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. 2006 Apr 26;34(7):2128-36.
doi: 10.1093/nar/gkl220. Print 2006.

Specific RNA binding to ordered phospholipid bilayers

Tadeusz Janas  1 , Teresa Janas, Michael Yarus
Affiliations

Specific RNA binding to ordered phospholipid bilayers

Tadeusz Janas et al. Nucleic Acids Res. .

Abstract

We have studied RNA binding to vesicles bounded by ordered and disordered phospholipid membranes. A positive correlation exists between bilayer order and RNA affinity. In particular, structure-dependent RNA binding appears for rafted (liquid-ordered) domains in sphingomyelin-cholesterol-1,2-dioleoyl-sn-glycero-3-phosphocholine vesicles. Binding to more highly ordered gel phase membranes is stronger, but much less RNA structure-dependent. All modes of RNA-membrane association seem to be electrostatic and headgroup directed. Fluorometry on 1,2-dimyristoyl-sn-glycero-3-phosphocholine liposomes indicates that bound RNA broadens the gel-fluid melting transition, and reduces lipid headgroup order, as detected via fluorometric measurement of intramembrane electric fields. RNA preference for rafted lipid was visualized and confirmed using multiple fluorophores that allow fluorescence and fluorescence resonance energy transfer microscopy on RNA molecules closely associated with ordered lipid patches within giant vesicles. Accordingly, both RNA structure and membrane order could modulate biological RNA-membrane interactions.

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Figures

Figure 1
Figure 1
Temperature-dependence of RNA 10 binding to DMPC liposomes measured by gel filtration. Elution profiles at 18°C (A), 23°C (B), 36°C (C); triangles—RNA without liposomes; circles—RNA reacted with liposomes; squares—liposomes with RNA. RNA concentration 0.25 µM, liposome concentration 10 mg/ml.
Figure 2
Figure 2
Effect of lipid bilayer order on RNA 10 affinity for large unilamellar phosphatidylcholine liposomes at 18°C (A), 23°C (B), 36°C (C); Pβ, lipid bilayer in ripple-gel phase; Lα, lipid bilayer in liquid-disordered phase; Pβ' + Lα, lipid bilayer at the main phase transition temperature. RNA concentration 0.25 µM, liposome concentration 10 mg/ml.
Figure 3
Figure 3
Effect of (A) temperature and (B) the ionic strength on RNA 80N binding to large unilamellar phosphatidylcholine liposomes. Pβ, lipid bilayer in ripple-gel phase; Lα, lipid bilayer in liquid-disordered phase; Pβ' + Lα, lipid bilayer at the main phase transition temperature. RNA concentration 0.25 µM, liposome concentration 10 mg/ml.
Figure 4
Figure 4
(A) Fluorescence anisotropy of DPH-HPC in DMPC liposomes in the presence (gray) and in the absence (black) of RNA 10; (B) Dipole-field-dependent ratio of fluorescence intensities, 440/540, of RH-421 in DMPC liposomes in the presence (gray) and in the absence (black) of RNA 10. Data were collected by temperature scanning at 1°C/min. RNA concentration 1 µM, liposome concentration 75 µM.
Figure 5
Figure 5
Cholesterol modulation of RNA 10 binding to LUV composed of DOPC/SM/CHOL. (A) Elution profiles obtained by gel filtration for DOPC/SM/CHOL (60:30:10 mol%, respectively) liposomes; triangles, RNA without liposomes; circles, RNA reacted with liposomes; squares, liposomes. (B) Cholesterol-dependence of RNA 10 binding to DOPC/SM/CHOL liposomes at constant (30 mol%) SM concentration. RNA concentration 0.75 µM, liposome concentration 2.5 mg/ml.
Figure 6
Figure 6
RNA 10 binding to liquid-ordered rafts visualized by FRET microscopy. (A) Blue, Pacific Blue-PE (PacBlue-PE) in giant lipid vesicles composed of DOPC/SM/CHOL (60:30:10 mol%, respectively) and 0.1 mol% of ganglioside GM1; green, YOYO-1 intercalated within RNA 10; red, AlexaFluor555 attached to cholera toxin B (AleFluor-CTB) bound to the ganglioside GM1 within liquid-ordered regions of giant lipid vesicles. (B) Blue, perylene in the liquid-ordered regions of giant lipid vesicles composed of DOPC/SM/CHOL (60:30:10 mol%, respectively); green, YOYO-1 intercalated in RNA 10; red, LissamineRhodamine-PE in giant lipid vesicle membranes. The width of the images is 8 µm.
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