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The role of RNA in prion aggregation and disease
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.ORCID iD: 0000-0003-4448-6447
2018 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

As humanity evolved to witness an exceptionally high standard of living, Alzheimer’s, cancer and diabetes gradually replaced infections as the main limiting factors in longevity. It is both disturbing and captivating that such degenerative conditions are caused by the most ubiquitous biomolecule – the protein. Indeed, proteins are not only the most functional, but also the least understood of the cellular biopolymers. It is then not surprising that many severe human ailments are associated with aberrant proteostasis. The key, causative mechanism of proteinopathy is protein aggregation. Naturally occurring and sometimes functional, aggregation is an auxiliary pathway in protein folding. In the context of a crowded cellular environment, folding and aggregation are the least and one of the least understood molecular processes, respectively. Unravelling one can help deconstruct the other and vice versa, but also can provide mechanistic insight on degenerative proteinopathies. A special class of proteins, which appear to propagate their own aggregation, occupy center-stage in the scientific field devoted to this goal. These proteins known as prions, can exist in at least two distinct forms. With the human prion, one of those is functional and benign and the other is infectious, aggregation prone, self-replicating and fatally pathogenic. As it happens, prion disease shares many of the descriptive features of other proteinaceous neuropathies. That, and the seductive idea that prions dwell in the twilight zone between folding and aggregation, have made the prion phenomenon a fixation for many molecular biologists. This thesis, although not the product of fixation, deals with one aspect of the prion process – the involvement of a molecular cofactor.

Of all plausible adjuvants, RNAs have been proposed as likely participants in the prion process. Their prominent secondary structures and attractive polyanionic surfaces allow RNAs to freely engage in interactions, at times transmitting conformational information through induced fit effects. The present work summarizes the influence of various RNAs on the aggregation profiles of three prionogenic model systems. The produced results indicate a generic role for RNA in the molecular processes prion propagation and aggregation. Altogether, this study illustrates a previously overlooked RNA function, of potential relevance for protein-based disease. 

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2018. , p. 81
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 1620
National Category
Biochemistry and Molecular Biology
Research subject
Molecular Cellbiology
Identifiers
URN: urn:nbn:se:uu:diva-338857ISBN: 978-91-513-0208-9 (print)OAI: oai:DiVA.org:uu-338857DiVA, id: diva2:1173858
Public defence
2018-03-02, A1:107a, BMC, Husargatan 3, Uppsala, 09:00 (English)
Opponent
Supervisors
Available from: 2018-02-07 Created: 2018-01-14 Last updated: 2018-03-08
List of papers
1. Distinct modulatory role of RNA in the aggregation of the tumor suppressor protein p53 core domain.
Open this publication in new window or tab >>Distinct modulatory role of RNA in the aggregation of the tumor suppressor protein p53 core domain.
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2017 (English)In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 292, no 22, p. 9345-9357Article in journal (Refereed) Published
Abstract [en]

Inactivation of the tumor suppressor protein p53 by mutagenesis, chemical modification, protein-protein interaction, or aggregation has been associated with different human cancers. Although DNA is the typical substrate of p53, numerous studies have reported p53 interactions with RNA. Here, we have examined the effects of RNA of varied sequence, length, and origin on the mechanism of aggregation of the core domain of p53 (p53C) using light scattering, intrinsic fluorescence, transmission electron microscopy, thioflavin-T binding, seeding, and immunoblot assays. Our results are the first to demonstrate that RNA can modulate the aggregation of p53C and full-length p53. We found bimodal behavior of RNA in p53C aggregation. A low RNA:protein ratio (∼1:50) facilitates the accumulation of large amorphous aggregates of p53C. By contrast, at a high RNA:protein ratio (≥1:8), the amorphous aggregation of p53C is clearly suppressed. Instead, amyloid p53C oligomers are formed that can act as seeds nucleating de novo aggregation of p53C. We propose that structured RNAs prevent p53C aggregation through surface interaction and play a significant role in the regulation of the tumor suppressor protein.

