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Patrick Linder - One of the best experts on this subject based on the ideXlab platform.

  • motif iii in superfamily 2 helicases helps convert the binding energy of atp into a high affinity rna binding site in the yeast DEAD Box Protein ded1
    Journal of Molecular Biology, 2010
    Co-Authors: Josette Banroques, Patrick Linder, Monique Doere, Marc Dreyfus, Kyle N Tanner
    Abstract:

    Motif III in the putative helicases of superfamily 2 is highly conserved in both its sequence and its structural context. It typically consists of the sequence alcohol-alanine-alcohol (S/T-A-S/T). Historically, it was thought to link ATPase activity with a "helicase" strand displacement activity that disrupts RNA or DNA duplexes. DEAD-Box Proteins constitute the largest family of superfamily 2; they are RNA-dependent ATPases and ATP-dependent RNA binding Proteins that, in some cases, are able to disrupt short RNA duplexes. We made mutations of motif III (S-A-T) in the yeast DEAD-Box Protein Ded1 and analyzed in vivo phenotypes and in vitro properties. Moreover, we made a tertiary model of Ded1 based on the solved structure of Vasa. We used Ded1 because it has relatively high ATPase and RNA binding activities; it is able to displace moderately stable duplexes at a large excess of substrate. We find that the alanine and the threonine in the second and third positions of motif III are more important than the serine, but that mutations of all three residues have strong phenotypes. We purified the wild-type and various mutants expressed in Escherichia coli. We found that motif III mutations affect the RNA-dependent hydrolysis of ATP (k(cat)), but not the affinity for ATP (K(m)). Moreover, mutations alter and reduce the affinity for single-stranded RNA and subsequently reduce the ability to disrupt duplexes. We obtained intragenic suppressors of the S-A-C mutant that compensate for the mutation by enhancing the affinity for ATP and RNA. We conclude that motif III and the binding energy of gamma-PO(4) of ATP are used to coordinate motifs I, II, and VI and the two RecA-like domains to create a high-affinity single-stranded RNA binding site. It also may help activate the beta,gamma-phosphoanhydride bond of ATP.

  • mRNA export: RNP remodeling by DEAD-Box Proteins.
    Current biology : CB, 2008
    Co-Authors: Patrick Linder
    Abstract:

    The DEAD-Box Protein Dbp5 was thought to remodel ribonucleoProteins and displace Proteins from these complexes in an ATP-dependent fashion to allow for mRNA export from the nucleus. A recent study on Dbp5 shows, however, that its ADP-bound form may also perform an important function in displacing export factors from mRNA.

  • the DEAD Box Protein family of rna helicases
    Gene, 2006
    Co-Authors: Olivier Cordin, Josette Banroques, Kyle N Tanner, Patrick Linder
    Abstract:

    RNA helicases of the DEAD-Box Protein family have been shown to participate in every aspect of RNA metabolism. They are present in most organisms where they work as RNA helicases or RNPases. The properties of these enzymes in vivo remains poorly described, however some were extensively characterized in vitro, and the solved crystal structures of a few are now available. Taken together, this information gives insight into the regulation of ATP and RNA binding as well as in the ATPase and helicase activities. This review will focus on the description of the molecular characteristics of members of the DEAD-Box Protein family and on the enzymatic activities they possess.

  • Belle is a Drosophila DEAD-Box Protein required for viability and in the germ line
    Developmental biology, 2005
    Co-Authors: Oona Johnstone, Patrick Linder, Renate Deuring, Ronald Bock, Margaret T. Fuller, Paul Lasko
    Abstract:

    DEAD-Box Proteins are ATP-dependent RNA helicases that function in various stages of RNA processing and in RNP remodeling. Here, we report identification and characterization of the Drosophila Protein Belle (Bel), which belongs to a highly conserved subfamily of DEAD-Box Proteins including yeast Ded1p, Xenopus An3, mouse PL10, human DDX3/DBX, and human DBY. Mutations in DBY are a frequent cause of male infertility in humans. Bel can substitute in vivo for Ded1p, an essential yeast translation factor, suggesting a requirement for Bel in translation initiation. Consistent with an essential cellular function, strong loss of function mutations in bel are recessive lethal with a larval growth defect phenotype. Hypomorphic bel mutants are male-sterile. Bel is also closely related to the Drosophila DEAD-Box Protein Vasa (Vas), a germ line-specific translational regulator. We find that Bel and Vas colocalize in nuage and at the oocyte posterior during oogenesis, and that bel function is required for female fertility. However, unlike Vas, Bel is not specifically enriched in embryonic pole cells. We conclude that the DEAD-Box Protein Bel has evolutionarily conserved roles in fertility and development.

