In vivo mapping of the functional regions of the DEAD-box helicase Vasa

AbstractProcessing bodies (PBs) are cytoplasmic mRNP granules that assemble via liquid-liquid phase separation and are implicated in the decay or storage of mRNAs. How PB assembly is regulated in cells remains unclear. We recently identified the ATPase activity of the DEAD-box protein Dhh1 as a key regulator of PB dynamics and demonstrated that Not1, an activator of the Dhh1 ATPase and member of the CCR4-NOT deadenylase complex inhibits PB assembly in vivo [Mugler et al., 2016]. Here, we show that the PB component Pat1 antagonizes Not1 and promotes PB assembly via its direct interaction with Dhh1. Intriguingly, in vivo PB dynamics can be recapitulated in vitro, since Pat1 enhances the phase separation of Dhh1 and RNA into liquid droplets, whereas Not1 reverses Pat1-Dhh1-RNA condensation. Overall, our results uncover a function of Pat1 in promoting the multimerization of Dhh1 on mRNA, thereby aiding the assembly of large multivalent mRNP granules that are PBs.

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ATP hydrolysis activity of the DEAD box protein Rok1p is required for in vivo ROK1 function

Nucleic Acids Research ◽

10.1093/nar/27.13.2753 ◽

1999 ◽

Vol 27 (13) ◽

pp. 2753-2759 ◽

Cited By ~ 17

Author(s):

J.-Y. Oh ◽

J. Kim

Keyword(s):

Atp Hydrolysis ◽

Dead Box ◽

The Dead ◽

Hydrolysis Activity

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Analyses of the Functional Regions of DEAD-Box RNA “Helicases” with Deletion and Chimera Constructs Tested In Vivo and In Vitro

Journal of Molecular Biology ◽

10.1016/j.jmb.2011.08.032 ◽

2011 ◽

Vol 413 (2) ◽

pp. 451-472 ◽

Cited By ~ 25

Author(s):

Josette Banroques ◽

Olivier Cordin ◽

Monique Doère ◽

Patrick Linder ◽

N. Kyle Tanner

Keyword(s):

Rna Helicases ◽

Functional Regions ◽

Dead Box

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Pat1 promotes processing body assembly by enhancing the phase separation of the DEAD-box ATPase Dhh1 and RNA

eLife ◽

10.7554/elife.41415 ◽

2019 ◽

Vol 8 ◽

Cited By ~ 20

Author(s):

Ruchika Sachdev ◽

Maria Hondele ◽

Miriam Linsenmeier ◽

Pascal Vallotton ◽

Christopher F Mugler ◽

...

Keyword(s):

Phase Separation ◽

Direct Interaction ◽

Liquid Droplets ◽

Processing Bodies ◽

Dead Box ◽

The Dead ◽

Processing Body ◽

Liquid Liquid Phase Separation

Processing bodies (PBs) are cytoplasmic mRNP granules that assemble via liquid–liquid phase separation and are implicated in the decay or storage of mRNAs. How PB assembly is regulated in cells remains unclear. Previously, we identified the ATPase activity of the DEAD-box protein Dhh1 as a key regulator of PB dynamics and demonstrated that Not1, an activator of the Dhh1 ATPase and member of the CCR4-NOT deadenylase complex inhibits PB assembly in vivo (Mugler et al., 2016). Here, we show that the PB component Pat1 antagonizes Not1 and promotes PB assembly via its direct interaction with Dhh1. Intriguingly, in vivo PB dynamics can be recapitulated in vitro, since Pat1 enhances the phase separation of Dhh1 and RNA into liquid droplets, whereas Not1 reverses Pat1-Dhh1-RNA condensation. Overall, our results uncover a function of Pat1 in promoting the multimerization of Dhh1 on mRNA, thereby aiding the assembly of large multivalent mRNP granules that are PBs.