Place, publisher, year, edition, pages
American Society for Biochemistry and Molecular Biology, 2017
Keywords
RNA, amyloid, domain V of 23S rRNA, fluorescence, kinetics, p53, p53C, prion, protein aggregation, protein folding
National Category
Cell Biology
Identifiers
urn:nbn:se:uu:diva-327976 (URN)10.1074/jbc.M116.762096 (DOI)000402538900028 ()28420731 (PubMedID)
Available from: 2017-08-14 Created: 2017-08-14 Last updated: 2018-01-14
2. Direct involvement of RNA in mammalian prion protein aggregation: Involvement of RNA in rPrP aggregation
Open this publication in new window or tab >>Direct involvement of RNA in mammalian prion protein aggregation: Involvement of RNA in rPrP aggregation
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(English)In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351XArticle in journal (Other academic) Submitted
Abstract [en]

Whether nucleic acids act as cofactors in the aggregation of prion proteins is still under debate. By employing RNAs of various source and size, we have studied the role of RNA in the aggregation of murine recombinant prion protein (rPrP23-231) using Rayleigh light scattering, dynamic light scattering, sedimentation, transmission electron microscopy, circular dichroism and isothermal titration calorimetry. We find that RNA modulates rPrP23-231 aggregation in a concentration dependent manner, affecting both the extent and rate of the process; the latter evident from fast kinetics measurements of rPrP23-231 aggregation using stopped-flow technique. At lower concentration, RNA stimulates amorphous aggregation of rPrP23-231, and at higher concentration it, instead, facilitates formation of oligomeric species capable of seeding de novo aggregation of rPrP23-231. Furthermore, RNA co-sediments with rPrP23-231. This leads to partial RNase resistance of RNA and secondary structure alterations in the protein, indicating a direct interaction between the two. Sequence analysis of the RNA co-aggregated with rPrP23-231 suggests that the interaction is not specific to RNA sequence. Alternatively, rPrP23-231 interaction with RNA appears site-specific and mediated by the N-terminus. Our study demonstrates the effective modulation of rPrP23-231 aggregation by RNA and puts forward the idea of the potential role of RNA in protein aggregation as a whole.

Keywords
prion, RNA, cofactor, RNA-protein interaction, aggregation, kinetics
National Category
Biochemistry and Molecular Biology
Research subject
Molecular Cellbiology
Identifiers
urn:nbn:se:uu:diva-338856 (URN)
Available from: 2018-01-14 Created: 2018-01-14 Last updated: 2018-01-14
3. Intervention of ribosomal RNA in HET-s prion aggregation Intervention of ribosomal RNA in HET-s prion aggregation
Open this publication in new window or tab >>Intervention of ribosomal RNA in HET-s prion aggregation Intervention of ribosomal RNA in HET-s prion aggregation
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(English)Manuscript (preprint) (Other academic)
Abstract [en]

The role of nucleic acids in prion aggregation / disaggregation has remained unclear. Here, using HET-s prion from Podospora anserina as a model system, we have studied the role of RNA, particularly different domains of ribosomal RNA, in its aggregation process. Our results show that domain V rRNA, from the large subunit of the ribosome, substantially prevents amyloid aggregation of the HET-s prion in a concentration dependent manner. Instead, it promotes the formation of oligomeric seeds, which facilitate de novo HET-s aggregation. The interaction sites for the HET-s prion on domain V rRNA were also identified and shown to overlap with the sites previously found to responsible for the protein folding activity of the ribosome (PFAR). This study provides a missing link between the role of rRNA-based PFAR and prion propagation.

National Category
Biochemistry and Molecular Biology
Research subject
Molecular Cellbiology
Identifiers
urn:nbn:se:uu:diva-338605 (URN)
Available from: 2018-01-14 Created: 2018-01-14 Last updated: 2018-01-14

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