  • characterization of the atpase and unwinding activities of the yeast DEAD Box Protein has1p and the analysis of the roles of the conserved motifs
    Nucleic Acids Research, 2005
    Co-Authors: Sanda Rocak, Kyle N Tanner, Bertrand Emery, Patrick Linder
    Abstract:

    The yeast DEAD-Box Protein Has1p is required for the maturation of 18S rRNA, the biogenesis of 40S r-subunits and for the processing of 27S pre-rRNAs during 60S r-subunit biogenesis. We purified recombinant Has1p and characterized its biochemical activities. We show that Has1p is an RNA-dependent ATPase in vitro and that it is able to unwind RNA/DNA duplexes in an ATP-dependent manner. We also report a mutational analysis of the conserved residues in motif I (86AKTGSGKT93), motif III (228SAT230) and motif VI (375HRVGRTARG383). The in vivo lethal K92A substitution in motif I abolishes ATPase activity in vitro. The mutations S228A and T230A partially dissociate ATPase and helicase activities, and they have cold-sensitive and lethal growth phenotypes, respectively. The H375E substitution in motif VI significantly decreased helicase but not ATPase activity and was lethal in vivo. These results suggest that both ATPase and unwinding activities are required in vivo. Has1p possesses a Walker A-like motif downstream of motif VI (383GTKGKGKS390). K389A substitution in this motif significantly increases the Has1p activity in vitro, which indicates it potentially plays a role as a negative regulator. Finally, rRNAs and poly(A) RNA serve as the best stimulators of the ATPase activity of Has1p among the tested RNAs.

Alan M. Lambowitz - One of the best experts on this subject based on the ideXlab platform.

  • Insights into Helicase Evolution from the Specificity and Mechanism of a DEAD-Box Protein
    Biophysical Journal, 2015
    Co-Authors: Anna L. Mallam, David J. Sidote, Alan M. Lambowitz
    Abstract:

    How helicase families with a conserved catalytic ‘helicase core’ evolved to function on varied RNA and DNA substrates by diverse mechanisms remains unclear. Here, we used the helicase core of Mss116, a DEAD-Box Protein that utilizes ATP to locally unwind dsRNA, to investigate helicase specificity and mechanism. Previously, we found that the two RecA-like domains of the helicase core of Mss116 are in an extended ‘open state’ in the absence of substrates and recognize ATP and duplex RNA in a modular manner. Upon formation of a compact ‘closed state’ containing an ATPase active site, conserved motifs in the first domain promote the nonprocessive unwinding of short duplex substrates bound to the second domain by excluding one RNA strand and bending the other. In the present work, we define the molecular basis for the specificity of DEAD-Box Proteins. However, we also find that Mss116 has ambiguous substrate unwinding properties and interacts with a variety of NTPs and nucleic acids. The efficiency of unwinding correlates with the stability of the closed-state helicase core, a complex with nucleotide and nucleic acid that forms as duplexes are unwound. Crystal structures reveal that core stability is modulated by family-specific interactions that favor certain substrates. This suggests how present-day helicases diversified from an ancestral core with broad specificity by retaining core closure as a common catalytic mechanism while optimizing substrate-binding interactions for different cellular functions.

