Yeast Nst1 is a Novel Component of P-bodies and is a Specific Suppressor of Proteasome Base Assembly Defects

Proteasome assembly utilizes multiple dedicated assembly chaperones and is regulated by signaling pathways that respond to diverse stress conditions. To discover new factors influencing proteasome base assembly, we screened a tiled high-copy yeast genomic library to identify dosage suppressors of a temperature-sensitive proteasome regulatory particle (RP) base mutant. The screen identified Nst1, a protein that when overexpressed, specifically suppressed the temperature sensitivity and proteasome-assembly defects of multiple base mutants. Nst1 overexpression reduced cytosolic RP ATPase (Rpt) aggregates in nas6Δ rpn14Δ cells, which lack two RP assembly chaperones. Nst1 is highly polar and predicted to have numerous intrinsically disordered regions, characteristics commonly found in proteins that can segregate into membraneless condensates. In agreement with this, both endogenous and overexpressed Nst1 could form cytosolic puncta that co-localized with processing body (P-body) components. Consistent with the accumulation of translationally inactive mRNAs in P-bodies, Nst1 overexpression inhibited global protein translation in nas6Δ rpn14Δ cells. Translational inhibition is known to suppress aggregation and proteasome assembly defects in base mutants under heat stress . Our data indicate that Nst1 is a previously overlooked P-body component that when expressed at elevated levels inhibits translation, prevents Rpt subunit aggregation, and rescues proteasome assembly under stress conditions.

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The arrested state of processing bodies supports mRNA regulation in early development

10.1101/2021.03.16.435709 ◽

2021 ◽

Cited By ~ 1

Author(s):

M. Sankaranarayanan ◽

Ryan J. Emenecker ◽

Marcus Jahnel ◽

Irmela R. E. A. Trussina ◽

Matt Wayland ◽

...

Keyword(s):

Physical Properties ◽

Electrostatic Interactions ◽

Processing Bodies ◽

P Bodies ◽

Intrinsically Disordered ◽

Intrinsically Disordered Regions ◽

Dead Box ◽

Premature Release ◽

Liquid Liquid Phase Separation

ABSTRACTBiomolecular condensates that form via liquid-liquid phase separation can exhibit diverse physical states. Despite considerable progress, the relevance of condensate physical states forin vivobiological function remains limited. Here, we investigated the physical properties ofin vivoprocessing bodies (P bodies) and their impact on mRNA storage in matureDrosophilaoocytes. We show that the conserved DEAD-box RNA helicase Me31B forms P body condensates which adopt a less dynamic, arrested physical state. We demonstrate that structurally distinct proteins and hydrophobic and electrostatic interactions, together with RNA and intrinsically disordered regions, regulate the physical properties of P bodies. Finally, using live imaging, we show that the arrested state of P bodies is required to prevent the premature release ofbicoid(bcd) mRNA, a body axis determinant, and that P body dissolution leads tobcdrelease. Together, this work establishes a role for arrested states of biomolecular condensates in regulating cellular function in a developing organism.

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Novel LOTUS-domain proteins are organizational hubs that recruit C. elegans Vasa to germ granules

eLife ◽

10.7554/elife.60833 ◽

2021 ◽

Vol 10 ◽

Author(s):

Patricia Giselle Cipriani ◽

Olivia Bay ◽

John Zinno ◽

Michelle Gutwein ◽

Hin Hark Gan ◽

...

Keyword(s):

Rna Processing ◽

Germ Line ◽

Temperature Sensitive ◽

Developmental Transitions ◽

P Granules ◽

C Elegans ◽

Intrinsically Disordered ◽

Meiotic Progression ◽

Intrinsically Disordered Regions ◽

Granule Growth

We describe MIP-1 and MIP-2, novel paralogous C. elegans germ granule components that interact with the intrinsically disordered MEG-3 protein. These proteins promote P granule condensation, form granules independently of MEG-3 in the postembryonic germ line, and balance each other in regulating P granule growth and localization. MIP-1 and MIP-2 each contain two LOTUS domains and intrinsically disordered regions and form homo- and heterodimers. They bind and anchor the Vasa homolog GLH-1 within P granules and are jointly required for coalescence of MEG-3, GLH-1, and PGL proteins. Animals lacking MIP-1 and MIP-2 show temperature-sensitive embryonic lethality, sterility, and mortal germ lines. Germline phenotypes include defects in stem cell self-renewal, meiotic progression, and gamete differentiation. We propose that these proteins serve as scaffolds and organizing centers for ribonucleoprotein networks within P granules that help recruit and balance essential RNA processing machinery to regulate key developmental transitions in the germ line.

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The ArathEULS3 Lectin Ends up in Stress Granules and Can Follow an Unconventional Route for Secretion

International Journal of Molecular Sciences ◽

10.3390/ijms21051659 ◽

2020 ◽

Vol 21 (5) ◽

pp. 1659

Author(s):

Malgorzata Dubiel ◽

Tibo De De Coninck ◽

Vinicius Jose Silva Osterne ◽

Isabel Verbeke ◽

Daniël Van Van Damme ◽

...

