Proteomic analysis of Dhh1 complexes reveals a role for Hsp40 chaperone Ydj1 in yeast P-body assembly

Cary, Gregory A.; Vinh, Dani B.N.; Kuestner, Rolf E.; Dudley, Andrew T.

doi:10.1101/016402

Cited by 7 publications

(7 citation statements)

References 110 publications

(133 reference statements)

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“…Consistent with this localization, Hrr25 was detected in immunoprecipitates with several different P-body proteins ( Figure 1C; Figure S1B). Previous largerscale studies had identified potential interactions between Hrr25 and particular P-body constituents (Ito et al 2001;Ho et al 2002;Cary et al 2015). Moreover, we found that the mammalian ortholog of Hrr25, CK1d, was also found in P-body foci ( Figure 1D).…”

Section: Resultssupporting

confidence: 60%

The Activity-Dependent Regulation of Protein Kinase Stability by the Localization to P-Bodies

Zhang¹,

Shi²,

Varia³

et al. 2016

Genetics

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The eukaryotic cytoplasm contains a variety of ribonucleoprotein (RNP) granules in addition to the better-understood membrane-bound organelles. These granules form in response to specific stress conditions and contain a number of signaling molecules important for the control of cell growth and survival. However, relatively little is known about the mechanisms responsible for, and the ultimate consequences of, this protein localization. Here, we show that the Hrr25/CK1d protein kinase is recruited to cytoplasmic processing bodies (P-bodies) in an evolutionarily conserved manner. This recruitment requires Hrr25 kinase activity and the Dcp2 decapping enzyme, a core constituent of these RNP granules. Interestingly, the data indicate that this localization sequesters active Hrr25 away from the remainder of the cytoplasm and thereby shields this enzyme from the degradation machinery during these periods of stress. Altogether, this work illustrates how the presence within an RNP granule can alter the ultimate fate of the localized protein.KEYWORDS ribonucleoprotein granules; processing bodies; protein kinase; protein stability; Dcp2 decapping enzyme; casein kinase 1 T HE eukaryotic cell is subdivided into distinct functional areas by the presence of a variety of organelles. The best understood of these are the membrane-bound structures, like the nucleus, endoplasmic reticulum, and mitochondria. These traditional compartments are relatively stable and essential for the proper compartmentalization of the different reactions occurring in the cytoplasm. However, the cell also contains a collection of nonmembraneous organelles that are more dynamic in nature and form in response to particular cellular and environmental stimuli. Perhaps the two best characterized of these are the centrosome and nucleolus, which act as a microtubule-organizing center and a subnuclear site of ribosome assembly, respectively (Greenan et al. 2010;Brangwynne 2011;Brangwynne et al. 2011). This latter class also includes a number of recently identified cytoplasmic ribonucleoprotein (RNP) granules like the processing body (P-body) and stress granule (Anderson and Kedersha 2009;Balagopal and Parker 2009;Thomas et al. 2011). These cytoplasmic structures have been conserved through evolution and have been linked to a variety of human diseases, including certain neurodegenerative disorders, cancers, and autoimmune conditions (Li et al. 2013;Anderson et al. 2015). Despite this importance to human health, relatively little is known about the manner in which these RNP granules influence biological processes in the cell.These cytoplasmic RNP granules typically assemble in response to environmental stress or particular developmental cues (Thomas et al. 2011). Granule formation occurs as a consequence of the regulated coalescence of specific sets of proteins and translationally repressed mRNAs at discrete sites in the cytoplasm (Anderson and Kedersha 2009;Balagopal and Parker 2009). The presence of these core proteins often depends upon specific RNA-...

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Section: Resultssupporting

confidence: 60%

The Activity-Dependent Regulation of Protein Kinase Stability by the Localization to P-Bodies

Zhang¹,

Shi²,

Varia³

et al. 2016

Genetics

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“…We conclude that multiple proteins of intermediary metabolism show substantially increased RNA association upon sorbic acid exposure. Notably, a set of metabolic enzymes were previously reported to accumulate in P‐bodies and stress granules during stress response (Cary et al , ; Jain et al , ). Seven of these enzymes showed increased RNA interactions in PAR‐TRAPP following sorbic acid treatment, while none showed reduced interactions.…”

