A Non-amyloid Prion Particle that Activates a Heritable Gene Expression Program - PubMed (original) (raw)
A Non-amyloid Prion Particle that Activates a Heritable Gene Expression Program
Anupam K Chakravarty et al. Mol Cell. 2020.
Abstract
Spatiotemporal gene regulation is often driven by RNA-binding proteins that harbor long intrinsically disordered regions in addition to folded RNA-binding domains. We report that the disordered region of the evolutionarily ancient developmental regulator Vts1/Smaug drives self-assembly into gel-like condensates. These proteinaceous particles are not composed of amyloid, yet they are infectious, allowing them to act as a protein-based epigenetic element: a prion [SMAUG+]. In contrast to many amyloid prions, condensation of Vts1 enhances its function in mRNA decay, and its self-assembly properties are conserved over large evolutionary distances. Yeast cells harboring [SMAUG+] downregulate a coherent network of mRNAs and exhibit improved growth under nutrient limitation. Vts1 condensates formed from purified protein can transform naive cells to acquire [SMAUG+]. Our data establish that non-amyloid self-assembly of RNA-binding proteins can drive a form of epigenetics beyond the chromosome, instilling adaptive gene expression programs that are heritable over long biological timescales.
Keywords: IDPs; RNA-binding proteins; epigenetics; non-amyloid prions; phase separation; post-transcriptional gene regulation.
Copyright © 2019 Elsevier Inc. All rights reserved.
Conflict of interest statement
DECLARATION OF INTERESTS
Authors declare no conflict of interest.
Figures
Figure 1:. Vts1 activity is enhanced in [SMAUG+] cells.
(A) Schematic of GFP-SRE+ reporter and genetic backgrounds used (top). Representative DIC and GFP images of cells expressing the GFP-SRE+ reporter. (B) Quantification of the micrographs. Data are means ± SEM from ~80 individual cells. (C) In vivo apparent degradation rate constants for GFP-SRE+ and GFP-SRE-mRNAs from single exponential fits. Data are means ± SEM from 3 biological replicates.
Figure 2.. IDR in Vts1 drive formation of condensates.
(A) Disorder probability plot of S. cerevisiae Vts1 and its domain architecture. Sequence and disorder conservation across 20 fungal species separated by ~200 MY of evolution. (B) Coomassie stained SDS-PAGE gel of IDR-Vts1, RBD-Vts1 and Vts1. (C) Size exclusion chromatography traces of Vts1, IDR-Vts1, and RBD-Vts1. Arrowheads indicate standards and their MW (in kDa) - Thyroglobulin (
); Ferritin (
); Aldolase (
), Conalbumin (
), Ovalbumin (
). (D) EMSA of fluorescein-labeled SRE+ RNA with Vts1 and RBD-Vts1. (E) Representative images of labeled Vts1, IDR-Vts1, RBD-Vts1, and SNAP tag alone in presence of crowder in DIC and SNAP549 channels. (F) Effect of concentration and time on Vts1 condensates.
Figure 3:. Properties of Vts1 condensates.
(A) FRAP curve of labeled Vts1 condensates. Trace depicts means ± SEM of 3 individual experiments. Insets show FRAP status at indicated times. Yellow dotted circle marks photobleached area. Scale bar is 1 μm. (B) Representative images showing SDS sensitivity of Vts1 condensates. (C) Seeding of SNAP488-labeled Vts1 (green signal) with pre-assembled SNAP549-labeled Vts1 (red signal). Buffer-matched controls with unassembled SNAP549 labeled Vts1 depicted on the left. (D) Seeding of Vts1 condensation by cell lysates from indicated yeast strains. (E) Reversibility of Vts1 condensates. (F) Vts1 condensates bind RNA. Representative images of Vts1 condensates (in red), SRE+ (top row) and SRE- RNA (bottom row) in blue, and their overlay (in magenta) are shown. (G) Quantification of fluorescein signal co-localized with Vts1 condensates. (H) Affinity precipitation of interactors with soluble and condensed Vts1. (I) Ratio of mean GFP intensity from GFP-SRE+ reporter in [SMAUG+] / naïve cells in wild-type and _ccr4_Δ strains. Dotted line marks the theoretical expectation if [SMAUG+] and CCR4 were genetic interactors.
