scholarly journals Dead or alive: DEAD-box ATPases as regulators of ribonucleoprotein complex condensation

2021 ◽  
Vol 402 (5) ◽  
pp. 653-661
Author(s):  
Karsten Weis
Keyword(s):  
Rna Processing ◽  
Atp Hydrolysis ◽  
Protein Complexes ◽  
Dependent Manner ◽  
Rna Helicases ◽  
Ribonucleoprotein Complexes ◽  
Dead Box ◽  
Rna Duplexes ◽  
Rna Protein Complexes

Abstract DEAD-box ATPase proteins are found in all clades of life and have been associated with a diverse array of RNA-processing reactions in eukaryotes, bacteria and archaea. Their highly conserved core enables them to bind RNA, often in an ATP-dependent manner. In the course of the ATP hydrolysis cycle, they undergo conformational rearrangements, which enable them to unwind short RNA duplexes or remodel RNA-protein complexes. Thus, they can function as RNA helicases or chaperones. However, when their conformation is locked, they can also clamp RNA and create ATP-dependent platforms for the formation of higher-order ribonucleoprotein complexes. Recently, it was shown that DEAD-box ATPases globally regulate the phase-separation behavior of RNA-protein complexes in vitro and control the dynamics of RNA-containing membraneless organelles in both pro- and eukaryotic cells. A role of these enzymes as regulators of RNA-protein condensates, or ‘condensases’, suggests a unifying view of how the biochemical activities of DEAD-box ATPases are used to keep cellular condensates dynamic and ‘alive’, and how they regulate the composition and fate of ribonucleoprotein complexes in different RNA processing steps.

scholarly journals Mutational Analysis of the Escherichia coli DEAD Box Protein CsdA

2007 ◽  
Vol 189 (7) ◽  
pp. 2769-2776 ◽  
Author(s):  
Anne-Marie W. Turner ◽  
Cheraton F. Love ◽  
Rebecca W. Alexander ◽  
Pamela G. Jones
Keyword(s):  
Escherichia Coli ◽  
Atp Hydrolysis ◽  
Mutational Analysis ◽  
Cold Shock Protein ◽  
Null Mutant ◽  
Rna Helicases ◽  
Dead Box ◽  
Rna Duplexes ◽  
The Dead

ABSTRACT The Escherichia coli cold shock protein CsdA is a member of the DEAD box family of ATP-dependent RNA helicases, which share a core of nine conserved motifs. The DEAD (Asp-Glu-Ala-Asp) motif for which this family is named has been demonstrated to be essential for ATP hydrolysis. We show here that CsdA exhibits in vitro ATPase and helicase activities in the presence of short RNA duplexes with either 3′ or 5′ extensions at 15°C. In contrast to wild-type CsdA, a DQAD variant of CsdA (Glu-157→Gln) had no detectible helicase or ATPase activity at 15°C in vitro. A plasmid encoding the DQAD variant was also unable to suppress the impaired growth of the csdA null mutant at 15°C. Plasmid-encoded CsdAΔ444, which lacks most of the carboxy-terminal extension, enhanced the growth of a csdA null mutant at 25°C but not at 15°C; this truncated protein also has limited in vitro activity at 15°C. These results support the physiological function of CsdA as a DEAD box ATP-dependent RNA helicase at low temperature.

Regulation of RNA helicase activity: principles and examples

2020 ◽  
Vol 0 (0) ◽  
Author(s):  
Pascal Donsbach ◽  
Dagmar Klostermeier
Keyword(s):  
Atp Hydrolysis ◽  
Wide Spectrum ◽  
Mrna Translation ◽  
Rna Degradation ◽  
Rna Helicases ◽  
Interaction Partners ◽  
Rna Duplexes ◽  
Self Association

Abstract RNA helicases are a ubiquitous class of enzymes involved in virtually all processes of RNA metabolism, from transcription, mRNA splicing and export, mRNA translation and RNA transport to RNA degradation. Although ATP-dependent unwinding of RNA duplexes is their hallmark reaction, not all helicases catalyze unwinding in vitro, and some in vivo functions do not depend on duplex unwinding. RNA helicases are divided into different families that share a common helicase core with a set of helicase signature motives. The core provides the active site for ATP hydrolysis, a binding site for the non-sequence-specific interactions with RNA, and in many cases a basal unwinding activity. Its activity is often regulated by flanking domains, by interaction partners, or by self-association. In this review, we summarize the regulatory mechanisms that modulate the activities of the helicase core. Case studies on selected helicases with functions in translation, splicing, and RNA sensing illustrate the various modes and layers of regulation in time and space that harness the helicase core for a wide spectrum of cellular tasks.