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ATP-Dependent Roles of the DEAD-Box Protein Mss116p in Group II Intron Splicing In Vitro and In Vivo

Journal of Molecular Biology ◽

10.1016/j.jmb.2011.05.047 ◽

2011 ◽

Vol 411 (3) ◽

pp. 661-679 ◽

Cited By ~ 23

Author(s):

Jeffrey P. Potratz ◽

Mark Del Campo ◽

Rachel Z. Wolf ◽

Alan M. Lambowitz ◽

Rick Russell

Keyword(s):

Group Ii Intron ◽

Intron Splicing ◽

Dead Box ◽

The Dead ◽

Group Ii

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The Immunodominant Antigen of an Ultraviolet-induced Regressor Tumor Is Generated by a Somatic Point Mutation in the DEAD Box Helicase p68

Journal of Experimental Medicine ◽

10.1084/jem.185.4.695 ◽

1997 ◽

Vol 185 (4) ◽

pp. 695-706 ◽

Cited By ~ 100

Author(s):

Purnima Dubey ◽

Ronald C. Hendrickson ◽

Stephen C. Meredith ◽

Christopher T. Siegel ◽

Jeffrey Shabanowitz ◽

...

Keyword(s):

Amino Acid ◽

Tumor Cells ◽

Amino Acid Substitution ◽

Single Amino Acid ◽

Coding Region ◽

Single Nucleotide Change ◽

Immunodominant Antigen ◽

Dead Box ◽

The Dead

The genetic origins of CD8+ T cell–recognized unique antigens to which mice respond when immunized with syngeneic tumor cells are unknown. The ultraviolet light-induced murine tumor 8101 expresses an H-2Kb-restricted immunodominant antigen, A, that induces cytolytic CD8+ T cells in vivo A+ 8101 cells are rejected by naive mice while A− 8101 tumor cells grow. To identify the antigen H-2Kb molecules were immunoprecipitated from A+ 8101 cells and peptides were eluted by acid. The sensitizing peptide was isolated by sequential reverse-phase HPLC and sequenced using microcapillary HPLC-triple quadruple mass spectrometry. The peptide, SNFVFAGI, matched the sequence of the DEAD box protein p68 RNA helicase except for a single amino acid substitution, caused by a single nucleotide change. This mutation was somatic since fibroblasts from the mouse of tumor origin expressed the wild-type sequence. The amino acid substitution created an anchor for binding of the mutant peptide to H-2Kb. Our results are consistent with mutant p68 being responsible for rejection of the tumor. Several functions of p68, which include nucleolar assembly and inhibition of DNA unwinding, may be mediated through its IQ domain, which was altered by the mutation. This is the first description of a somatic tumor–specific mutation in the coding region of a nucleic acid helicase.

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ATPase activity of the DEAD-box protein Dhh1 controls processing body formation

eLife ◽

10.7554/elife.18746 ◽

2016 ◽

Vol 5 ◽

Cited By ~ 50

Author(s):

Christopher Frederick Mugler ◽

Maria Hondele ◽

Stephanie Heinrich ◽

Ruchika Sachdev ◽

Pascal Vallotton ◽

...

Keyword(s):

Atpase Activity ◽

Atp Hydrolysis ◽

Liquid Droplets ◽

Processing Bodies ◽

Dead Box ◽

Posttranscriptional Gene Regulation ◽

The Dead ◽

Processing Body

Translational repression and mRNA degradation are critical mechanisms of posttranscriptional gene regulation that help cells respond to internal and external cues. In response to certain stress conditions, many mRNA decay factors are enriched in processing bodies (PBs), cellular structures involved in degradation and/or storage of mRNAs. Yet, how cells regulate assembly and disassembly of PBs remains poorly understood. Here, we show that in budding yeast, mutations in the DEAD-box ATPase Dhh1 that prevent ATP hydrolysis, or that affect the interaction between Dhh1 and Not1, the central scaffold of the CCR4-NOT complex and an activator of the Dhh1 ATPase, prevent PB disassembly in vivo. Intriguingly, this process can be recapitulated in vitro, since recombinant Dhh1 and RNA, in the presence of ATP, phase-separate into liquid droplets that rapidly dissolve upon addition of Not1. Our results identify the ATPase activity of Dhh1 as a critical regulator of PB formation.

