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RNA Binds to Tau Fibrils and Sustains Template-Assisted Growth.

Dinkel PD, Holden MR, Matin N, Margittai M - Biochemistry (2015)

Bottom Line: These structural features are similar to those previously observed for heparin-induced fibrils, indicating that basic conformational properties are conserved, despite their being molecular differences of the nucleating agents.Furthermore, RNA sustains template-assisted growth and binds to the fibril surface and can be exchanged by heparin.These findings suggest that, in addition to mediating fibrillization, cofactors decorating the surface of Tau fibrils may modulate biological interactions and thereby influence the spreading of Tau pathology in the human brain.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry and Biochemistry, University of Denver, Denver, Colorado 80208, United States.

ABSTRACT
Tau fibrils are the main proteinacious components of neurofibrillary lesions in Alzheimer disease. Although RNA molecules are sequestered into these lesions, their relationship to Tau fibrils is only poorly understood. Such understanding, however, is important, as short fibrils can transfer between neurons and nonproteinacious factors including RNA could play a defining role in modulating the latter process. Here, we used sedimentation assays combined with electron paramagnetic resonance (EPR), fluorescence, and absorbance spectroscopy to determine the effects of RNA on Tau fibril structure and growth. We observe that, in the presence of RNA, three-repeat (3R) and four-repeat (4R) Tau form fibrils with parallel, in-register arrangement of β-strands and exhibit an asymmetric seeding barrier in which 4R Tau grows onto 3R Tau seeds but not vice versa. These structural features are similar to those previously observed for heparin-induced fibrils, indicating that basic conformational properties are conserved, despite their being molecular differences of the nucleating agents. Furthermore, RNA sustains template-assisted growth and binds to the fibril surface and can be exchanged by heparin. These findings suggest that, in addition to mediating fibrillization, cofactors decorating the surface of Tau fibrils may modulate biological interactions and thereby influence the spreading of Tau pathology in the human brain.

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RNA binds to Tau fibrils. K18 and K19fibrils were formed for 2h at 37 °C from a mixture of Tau monomers (25 μM), respectiveseeds (10%), and polyA RNA (50 μg/mL). (A) Absorption spectraof solubilized K18 pellets (red), supernatants (blue), and total RNA(black). (B) Corresponding normalized intensities for experimentsperformed in triplicate. (C) Absorption spectra of solubilized K19pellets, supernatant, and total RNA (color coding as above). (D) Correspondingnormalized intensities for experiments performed in triplicate. Valuesrepresent mean ± SD. The data indicate that the majority of RNAis associated with K18 and K19 fibrils.
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fig4: RNA binds to Tau fibrils. K18 and K19fibrils were formed for 2h at 37 °C from a mixture of Tau monomers (25 μM), respectiveseeds (10%), and polyA RNA (50 μg/mL). (A) Absorption spectraof solubilized K18 pellets (red), supernatants (blue), and total RNA(black). (B) Corresponding normalized intensities for experimentsperformed in triplicate. (C) Absorption spectra of solubilized K19pellets, supernatant, and total RNA (color coding as above). (D) Correspondingnormalized intensities for experiments performed in triplicate. Valuesrepresent mean ± SD. The data indicate that the majority of RNAis associated with K18 and K19 fibrils.

Mentions: Next, we asked whether RNA physically associateswith Tau fibrils or whether the interactions are of transient nature.For this purpose, K18 and K19 fibrils were formed in the presenceof polyA with 10% fibril seeds present to expedite aggregation. Aftersedimentation, the pellets were dissolved in buffer containing 2%SDS. The supernatants were adjusted to the same SDS concentrationas that of the pellets. Absorption spectra were taken of the supernatant,the solubilized pellets, and control samples that contained RNA only(Figure 4A,C). Theabsorption maxima at 260 nm are indicative of RNA. The protein itselfhas negligible absorbance at this wavelength, as it harbors only asingle aromatic tyrosine. Quantification of three independent experimentsfor each K18 and K19 seeded growth reveals that the majority of RNA(∼90%) became incorporated into the fibrils (Figure 4B,D). On the basis of the sizedistribution of RNA (0.2–2 kb), one RNA molecule bound to ∼40–400Tau molecules. Notably, dilution of Tau fibrils into buffer lackingRNA did not lead to the dissociation of Tau fibrils (Figure S1A). Furthermore, elevated concentrations of salt(500 mM NaCl) caused only minor dissociation of RNA (Figure S1B) and had no effect on fibril mass (Figure S1C). The findings suggest that RNA isfirmly associated with the fibrils.


RNA Binds to Tau Fibrils and Sustains Template-Assisted Growth.