scholarly journals The mechanism of ATP-dependent RNA unwinding by DEAD box proteins

2009 ◽  
Vol 390 (12) ◽  
Author(s):  
Manuel Hilbert ◽  
Anne R. Karow ◽  
Dagmar Klostermeier
Keyword(s):  
Rna Binding ◽  
Atp Hydrolysis ◽  
Protein Complexes ◽  
Current Data ◽  
Protein Activity ◽  
Closed Conformation ◽  
Dead Box ◽  
Rna Duplexes ◽  
The Dead ◽  
Rna Unwinding

Abstract DEAD box proteins catalyze the ATP-dependent unwinding of double-stranded RNA (dsRNA). In addition, they facilitate protein displacement and remodeling of RNA or RNA/protein complexes. Their hallmark feature is local destabilization of RNA duplexes. Here, we summarize current data on the DEAD box protein mechanism and present a model for RNA unwinding that integrates recent data on the effect of ATP analogs and mutations on DEAD box protein activity. DEAD box proteins share a conserved helicase core with two flexibly linked RecA-like domains that contain all helicase signature motifs. Variable flanking regions contribute to substrate binding and modulate activity. In the presence of ATP and RNA, the helicase core adopts a compact, closed conformation with extensive interdomain contacts and high affinity for RNA. In the closed conformation, the RecA-like domains form a catalytic site for ATP hydrolysis and a continuous RNA binding site. A kink in the backbone of the bound RNA locally destabilizes the duplex. Rearrangement of this initial complex generates a hydrolysis- and unwinding-competent state. From this complex, the first RNA strand can dissociate. After ATP hydrolysis and phosphate release, the DEAD box protein returns to a low-affinity state for RNA. Dissociation of the second RNA strand and reopening of the cleft in the helicase core allow for further catalytic cycles.

scholarly journals Regulation of DEAH-box RNA helicases by G-patch proteins

2021 ◽  
Vol 0 (0) ◽  
Author(s):  
Katherine E. Bohnsack ◽  
Ralf Ficner ◽  
Markus T. Bohnsack ◽  
Stefanie Jonas
Keyword(s):  
Ribosome Biogenesis ◽  
Cellular Localization ◽  
Atp Hydrolysis ◽  
Protein Complexes ◽  
Adaptor Proteins ◽  
Innate Immune ◽  
Telomere Maintenance ◽  
Rna Helicases ◽  
Mechanistic Basis ◽  
Rna Protein Complexes

Abstract RNA helicases of the DEAH/RHA family form a large and conserved class of enzymes that remodel RNA protein complexes (RNPs) by translocating along the RNA. Driven by ATP hydrolysis, they exert force to dissociate hybridized RNAs, dislocate bound proteins or unwind secondary structure elements in RNAs. The sub-cellular localization of DEAH-helicases and their concomitant association with different pathways in RNA metabolism, such as pre-mRNA splicing or ribosome biogenesis, can be guided by cofactor proteins that specifically recruit and simultaneously activate them. Here we review the mode of action of a large class of DEAH-specific adaptor proteins of the G-patch family. Defined only by their eponymous short glycine-rich motif, which is sufficient for helicase binding and stimulation, this family encompasses an immensely varied array of domain compositions and is linked to an equally diverse set of functions. G-patch proteins are conserved throughout eukaryotes and are even encoded within retroviruses. They are involved in mRNA, rRNA and snoRNA maturation, telomere maintenance and the innate immune response. Only recently was the structural and mechanistic basis for their helicase enhancing activity determined. We summarize the molecular and functional details of G-patch-mediated helicase regulation in their associated pathways and their involvement in human diseases.

scholarly journals Alu RNA-protein complexes formed in vitro react with a novel lupus autoantibody.