  • structural basis for rna duplex recognition and unwinding by the DEAD Box helicase mss116p
    Nature, 2012
    Co-Authors: Anna L. Mallam, David J. Sidote, Benjamin Gilman, Mark Del Campo, Alan M. Lambowitz
    Abstract:

    Analysis of the yeast DEAD-Box nucleic acid helicase Mss116p provides a structural model for how DEAD-Box Proteins recognize and unwind RNA duplexes. Alan Lambowitz and colleagues have solved the structure of Mss116, a yeast DEAD-Box Protein, bound to double-stranded RNA and a DNA–RNA hybrid. DEAD-Box Proteins are nucleic acid helicases that function to unwind and remodel RNAs and RNA-Protein complexes. The structure shows the enzyme in a pre-unwound state, with ATP and RNA bound to different domains; it is proposed that a conformational change brings them together during unwinding. The structure also reveals how the enzyme discriminates between A-form RNA and B-form DNA. DEAD-Box Proteins are the largest family of nucleic acid helicases, and are crucial to RNA metabolism throughout all domains of life1,2. They contain a conserved ‘helicase core’ of two RecA-like domains (domains (D)1 and D2), which uses ATP to catalyse the unwinding of short RNA duplexes by non-processive, local strand separation3. This mode of action differs from that of translocating helicases and allows DEAD-Box Proteins to remodel large RNAs and RNA–Protein complexes without globally disrupting RNA structure4. However, the structural basis for this distinctive mode of RNA unwinding remains unclear. Here, structural, biochemical and genetic analyses of the yeast DEAD-Box Protein Mss116p indicate that the helicase core domains have modular functions that enable a novel mechanism for RNA-duplex recognition and unwinding. By investigating D1 and D2 individually and together, we find that D1 acts as an ATP-binding domain and D2 functions as an RNA-duplex recognition domain. D2 contains a nucleic-acid-binding pocket that is formed by conserved DEAD-Box Protein sequence motifs and accommodates A-form but not B-form duplexes, providing a basis for RNA substrate specificity. Upon a conformational change in which the two core domains join to form a ‘closed state’ with an ATPase active site, conserved motifs in D1 promote the unwinding of duplex substrates bound to D2 by excluding one RNA strand and bending the other. Our results provide a comprehensive structural model for how DEAD-Box Proteins recognize and unwind RNA duplexes. This model explains key features of DEAD-Box Protein function and affords a new perspective on how the evolutionarily related cores of other RNA and DNA helicases diverged to use different mechanisms.

  • High-Throughput Genetic Identification of Functionally Important Regions of the Yeast DEAD-Box Protein Mss116p
    Journal of molecular biology, 2011
    Co-Authors: Georg Mohr, Mark Del Campo, Kathryn G. Turner, Benjamin Gilman, Rachel Z. Wolf, Alan M. Lambowitz
    Abstract:

    The Saccharomyces cerevisiae DEAD-Box Protein Mss116p is a general RNA chaperone that functions in splicing mitochondrial group I and group II introns. Recent X-ray crystal structures of Mss116p in complex with ATP analogs and single-stranded RNA show that the helicase core induces a bend in the bound RNA, as in other DEAD-Box Proteins, while a C-terminal extension (CTE) induces a second bend, resulting in RNA crimping. Here, we illuminate these structures by using high-throughput genetic selections, unigenic evolution, and analyses of in vivo splicing activity to comprehensively identify functionally important regions and permissible amino acid substitutions throughout Mss116p. The functionally important regions include those containing conserved sequence motifs involved in ATP and RNA binding or interdomain interactions, as well as previously unidentified regions, including surface loops that may function in Protein-Protein interactions. The genetic selections recapitulate major features of the conserved helicase motifs seen in other DEAD-Box Proteins but also show surprising variations, including multiple novel variants of motif III (SAT). Patterns of amino acid substitutions indicate that the RNA bend induced by the helicase core depends on ionic and hydrogen-bonding interactions with the bound RNA; identify a subset of critically interacting residues; and indicate that the bend induced bymore » the CTE results primarily from a steric block. Finally, we identified two conserved regions - one the previously noted post II region in the helicase core and the other in the CTE - that may help displace or sequester the opposite RNA strand during RNA unwinding.« less