Figure 4:. Vts1 condensates transform naïve cells into [_SMAUG+_] cells.
(A) Endogenous reporter used to assay [SMAUG+] (left). Lag times of strains with indicated genotype and prion status in medium lacking uracil (right). Bar depicts mean of 4 biological replicates. (B & C) (top) Schematic of protein transformation in WT-naïve and _vts1_Δ cells. (bottom) Histogram of lag times of individual transformants after incubation with Vts1 condensates (blue bars, black borders in WT-naïve; gray bars, black borders in _vts1_Δ cells) or BSA (gray bars in both) in WT naïve and _vts1_Δ cells respectively.
Figure 5:. [_SMAUG+_] drives a prion-based regulon.
[ (A) PCA of biological replicate transcriptomes from naïve, two independent [SMAUG+] inductants, and _vts1_Δ strains. (B) Volcano plot of -log10(adjusted p-values) vs. log2(fold change) of transcriptome-wide mRNA abundances in [SMAUG+] relative to naïve cells. The red dotted line indicates the significance cutoff (FDR=1%; Benjamini-Hochberg corrected). Teal square depicts ratio of RNA abundance in [SMAUG+] vs. naïve cells for ACT1 mRNA. (C) Network of physical and genetic interactions for transcripts uniquely downregulated in [SMAUG+] cells. Target RNAs were clustered by k-means. (D) Top Gene Ontology terms and associated genes that were downregulated in [SMAUG+] cells.
Figure 6:. [_SMAUG+_] provides an adaptive advantage.
[ (A) Growth curves for naïve and [SMAUG+] strains in different glucose concentrations. Each point depicts mean ± SEM of 3 biological replicates. (B) Competition experiment schematic. Normalized fluorescence intensities (Neon/Kate) when naïve and [SMAUG+] cells are co-cultured are depicted. Blue plot represents data obtained when [SMAUG+] cells were Neon-marked and naïve cells were Kate-marked; Gray plot represents data from the marker-swap experiment. Blue and gray solid lines depict linear fits of (Neon/Kate) intensities vs. time, and the shaded region bounded by dashed lines represents the 95% confidence interval over 3 biological replicates.
Figure 7:. Metazoan Vts1/Smaug homolog can form condensates and self-template.
(A) Disorder profile and domain architecture of hSmaug1. (B) Condensates formed by purified hSmaug1 in presence of crowder. (C) Transient overexpression experiment schematic. (D) Representative micrographs of strains harboring GFP-tagged hSmaug1 or GFP alone at different experimental stages. White arrows highlight puncta. (E & F) Quantification of micrographs from experimental regimen. (E) Plot of percent of cells with puncta; p-value represents the statistical significance of difference in pre-induction and withdrawal samples by Fisher’s exact test. (F) Scatter dot plot of number of puncta per cell; green bars represent mean±95% confidence interval of this distribution.
Comment in
- Yeast Sporulation and [SMAUG+] Prion: Faster Is Not Always Better.
Parfenova I, Barral Y. Parfenova I, et al. Mol Cell. 2020 Jan 16;77(2):203-204. doi: 10.1016/j.molcel.2019.12.017. Mol Cell. 2020. PMID: 31951543
References
- Aigle M, and Lacroute F (1975). Genetical aspects of [URE3], a non-mitochondrial, cytoplasmically inherited mutation in yeast. Mol Gen Genet 136, 327–335. - PubMed
- Alberti S (2017). Phase separation in biology. Curr Biol 27, R1097–R1102. - PubMed
Publication types
MeSH terms
Substances
Grants and funding
- DP2 GM119140/GM/NIGMS NIH HHS/United States
- K99 GM128180/GM/NIGMS NIH HHS/United States
- R01 AG063418/AG/NIA NIH HHS/United States
- S10 RR026780/RR/NCRR NIH HHS/United States
LinkOut - more resources
Full Text Sources
Molecular Biology Databases