Dinkel PD, Holden MR, Matin N, Margittai M - Biochemistry (2015)

RNA binds to Tau fibrils. K18 and K19fibrils were formed for 2h at 37 °C from a mixture of Tau monomers (25 μM), respectiveseeds (10%), and polyA RNA (50 μg/mL). (A) Absorption spectraof solubilized K18 pellets (red), supernatants (blue), and total RNA(black). (B) Corresponding normalized intensities for experimentsperformed in triplicate. (C) Absorption spectra of solubilized K19pellets, supernatant, and total RNA (color coding as above). (D) Correspondingnormalized intensities for experiments performed in triplicate. Valuesrepresent mean ± SD. The data indicate that the majority of RNAis associated with K18 and K19 fibrils.
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Related In: Results  -  Collection

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Show All Figures
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fig4: RNA binds to Tau fibrils. K18 and K19fibrils were formed for 2h at 37 °C from a mixture of Tau monomers (25 μM), respectiveseeds (10%), and polyA RNA (50 μg/mL). (A) Absorption spectraof solubilized K18 pellets (red), supernatants (blue), and total RNA(black). (B) Corresponding normalized intensities for experimentsperformed in triplicate. (C) Absorption spectra of solubilized K19pellets, supernatant, and total RNA (color coding as above). (D) Correspondingnormalized intensities for experiments performed in triplicate. Valuesrepresent mean ± SD. The data indicate that the majority of RNAis associated with K18 and K19 fibrils.
Mentions: Next, we asked whether RNA physically associateswith Tau fibrils or whether the interactions are of transient nature.For this purpose, K18 and K19 fibrils were formed in the presenceof polyA with 10% fibril seeds present to expedite aggregation. Aftersedimentation, the pellets were dissolved in buffer containing 2%SDS. The supernatants were adjusted to the same SDS concentrationas that of the pellets. Absorption spectra were taken of the supernatant,the solubilized pellets, and control samples that contained RNA only(Figure 4A,C). Theabsorption maxima at 260 nm are indicative of RNA. The protein itselfhas negligible absorbance at this wavelength, as it harbors only asingle aromatic tyrosine. Quantification of three independent experimentsfor each K18 and K19 seeded growth reveals that the majority of RNA(∼90%) became incorporated into the fibrils (Figure 4B,D). On the basis of the sizedistribution of RNA (0.2–2 kb), one RNA molecule bound to ∼40–400Tau molecules. Notably, dilution of Tau fibrils into buffer lackingRNA did not lead to the dissociation of Tau fibrils (Figure S1A). Furthermore, elevated concentrations of salt(500 mM NaCl) caused only minor dissociation of RNA (Figure S1B) and had no effect on fibril mass (Figure S1C). The findings suggest that RNA isfirmly associated with the fibrils.

Bottom Line: These structural features are similar to those previously observed for heparin-induced fibrils, indicating that basic conformational properties are conserved, despite their being molecular differences of the nucleating agents.Furthermore, RNA sustains template-assisted growth and binds to the fibril surface and can be exchanged by heparin.These findings suggest that, in addition to mediating fibrillization, cofactors decorating the surface of Tau fibrils may modulate biological interactions and thereby influence the spreading of Tau pathology in the human brain.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry and Biochemistry, University of Denver, Denver, Colorado 80208, United States.

ABSTRACT
Tau fibrils are the main proteinacious components of neurofibrillary lesions in Alzheimer disease. Although RNA molecules are sequestered into these lesions, their relationship to Tau fibrils is only poorly understood. Such understanding, however, is important, as short fibrils can transfer between neurons and nonproteinacious factors including RNA could play a defining role in modulating the latter process. Here, we used sedimentation assays combined with electron paramagnetic resonance (EPR), fluorescence, and absorbance spectroscopy to determine the effects of RNA on Tau fibril structure and growth. We observe that, in the presence of RNA, three-repeat (3R) and four-repeat (4R) Tau form fibrils with parallel, in-register arrangement of β-strands and exhibit an asymmetric seeding barrier in which 4R Tau grows onto 3R Tau seeds but not vice versa. These structural features are similar to those previously observed for heparin-induced fibrils, indicating that basic conformational properties are conserved, despite their being molecular differences of the nucleating agents. Furthermore, RNA sustains template-assisted growth and binds to the fibril surface and can be exchanged by heparin. These findings suggest that, in addition to mediating fibrillization, cofactors decorating the surface of Tau fibrils may modulate biological interactions and thereby influence the spreading of Tau pathology in the human brain.

Show MeSH
Related in: MedlinePlus