Section: Resultsmentioning

confidence: 99%

Defining the RNA interactome by total RNA ‐associated protein purification

Shchepachev

Bresson

Spanos

et al. 2019

Molecular Systems Biology

130

139

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The RNA binding proteome ( RBP ome) was previously investigated using UV crosslinking and purification of poly(A)‐associated proteins. However, most cellular transcripts are not polyadenylated. We therefore developed total RNA ‐associated protein purification ( TRAPP ) based on 254 nm UV crosslinking and purification of all RNA –protein complexes using silica beads. In a variant approach ( PAR ‐ TRAPP ), RNA s were labelled with 4‐thiouracil prior to 350 nm crosslinking. PAR ‐ TRAPP in yeast identified hundreds of RNA binding proteins, strongly enriched for canonical RBP s. In comparison, TRAPP identified many more proteins not expected to bind RNA , and this correlated strongly with protein abundance. Comparing TRAPP in yeast and E. coli showed apparent conservation of RNA binding by metabolic enzymes. Illustrating the value of total RBP purification, we discovered that the glycolytic enzyme enolase interacts with tRNA s. Exploiting PAR ‐ TRAPP to determine the effects of brief exposure to weak acid stress revealed specific changes in late 60S ribosome biogenesis. Furthermore, we identified the precise sites of crosslinking for hundreds of RNA –peptide conjugates, using iTRAPP , providing insights into potential regulation. We conclude that TRAPP is a widely applicable tool for RBP ome characterization.

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“…We conclude that multiple proteins of intermediary metabolism show substantially increased RNA association upon sorbic acid exposure. Notably, a set of metabolic enzymes were previously reported to accumulate in P-bodies and stress granules during stress response (Cary et al, 2015;Jain et al, 2016). Seven of these enzymes showed increased RNA interactions in PAR-TRAPP following sorbic acid treatment, while none showed reduced interactions.…”

Section: Trapp Reveals Dynamic Changes In Rna-protein Interaction Folmentioning

confidence: 97%

Defining the RNA Interactome by Total RNA-Associated Protein Purification

Shchepachev

Bresson

Spanos

et al. 2018

Preprint

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UV crosslinking can be used to identify precise RNA targets for individual proteins, transcriptome-wide. We sought to develop a technique to generate reciprocal data, identifying precise sites of RNA-binding proteome-wide. The resulting technique, total RNA-associated protein purification (TRAPP), was applied to yeast (S. cerevisiae) and bacteria (E. coli). In all analyses, SILAC labelling was used to quantify protein recovery in the presence and absence of irradiation. For S. cerevisiae, we also compared crosslinking using 254 nm (UVC) irradiation (TRAPP) with 4-thiouracil (4tU) labelling combined with ~350 nm (UVA) irradiation (PAR-TRAPP). Recovery of proteins not anticipated to show RNA-binding activity was substantially higher in TRAPP compared to PAR-TRAPP. As an example of preferential TRAPP-crosslinking, we tested enolase (Eno1) and demonstrated its binding to tRNA loops in vivo. We speculate that many protein-RNA interactions have biophysical effects on localization and/or accessibility, by opposing or promoting phase separation for highly abundant protein. Homologous metabolic enzymes showed RNA crosslinking in S. cerevisiae and E. coli, indicating conservation of this property. TRAPP allows alterations in RNA interactions to be followed and we initially analyzed the effects of weak acid stress. This revealed specific alterations in RNA-protein interactions; for example, during late 60S ribosome subunit maturation. Precise sites of crosslinking at the level of individual amino acids (iTRAPP) were identified in 395 peptides from 155 unique proteins, following phospho-peptide enrichment combined with a bioinformatics pipeline (Xi). TRAPP is quick, simple and scalable, allowing rapid characterization of the RNA-bound proteome in many systems.

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Proteomic analysis of Dhh1 complexes reveals a role for Hsp40 chaperone Ydj1 in yeast P-body assembly

Cited by 7 publications

References 110 publications

The Activity-Dependent Regulation of Protein Kinase Stability by the Localization to P-Bodies

The Activity-Dependent Regulation of Protein Kinase Stability by the Localization to P-Bodies

Defining the RNA interactome by total RNA ‐associated protein purification

Defining the RNA Interactome by Total RNA-Associated Protein Purification

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