1985 ◽  
Vol 260 (21) ◽  
pp. 11781-11786
Author(s):  
R Kole ◽  
L D Fresco ◽  
J D Keene ◽  
P L Cohen ◽  
R A Eisenberg ◽  
...  
Keyword(s):  
Protein Complexes ◽  
Alu Rna ◽  
Rna Protein Complexes

scholarly journals Structural basis for DEAH-helicase activation by G-patch proteins

2020 ◽  
Vol 117 (13) ◽  
pp. 7159-7170 ◽  
Author(s):  
Michael K. Studer ◽  
Lazar Ivanović ◽  
Marco E. Weber ◽  
Sabrina Marti ◽  
Stefanie Jonas
Keyword(s):  
Conformational Changes ◽  
Ribosome Biogenesis ◽  
Rna Binding ◽  
Atp Hydrolysis ◽  
Protein Complexes ◽  
Adenosine Diphosphate ◽  
Structural Basis ◽  
Rna Helicases ◽  
Catalytic Core ◽  
Terminal Domains

RNA helicases of the DEAH/RHA family are involved in many essential cellular processes, such as splicing or ribosome biogenesis, where they remodel large RNA–protein complexes to facilitate transitions to the next intermediate. DEAH helicases couple adenosine triphosphate (ATP) hydrolysis to conformational changes of their catalytic core. This movement results in translocation along RNA, which is held in place by auxiliary C-terminal domains. The activity of DEAH proteins is strongly enhanced by the large and diverse class of G-patch activators. Despite their central roles in RNA metabolism, insight into the molecular basis of G-patch–mediated helicase activation is missing. Here, we have solved the structure of human helicase DHX15/Prp43, which has a dual role in splicing and ribosome assembly, in complex with the G-patch motif of the ribosome biogenesis factor NKRF. The G-patch motif binds in an extended conformation across the helicase surface. It tethers the catalytic core to the flexibly attached C-terminal domains, thereby fixing a conformation that is compatible with RNA binding. Structures in the presence or absence of adenosine diphosphate (ADP) suggest that motions of the catalytic core, which are required for ATP binding, are still permitted. Concomitantly, RNA affinity, helicase, and ATPase activity of DHX15 are increased when G-patch is bound. Mutations that detach one end of the tether but maintain overall binding severely impair this enhancement. Collectively, our data suggest that the G-patch motif acts like a flexible brace between dynamic portions of DHX15 that restricts excessive domain motions but maintains sufficient flexibility for catalysis.

scholarly journals A Novel Helicase-Type Protein in the Nucleolus: Protein NOH61

2000 ◽  
Vol 11 (4) ◽  
pp. 1153-1167 ◽  
Author(s):  
Rudolf F. Zirwes ◽  
Jens Eilbracht ◽  
Sandra Kneissel ◽  
Marion S. Schmidt-Zachmann
Keyword(s):  
Cultured Cells ◽  
Gradient Centrifugation ◽  
Intracellular Localization ◽  
Ribosomal Subunit ◽  
Rnase A ◽  
Rna Helicases ◽  
Dead Box ◽  
Transcription Inhibitor ◽  
Calculated Mass

We report the identification, cDNA cloning, and molecular characterization of a novel, constitutive nucleolar protein. The cDNA-deduced amino acid sequence of the human protein defines a polypeptide of a calculated mass of 61.5 kDa and an isoelectric point of 9.9. Inspection of the primary sequence disclosed that the protein is a member of the family of “DEAD-box” proteins, representing a subgroup of putative ATP-dependent RNA helicases. ATPase activity of the recombinant protein is evident and stimulated by a variety of polynucleotides tested. Immunolocalization studies revealed that protein NOH61 (nucleolar helicase of 61 kDa) is highly conserved during evolution and shows a strong accumulation in nucleoli. Biochemical experiments have shown that protein NOH61 synthesized in vitro sediments with ∼11.5 S, i.e., apparently as homo-oligomeric structures. By contrast, sucrose gradient centrifugation analysis of cellular extracts obtained with buffers of elevated ionic strength (600 mM NaCl) revealed that the solubilized native protein sediments with ∼4 S, suggestive of the monomeric form. Interestingly, protein NOH61 has also been identified as a specific constituent of free nucleoplasmic 65S preribosomal particles but is absent from cytoplasmic ribosomes. Treatment of cultured cells with 1) the transcription inhibitor actinomycin D and 2) RNase A results in a complete dissociation of NOH61 from nucleolar structures. The specific intracellular localization and its striking sequence homology to other known RNA helicases lead to the hypothesis that protein NOH61 might be involved in ribosome synthesis, most likely during the assembly process of the large (60S) ribosomal subunit.

scholarly journals Distinct RNA-unwinding mechanisms of DEAD-box and DEAH-box RNA helicase proteins in remodeling structured RNAs and RNPs

2017 ◽  
Vol 45 (6) ◽  
pp. 1313-1321 ◽  
Author(s):  
Benjamin Gilman ◽  
Pilar Tijerina ◽  
Rick Russell
Keyword(s):  
Conformational Changes ◽  
Protein Complexes ◽  
Rna Helicase ◽  
Conformational Transitions ◽  
Messenger Rnas ◽  
Base Pairs ◽  
Dead Box ◽  
Evolutionarily Conserved ◽  
Partially Unfolded ◽  
Rna Protein Complexes

