Discrimination between Purine and Pyrimidine-rich RNA in Liquid-Liquid Phase Separated Condensates with Cationic Peptides and the Effect of Artificial Crowding Agents

05 September 2024, Version 2
This content is a preprint and has not undergone peer review at the time of posting.

Abstract

Membraneless organelles – often referred to as condensates or coacervates – are liquid-liquid phase separated systems formed between non-coding RNAs and intrinsically disordered proteins. While the importance of different amino acid residues in short peptide-based condensates have been investigated, the role of the individual nucleobases or the type of hetero-cyclic structures; the purine viz pyrimidine nucleobases, is less researched. The cell’s crowded environment has been mimicked in vitro to demonstrate its ability to induce the formation of condensates, but more research in this area is required, especially with respect to RNA-facilitated phase separation, and the properties of the crowding agent, (poly)ethylene glycol (PEG). Herein we have shown that whether the nucleotide base sequence of RNA, can greatly influence its propensity to undergo phase separation with cationic peptides, with the purine-only RNA decamer (AG)5 readily do so while the pyrimidine-only (CU)5 does not. Furthermore, we show that the presence and size of a PEG macromolecular crowder affects both the ability to phase separate, and the stability of condensates formed. This work may shed light on the presence of low complexity long purine- or pyrimidine-rich non-complementary repeat (AG or CU) sequences in various non-coding RNAs found in bi-ology.

Keywords

RNA
Condensates
macromolecular crowding
coacervates
low-complexity long non-coding RNA
peptides
turbidity

Supplementary materials

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Description
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Supporting Information
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Experimental procedures, brightfield and confocal microscopy images, NMR spectra and other characterization data
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NMR data
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Raw NMR data files
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IR spectra of peptide
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IR spectra (TIF file)
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Turbidity data
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Turbidity data (excel format) from plate reader assay as a function of time.
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FRAP data
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FRAP data (excel format).
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