  • ATP-Dependent Roles of the DEAD-Box Protein Mss116p in Group II Intron Splicing In Vitro and In Vivo
    Journal of molecular biology, 2011
    Co-Authors: Jeffrey P. Potratz, Mark Del Campo, Alan M. Lambowitz, Rachel Z. Wolf, Rick Russell
    Abstract:

    The yeast DEAD-Box Protein Mss116p functions as a general RNA chaperone in splicing mitochondrial group I and group II introns. For most of its functions, Mss116p is thought to use ATP-dependent RNA unwinding to facilitate RNA structural transitions, but it has been suggested to assist in the folding of one group II intron (aI5γ) primarily by stabilizing a folding intermediate. Here we compare three aI5γ constructs: one with long exons, one with short exons, and a ribozyme construct lacking exons. The long exons result in slower splicing, suggesting that they misfold and/or stabilize nonnative intronic structures. Nevertheless, Mss116p acceleration of all three constructs depends on ATP and is inhibited by mutations that compromise RNA unwinding, suggesting similar mechanisms. Results of splicing assays and a new two-stage assay that separates ribozyme folding and catalysis indicate that maximal folding of all three constructs by Mss116p requires ATP-dependent RNA unwinding. ATP-independent activation is appreciable for only a subpopulation of the minimal ribozyme construct and not for constructs containing exons. As expected for a general RNA chaperone, Mss116p can also disrupt the native ribozyme, which can refold after Mss116p removal. Finally, using yeast strains with mitochondrial DNA containing only the single intron aI5γ, we show that Mss116p mutants promote splicing in vivo to degrees that correlate with their residual ATP-dependent RNA-unwinding activities. Together, our results indicate that, although DEAD-Box Proteins play multiple roles in RNA folding, the physiological function of Mss116p in aI5γ splicing includes a requirement for ATP-dependent local unfolding, allowing the conversion of nonfunctional RNA structure into functional RNA structure.

  • Structure of the Yeast DEAD Box Protein Mss116p reveals two wedges that crimp RNA
    Molecular cell, 2009
    Co-Authors: Mark Del Campo, Alan M. Lambowitz
    Abstract:

    Summary The yeast DEAD Box Protein Mss116p is a general RNA chaperone that functions in mitochondrial group I and II intron splicing, translational activation, and RNA end processing. Here we determined high-resolution X-ray crystal structures of Mss116p complexed with an RNA oligonucleotide and ATP analogs AMP-PNP, ADP-BeF 3 − , or ADP-AlF 4 − . The structures show the entire helicase core acting together with a functionally important C-terminal extension. In all structures, the helicase core is in a closed conformation with a wedge α helix bending RNA 3′ of the central bound nucleotides, as in previous DEAD Box Protein structures. Notably, Mss116p's C-terminal extension also bends RNA 5′ of the central nucleotides, resulting in RNA crimping. Despite reported functional differences, we observe few structural changes in ternary complexes with different ATP analogs. The structures constrain models of DEAD Box Protein function and reveal a strand separation mechanism in which a Protein uses two wedges to act as a molecular crimper.

Elizabeth J. Tran - One of the best experts on this subject based on the ideXlab platform.

  • Characterization of the mammalian DEAD-Box Protein DDX5 reveals functional conservation with S. cerevisiae ortholog Dbp2 in transcriptional control and glucose metabolism
    RNA (New York N.Y.), 2017
    Co-Authors: Zheng Xing, Siwen Wang, Elizabeth J. Tran
    Abstract:

    DEAD-Box Proteins are a class of nonprocessive RNA helicases that dynamically modulate the structure of RNA and ribonucleoProtein complexes (RNPs). However, the precise roles of individual members are not well understood. Work from our laboratory revealed that the DEAD-Box Protein Dbp2 in Saccharomyces cerevisiae is an active RNA helicase in vitro that functions in transcription by promoting mRNP assembly, repressing cryptic transcription initiation, and regulating long noncoding RNA activity. Interestingly, Dbp2 is also linked to glucose sensing and hexose transporter gene expression. DDX5 is the mammalian ortholog of Dbp2 that has been implicated in cancer and metabolic syndrome, suggesting that the role of Dbp2 and DDX5 in glucose metabolic regulation is conserved. Herein, we present a refined biochemical and biological comparison of yeast Dbp2 and human DDX5 enzymes. We find that human DDX5 possesses a 10-fold higher unwinding activity than Dbp2, which is partially due to the presence of a mammalian/avian specific C-terminal extension. Interestingly, ectopic expression of DDX5 rescues the cold sensitivity, cryptic initiation defects, and impaired glucose import in dbp2Δ cells, suggesting functional conservation. Consistently, we show that DDX5 promotes glucose uptake and glycolysis in mouse AML12 hepatocyte cells, suggesting that mammalian DDX5 and S. cerevisiae Dbp2 share conserved roles in cellular metabolism.