Keyword(s):

Stress Granules ◽

Protein Translation ◽

Bioinformatics Analyses ◽

Lectin Domain ◽

Intrinsically Disordered ◽

Intrinsically Disordered Regions ◽

Terminal Domain ◽

Unknown Structure ◽

Stressed Cells ◽

And Function

Stress granules are cytoplasmic compartments, which serve as mRNA storage units during stress, therefore regulating translation. The Arabidopsis thaliana lectin ArathEULS3 has been widely described as a stress inducible gene. This study aimed to examine in detail the localization of ArathEULS3 lectin in normal and stressed cells. Colocalization experiments revealed that the nucleo-cytoplasmic lectin ArathEULS3 relocates to stress granules after stress. The ArathEULS3 sequence encodes a protein with a EUL lectin domain and an N-terminal domain with unknown structure and function. Bioinformatics analyses showed that the N-terminal domain sequence contains intrinsically disordered regions and likely does not exhibit a stable protein fold. Plasmolysis experiments indicated that ArathEULS3 also localizes to the apoplast, suggesting that this protein might follow an unconventional route for secretion. As part of our efforts we also investigated the interactome of ArathEULS3 and identified several putative interaction partners important for the protein translation process.

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P-Body Components Are Required for Ty1 Retrotransposition during Assembly of Retrotransposition-Competent Virus-Like Particles

Molecular and Cellular Biology ◽

10.1128/mcb.00251-09 ◽

2009 ◽

Vol 30 (2) ◽

pp. 382-398 ◽

Cited By ~ 52

Author(s):

Mary Ann Checkley ◽

Kunio Nagashima ◽

Stephen J. Lockett ◽

Katherine M. Nyswaner ◽

David J. Garfinkel

Keyword(s):

Saccharomyces Cerevisiae ◽

Immunofluorescence Microscopy ◽

Wild Type ◽

Virus Like Particles ◽

P Bodies ◽

Cellular Processes ◽

Body Component ◽

Body Components ◽

Ty1 Retrotransposition

ABSTRACT Ty1 is a retrovirus-like retrotransposon whose replication is influenced by diverse cellular processes in Saccharomyces cerevisiae. We have identified cytoplasmic P-body components encoded by DHH1, KEM1, LSM1, and PAT1 as cofactors that posttranscriptionally enhance Ty1 retrotransposition. Using fluorescent in situ hybridization and immunofluorescence microscopy, we found that Ty1 mRNA and Gag colocalize to discrete cytoplasmic foci in wild-type cells. These foci, which are distinct from P-bodies, do not form in P-body component mutants or under conditions suboptimal for retrotransposition. Our immunoelectron microscopy (IEM) data suggest that mRNA/Gag foci are sites where virus-like particles (VLPs) cluster. Overexpression of Ty1 leads to a large increase in retrotransposition in wild-type cells, which allows VLPs to be detected by IEM. However, retrotransposition is still reduced in P-body component mutants under these conditions. Moreover, the percentage of Ty1 mRNA/Gag foci and VLP clusters and levels of integrase and reverse transcriptase are reduced in these mutants. Ty1 antisense RNAs, which have been reported to inhibit Ty1 transposition, are more abundant in the kem1Δ mutant and colocalize with Ty1 mRNA in the cytoplasm. Therefore, Kem1p may prevent the aggregation of Ty1 antisense and mRNAs. Overall, our results suggest that P-body components enhance the formation of retrotransposition-competent Ty1 VLPs.

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Novel LOTUS-domain proteins are organizational hubs that recruit C. elegans Vasa to germ granules

10.1101/2021.06.17.448425 ◽

2021 ◽

Author(s):

Patricia Giselle Cipriani ◽

Olivia Bay ◽

John Peter Zinno ◽

Michelle Gutwein ◽

Hin Hark Gan ◽

...

Keyword(s):

Rna Processing ◽

Germ Line ◽

Temperature Sensitive ◽

Developmental Transitions ◽

P Granules ◽

C Elegans ◽

Intrinsically Disordered ◽

Meiotic Progression ◽

Intrinsically Disordered Regions ◽

Granule Growth

We describe MIP-1 and MIP-2, novel paralogous C. elegans germ granule components that interact with the intrinsically disordered MEG-3 protein. These proteins promote P granule condensation, form granules independently of MEG-3 in the postembryonic germ line and balance each other in regulating P granule growth and localization. MIP-1 and MIP-2 each contain two LOTUS domains and intrinsically disordered regions and form homo- and heterodimers. They bind and anchor the Vasa homolog GLH-1 within P granules and are jointly required for coalescence of MEG-3, GLH-1, and PGL proteins. Animals lacking MIP-1 and MIP-2 show temperature-sensitive embryonic lethality, sterility, and mortal germ lines. Germline phenotypes include defects in stem cell self-renewal, meiotic progression, and gamete differentiation. We propose that these proteins serve as scaffolds and organizing centers for ribonucleoprotein networks within P granules that help recruit and balance essential RNA processing machinery to regulate key developmental transitions in the germ line.

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