5, 0.6, 0.8 and 1 mM MgCl₂). n = 3 experiments; S.D. E_FRET at Conditions I, II, III and IV are circled (Condition I: 2.6 mM total monovalent salt, 1 mM MgCl₂; Condition II: 2.6 mM total monovalent salt, 0.1 mM MgCl₂; Condition III: 52.6 mM total monovalent salt, 0.1 mM MgCl₂; Condition IV: ∼150 mM total monovalent salt, 1 mM MgCl₂). Condition IV represents near-physiological ionic conditions. Maximal E_FRET is observed in high MgCl₂ concentration and low monovalent concentration (Condition I, top left of each panel). Minimal E_FRET is recorded in low MgCl₂ concentration and medium monovalent concentration (Condition III, bottom middle of each panel).

Figure 5.

3′-5′ exonucleolytic degradation of the MALAT1 ENE triplex. (A) Schematic of exonucleolytic triplex degradation by RNase R. Sequestration of the 3′ end precludes degradation by 3′-5′ exonuclease RNase R while a highly dynamic, disrupted 3′ end leads to degradation of the entire RNA. (B) Secondary structure of M1^polyA, which includes two mutations, G88A and C89A (boxed) in middle of the triple helix. In (C) and (D) 0.5 μg RNA was incubated in 20 mM HEPES, pH 7.4 in ionic Conditions I-IV with 5 units of RNase R for 5 hours prior to analysis using 6% denaturing PAGE stained with ethidium bromide. (Condition I: 2.6 mM total monovalent salt, 1 mM MgCl₂; Condition II: 2.6 mM total monovalent salt, 0.1 mM MgCl₂; Condition III: 52.6 mM total monovalent salt, 0.1 mM MgCl₂; Condition IV: 152.6 mM total monovalent salt, 1 mM MgCl₂). (C) A representative denaturing PAGE image of the M1^polyA mutant reveals that the RNA is completely degraded under all four conditions tested. (D) A representative denaturing PAGE image demonstrates that M1^TH is not appreciably degraded in Conditions I, II, and IV but is completely degraded in Condition III (for full gel image, see Supplementary Figure S7). Additional replicates of experiments in C and D containing 1 U RNase R are shown in Supplementary Figure S6F and G.

Figure 6.

Maturation, folding, and degradation of the 3′ terminus of lncRNA MALAT1. The schematic depicts the ENE triplex region; faded lines indicate 5′ and 3′ ends not to scale. (1) The P2 helix is stably formed near a 3′ terminal tRNA-like domain in the nascent transcript (not shown). Transient, weak formation of putative U•U base pairs and a short P1 helix in the ENE motif occurs prior to triplex formation (dotted lines). Highly dynamic regions of the RNA structure are denoted by two curved lines. (2) Cleavage by RNase P produces the mature 3′ end containing an A-rich tail (dark gray). (3) In a concerted step, the A-rich tail forms triplex interactions with the U-rich regions and A-minor interactions (dashed lines) with the P1 helix. The continuous duplex-triplex-duplex stacking interactions (P1–triplex–P2) significantly restrict 3′ end dynamics. (4) 3′-5′ exonucleolytic degradation is slow when the triplex is well-formed. However, structural disruption or increased triplex dynamics lead to rapid degradation of the RNA.