  • RNA helicase DEAD Box Protein 5 regulates Polycomb repressive complex 2/Hox transcript antisense intergenic RNA function in hepatitis B virus infection and hepatocarcinogenesis
    Hepatology, 2016
    Co-Authors: Hao Zhang, Elizabeth J. Tran, Zheng Xing, Saravana Kumar Kailasam Mani, Brigitte Bancel, David Durantel, Fabien Zoulim, Philippe Merle, Ourania Andrisani
    Abstract:

    Chronic hepatitis B virus (HBV) infection is a major factor in hepatocellular carcinoma (HCC) pathogenesis by a mechanism not yet understood. Elucidating mechanisms of HBV-mediated hepatocarcinogenesis is needed to gain insights into classification and treatment of HCC. In HBV replicating cells, including virus-associated HCCs, suppressor of zeste 12 homolog (SUZ12), a core subunit of Polycomb repressive complex2 (PRC2), undergoes proteasomal degradation. This process requires the long noncoding RNA, Hox transcript antisense intergenic RNA (HOTAIR). Intriguingly, HOTAIR interacts with PRC2 and also binds RNA-binding E3 ligases, serving as a ubiquitination scaffold. Herein, we identified the RNA helicase, DEAD Box Protein 5 (DDX5), as a regulator of SUZ12 stability and PRC2-mediated gene repression, acting by regulating RNA-Protein complexes formed with HOTAIR. Specifically, knockdown of DDX5 and/or HOTAIR enabled reexpression of PRC2-repressed genes epithelial cell adhesion molecule (EpCAM) and pluripotency genes. Also, knockdown of DDX5 enhanced transcription from the HBV minichromosome. The helicase activity of DDX5 stabilized S

  • rna helicase DEAD Box Protein 5 regulates polycomb repressive complex 2 hox transcript antisense intergenic rna function in hepatitis b virus infection and hepatocarcinogenesis
    Hepatology, 2016
    Co-Authors: Hao Zhang, Elizabeth J. Tran, Zheng Xing, Saravana Kumar Kailasam Mani, Brigitte Bancel, David Durantel, Fabien Zoulim, Philippe Merle, Ourania M Andrisani
    Abstract:

    Chronic hepatitis B virus (HBV) infection is a major factor in hepatocellular carcinoma (HCC) pathogenesis by a mechanism not yet understood. Elucidating mechanisms of HBV-mediated hepatocarcinogenesis is needed to gain insights into classification and treatment of HCC. In HBV replicating cells, including virus-associated HCCs, suppressor of zeste 12 homolog (SUZ12), a core subunit of Polycomb repressive complex2 (PRC2), undergoes proteasomal degradation. This process requires the long noncoding RNA, Hox transcript antisense intergenic RNA (HOTAIR). Intriguingly, HOTAIR interacts with PRC2 and also binds RNA-binding E3 ligases, serving as a ubiquitination scaffold. Herein, we identified the RNA helicase, DEAD Box Protein 5 (DDX5), as a regulator of SUZ12 stability and PRC2-mediated gene repression, acting by regulating RNA-Protein complexes formed with HOTAIR. Specifically, knockdown of DDX5 and/or HOTAIR enabled reexpression of PRC2-repressed genes epithelial cell adhesion molecule (EpCAM) and pluripotency genes. Also, knockdown of DDX5 enhanced transcription from the HBV minichromosome. The helicase activity of DDX5 stabilized SUZ12- and PRC2-mediated gene silencing, by displacing the RNA-binding E3 ligase, Mex-3 RNA-binding family member B (Mex3b), from HOTAIR. Conversely, ectopic expression of Mex3b ubiquitinated SUZ12, displaced DDX5 from HOTAIR, and induced SUZ12 down-regulation. In G2 phase of cells expressing the HBV X Protein (HBx), SUZ12 preferentially associated with Mex3b, but not DDX5, resulting in de-repression of PRC2 targets, including EpCAM and pluripotency genes. Significantly, liver tumors from HBx/c-myc bitransgenic mice and chronically HBV-infected patients exhibited a strong negative correlation between DDX5 messenger RNA levels, pluripotency gene expression, and liver tumor differentiation. Notably, chronically infected HBV patients with HCC expressing reduced DDX5 exhibited poor prognosis after tumor resection, identifying DDX5 as an important player in poor prognosis HCC. Conclusion: The RNA helicase DDX5, and E3 ligase Mex3b, are important cellular targets for the design of novel, epigenetic therapies to combat HBV infection and poor prognosis HBV-associated liver cancer. (Hepatology 2016;64:1033-1048)