Structured RNAs and RNA–protein complexes (RNPs) fold through complex pathways that are replete with misfolded traps, and many RNAs and RNPs undergo extensive conformational changes during their functional cycles. These folding steps and conformational transitions are frequently promoted by RNA chaperone proteins, notably by superfamily 2 (SF2) RNA helicase proteins. The two largest families of SF2 helicases, DEAD-box and DEAH-box proteins, share evolutionarily conserved helicase cores, but unwind RNA helices through distinct mechanisms. Recent studies have advanced our understanding of how their distinct mechanisms enable DEAD-box proteins to disrupt RNA base pairs on the surfaces of structured RNAs and RNPs, while some DEAH-box proteins are adept at disrupting base pairs in the interior of RNPs. Proteins from these families use these mechanisms to chaperone folding and promote rearrangements of structured RNAs and RNPs, including the spliceosome, and may use related mechanisms to maintain cellular messenger RNAs in unfolded or partially unfolded conformations.

scholarly journals Granule regulation by phase separation during Drosophila oogenesis

2020 ◽  
Vol 4 (3) ◽  
pp. 355-364
Author(s):  
M. Sankaranarayanan ◽  
Timothy T. Weil
Keyword(s):  
Phase Separation ◽  
Translational Control ◽  
Protein Complexes ◽  
Physiological Role ◽  
Drosophila Oogenesis ◽  
And Function ◽  
Rna Protein Complexes ◽  
Liquid Liquid Phase Separation

Drosophila eggs are highly polarised cells that use RNA–protein complexes to regulate storage and translational control of maternal RNAs. Ribonucleoprotein granules are a class of biological condensates that form predominantly by intracellular phase separation. Despite extensive in vitro studies testing the physical principles regulating condensates, how phase separation translates to biological function remains largely unanswered. In this perspective, we discuss granules in Drosophila oogenesis as a model system for investigating the physiological role of phase separation. We review key maternal granules and their properties while highlighting ribonucleoprotein phase separation behaviours observed during development. Finally, we discuss how concepts and models from liquid–liquid phase separation could be used to test mechanisms underlying granule assembly, regulation and function in Drosophila oogenesis.

scholarly journals Targeted knock-in mice with a human mutation in GRTH/DDX25 reveals the essential role of phosphorylated GRTH in spermatid development during spermatogenesis

2019 ◽  
Vol 28 (15) ◽  
pp. 2561-2572 ◽  
Author(s):  
Raghuveer Kavarthapu ◽  
Rajakumar Anbazhagan ◽  
Murugananthkumar Raju ◽  
Chon-Hwa Tsai Morris ◽  
James Pickel ◽  
...  
Keyword(s):  
Essential Role ◽  
Round Spermatid ◽  
Dependent Manner ◽  
Testis Size ◽  
Rna Helicases ◽  
Dead Box ◽  
Chromatoid Bodies ◽  
Human Mutation ◽  
Related Proteins

Abstract Gonadotropin-regulated testicular RNA helicase (GRTH/DDX25) is a testis specific member of the DEAD-box family of RNA helicases expressed in meiotic and haploid germ cells which plays an essential role in spermatogenesis. There are two species of GRTH the 56 kDa non-phospho and 61 kDa phospho forms. Our early studies revealed a missense mutation (R242H) of GRTH in azoospermic men that when expressed in COS1-cells lack the phospho-form of GRTH. To investigate the role of the phospho-GRTH species in spermatogenesis, we generated a GRTH knock-in (KI) transgenic mice with the R242H mutation. GRTH-KI mice are sterile with reduced testis size, lack sperm with spermatogenic arrest at round spermatid stage and loss of the cytoplasmic phospho-GRTH species. Electron microscopy studies revealed reduction in the size of chromatoid bodies (CB) of round spermatids (RS) and germ cell apoptosis. We observed absence of phospho-GRTH in the CB of RS. Complete loss of chromatin remodeling and related proteins such as TP2, PRM2, TSSK6 and marked reduction of their respective mRNAs and half-lives were observed in GRTH-KI mice. We showed that phospho-GRTH has a role in TP2 translation and revealed its occurrence in a 3′ UTR dependent manner. These findings demonstrate the relevance of phospho-GRTH in the structure of the chromatoid body, spermatid development and completion of spermatogenesis and provide an avenue for the development of a male contraceptive.

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