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Function of the C-terminal Domain of the DEAD-box Protein Mss116p Analyzed in Vivo and in Vitro

Journal of Molecular Biology ◽

10.1016/j.jmb.2007.11.041 ◽

2008 ◽

Vol 375 (5) ◽

pp. 1344-1364 ◽

Cited By ~ 56

Author(s):

Georg Mohr ◽

Mark Del Campo ◽

Sabine Mohr ◽

Quansheng Yang ◽

Huijue Jia ◽

...

Keyword(s):

Terminal Domain ◽

Dead Box ◽

The Dead

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Blocking a dead-end assembly pathway creates a point of regulation for the DEAD-box ATPase Has1 and prevents platform misassembly

10.1101/2021.09.06.459192 ◽

2021 ◽

Author(s):

Xin Liu ◽

Haina Huang ◽

Katrin Karbstein

Keyword(s):

Rrna Processing ◽

Ribosomal Subunits ◽

Dead Box ◽

Assembly Pathway ◽

Dead End ◽

Biochemical Genetic ◽

The Dead ◽

Assembly Pathways

AbstractAssembly of ribosomal subunits occurs via parallel pathways, which accelerate the process and render it more robust. Nonetheless, in vitro analyses have also demonstrated that some assembly pathways are dead-ends, presumably due to rRNA misfolding. If and how these non-productive pathways are avoided during assembly in vivo remains unknown. Here we use a combination of biochemical, genetic, proteomic and structural analyses to demonstrate a role for assembly factors in biasing the folding landscape away from non-productive intermediates. By binding Rrp36, Rrp5 is prevented from forming a premature interaction with the platform, which leads to a dead-end intermediate, and a misassembled platform that is functionally defective. The DEAD-box ATPase Has1 separates Rrp5 and Rrp36, allowing Rrp5 to reposition to the platform, thereby promoting ribosome assembly and enabling rRNA processing. Thus, Rrp36 establishes an ATP-dependent regulatory point that ensures correct platform assembly by opening a new folding channel that avoids funnels to misfolding.

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The DEAD-box RNA helicase Ded1 from yeast is associated with the signal recognition particle (SRP), and its enzymatic activity is regulated by SRP21

10.1101/2020.11.08.373522 ◽

2020 ◽

Author(s):

Hilal Yeter-Alat ◽

Naïma Belgareh-Touzé ◽

Emmeline Huvelle ◽

Molka Mokdadi ◽

Josette Banroques ◽

...

Keyword(s):

Enzymatic Activity ◽

Rna Binding ◽

Signal Recognition Particle ◽

Rna Helicase ◽

Signal Recognition ◽

Dead Box ◽

Enzymatic Regulation ◽

The Dead

ABSTRACTThe DEAD-box RNA helicase Ded1 is an essential yeast protein involved in translation initiation. It belongs to the DDX3 subfamily of proteins implicated in developmental and cell-cycle regulation. In vitro, the purified Ded1 protein is an ATP-dependent RNA binding protein and an RNA-dependent ATPase, but it lacks RNA substrate specificity and enzymatic regulation. Here we demonstrate by yeast genetics, in situ localization and in vitro biochemical approaches that Ded1 is associated with, and regulated by, the signal recognition particle (SRP), which is a universally conserved ribonucleoprotein complex required for the co-translational translocation of polypeptides into the endoplasmic reticulum lumen and membrane. Ded1 is physically associated with SRP components in vivo and in vitro. Ded1 is genetically linked with SRP proteins. Finally, the enzymatic activity of Ded1 is inhibited by SRP21 with SCR1 RNA. We propose a model where Ded1 actively participates in the translocation of proteins during translation. Our results open a new comprehension of the cellular role of Ded1 during translation.

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