  • Measuring helicase inhibition of the DEAD-Box Protein Dbp2 by Yra1.
    Methods in molecular biology (Clifton N.J.), 2014
    Co-Authors: Wai Kit, Elizabeth J. Tran
    Abstract:

    Despite the highly conserved helicase core, individual DEAD-Box Proteins are specialized in diverse RNA metabolic processes. One mechanism that determines DEAD-Box Protein specificity is enzymatic regulation by other Protein cofactors. In this chapter, we describe a protocol for purifying the Saccharomyces cerevisiae DEAD-Box RNA helicase Dbp2 and RNA-binding Protein Yra1 and subsequent analysis of helicase regulation. The experiments described here can be adapted to other RNA helicases and their purified cofactor(s).

  • The DEAD-Box Protein Dbp2 functions with the RNA-binding Protein Yra1 to promote mRNP assembly.
    Journal of molecular biology, 2013
    Co-Authors: Wai Kit, Sara C. Cloutier, Elizabeth J. Tran
    Abstract:

    Eukaryotic gene expression involves numerous biochemical steps that are dependent on RNA structure and ribonucleoProtein (RNP) complex formation. The DEAD-Box class of RNA helicases plays fundamental roles in formation of RNA and RNP structure in every aspect of RNA metabolism. In an effort to explore the diversity of biological roles for DEAD-Box Proteins, our laboratory recently demonstrated that the DEAD-Box Protein Dbp2 associates with actively transcribing genes and is required for normal gene expression in Saccharomyces cerevisiae. We now provide evidence that Dbp2 interacts genetically and physically with the mRNA export factor Yra1. In addition, we find that Dbp2 is required for in vivo assembly of mRNA-binding Proteins Yra1, Nab2, and Mex67 onto poly(A)+ RNA. Strikingly, we also show that Dbp2 is an efficient RNA helicase in vitro and that Yra1 decreases the efficiency of ATP-dependent duplex unwinding. We provide a model whereby messenger ribonucleoProtein (mRNP) assembly requires Dbp2 unwinding activity and once the mRNP is properly assembled, inhibition by Yra1 prevents further rearrangements. Both Yra1 and Dbp2 are conserved in multicellular eukaryotes, suggesting that this constitutes a broadly conserved mechanism for stepwise assembly of mature mRNPs in the nucleus.

Eckhard Jankowsky - One of the best experts on this subject based on the ideXlab platform.

  • autoinhibitory interdomain interactions and subfamily specific extensions redefine the catalytic core of the human DEAD Box Protein ddx3
    Journal of Biological Chemistry, 2016
    Co-Authors: Stephen N. Floor, Kendall J. Condon, Eckhard Jankowsky, Deepak Sharma, Jennifer A. Doudna
    Abstract:

    DEAD-Box Proteins utilize ATP to bind and remodel RNA and RNA-Protein complexes. All DEAD-Box Proteins share a conserved core that consists of two RecA-like domains. The core is flanked by subfamily-specific extensions of idiosyncratic function. The Ded1/DDX3 subfamily of DEAD-Box Proteins is of particular interest as members function during Protein translation, are essential for viability, and are frequently altered in human malignancies. Here, we define the function of the subfamily-specific extensions of the human DEAD-Box Protein DDX3. We describe the crystal structure of the subfamily-specific core of wild-type DDX3 at 2.2 A resolution, alone and in the presence of AMP or nonhydrolyzable ATP. These structures illustrate a unique interdomain interaction between the two ATPase domains in which the C-terminal domain clashes with the RNA-binding surface. Destabilizing this interaction accelerates RNA duplex unwinding, suggesting that it is present in solution and inhibitory for catalysis. We use this core fragment of DDX3 to test the function of two recurrent medulloblastoma variants of DDX3 and find that both inactivate the Protein in vitro and in vivo. Taken together, these results redefine the structural and functional core of the DDX3 subfamily of DEAD-Box Proteins.

  • The DEAD-Box Protein Ded1 modulates translation by the formation and resolution of an eIF4F-mRNA complex.
    Molecular cell, 2011
    Co-Authors: Angela K. Hilliker, Zhaofeng Gao, Eckhard Jankowsky, Roy Parker
    Abstract:

    Summary The translation, localization, and degradation of cytoplasmic mRNAs are controlled by the formation and rearrangement of their mRNPs. The conserved Ded1/DDX3 DEAD-Box Protein functions in an unknown manner to affect both translation initiation and repression. We demonstrate that Ded1 first functions by directly interacting with eIF4G to assemble a Ded1-mRNA-eIF4F complex, which accumulates in stress granules. After ATP hydrolysis by Ded1, the mRNP exits stress granules and completes translation initiation. Thus, Ded1 functions both as a repressor of translation, by assembling an mRNP stalled in translation initiation, and as an ATP-dependent activator of translation, by resolving the stalled mRNP. These results identify Ded1 as a translation initiation factor that assembles and remodels an intermediate complex in translation initiation.

  • Do DEAD-Box Proteins Promote Group II Intron Splicing without Unwinding RNA?
    Molecular cell, 2007
    Co-Authors: Mark Del Campo, Sabine Mohr, Eckhard Jankowsky, Rick Russell, Quansheng Yang, Pilar Tijerina, Hari Bhaskaran, Alan M. Lambowitz
    Abstract:

    The DEAD-Box Protein Mss116p promotes group II intron splicing in vivo and in vitro. Here we explore two hypotheses for how Mss116p promotes group II intron splicing: by using its RNA unwinding activity to act as an RNA chaperone or by stabilizing RNA folding intermediates. We show that an Mss116p mutant in helicase motif III (SAT/AAA), which was reported to stimulate splicing without unwinding RNA, retains ATP-dependent unwinding activity and promotes unfolding of a structured RNA. Its unwinding activity increases sharply with decreasing duplex length and correlates with group II intron splicing activity in quantitative assays. Additionally, we show that Mss116p can promote ATP-independent RNA unwinding, presumably via single-strand capture, also potentially contributing to DEAD-Box Protein RNA chaperone activity. Our findings favor the hypothesis that DEAD-Box Proteins function in group II intron splicing as in other processes by using their unwinding activity to act as RNA chaperones.

  • discriminatory rnp remodeling by the DEAD Box Protein ded1
    RNA, 2006
    Co-Authors: Heath A Bowers, Patricia A Maroney, Margaret E Fairman, Berthold Kastner, Reinhard Luhrmann, Timothy W Nilsen, Eckhard Jankowsky
    Abstract:

    DExH/D Proteins catalyze NTP-driven rearrangements of RNA and RNA-Protein complexes during most aspects of RNA metabolism. Although the vast majority of DExH/D Proteins displays virtually no sequence-specificity when remodeling RNA complexes in vitro, the enzymes clearly distinguish between a large number of RNA and RNP complexes in a physiological context. It is unknown how this discrimination between potential substrates is achieved. Here we show one possible way by which a non-sequence specific DExH/D Protein can discriminately remodel similar RNA complexes. We have measured in vitro the disassembly of model RNPs by two distinct DExH/D Proteins, DED1 and NPH-II. Both enzymes displace the U1 snRNP from a tightly bound RNA in an active, ATP-dependent fashion. However, DED1 cannot actively displace the Protein U1A from its binding site, whereas NPH-II can. The dissociation rate of U1A dictates the rate by which DED1 remodels RNA complexes with U1A bound. We further show that DED1 disassembles RNA complexes with slightly altered U1A binding sites at different rates, but only when U1A is bound to the RNA. These findings suggest that the “inability” to actively displace other Proteins from RNA can provide non-sequence specific DExH/D Proteins with the capacity to disassemble similar RNA complexes in a discriminatory fashion. In addition, our study illuminates possible mechanisms for Protein displacement by DExH/D Proteins.

  • ATP- and ADP-Dependent Modulation of RNA Unwinding and Strand Annealing Activities by the DEAD-Box Protein DED1†
    Biochemistry, 2005
    Co-Authors: Quansheng Yang, Eckhard Jankowsky
    Abstract:

    DEAD-Box RNA helicases, which are involved in virtually all aspects of RNA metabolism, are generally viewed as enzymes that unwind RNA duplexes or disrupt RNA-Protein interactions in an ATP-dependent manner. Here, we show in vitro that the DEAD-Box Protein DED1 from Saccharomyces cerevisiae promotes not only RNA unwinding but also strand annealing, the latter in such a profound fashion that the physical limit for a bimolecular association rate constant is approached. We further demonstrate that DED1 establishes an ATP-dependent steady state between unwinding and annealing, which enables the enzyme to modulate the balance between the two opposing activities through ATP and ADP concentrations. The ratio between unwinding and annealing and the degree to which both activities are ATP- and ADP-modulated are strongly influenced by structured as well as unstructured regions in the RNA substrate. Collectively, these findings expand the known functional repertoire of DEAD-Box Proteins and reveal the capacity of DED1 to remodel RNA in response to ADP and ATP concentrations by facilitating not only disruption but also formation of RNA duplexes.

Rick Russell - One of the best experts on this subject based on the ideXlab platform.

  • atp utilization by a DEAD Box Protein during refolding of a misfolded group i intron ribozyme
    Journal of Biological Chemistry, 2020
    Co-Authors: Inga Jarmoskaite, Pilar Tijerina, Rick Russell
    Abstract:

    DEAD-Box helicase Proteins perform ATP-dependent rearrangements of structured RNAs throughout RNA biology. Short RNA helices are unwound in a single ATPase cycle, but the ATP requirement for more complex RNA structural rearrangements is unknown. Here we measure the amount of ATP used for native refolding of a misfolded group I intron ribozyme by CYT-19, a Neurospora crassa DEAD-Box Protein that functions as a general chaperone for mitochondrial group I introns. By comparing the rates of ATP hydrolysis and ribozyme refolding, we find that several hundred ATP molecules are hydrolyzed during refolding of each ribozyme molecule. After subtracting non-productive ATP hydrolysis that occurs in the absence of ribozyme refolding, we find that approximately 100 ATPs are hydrolyzed per refolded RNA as a consequence of interactions specific to the misfolded ribozyme. This value is insensitive to changes in ATP and CYT-19 concentration and decreases with decreasing ribozyme stability. Because of earlier findings that ~90% of global ribozyme unfolding cycles lead back to the kinetically preferred misfolded conformation and are not observed, we estimate that each global unfolding cycle consumes ~10 ATPs. Our results indicate that CYT-19 functions as a general RNA chaperone by using a stochastic, energy-intensive mechanism to promote RNA unfolding and refolding, suggesting an evolutionary convergence with Protein chaperones.

  • The DEAD-Box Protein CYT-19 Uses Arginine Residues in Its C-Tail To Tether RNA Substrates
    Biochemistry, 2017
    Co-Authors: Veronica F. Busa, Maxwell J. Rector, Rick Russell
    Abstract:

    DEAD-Box Proteins are nonprocessive RNA helicases that play diverse roles in cellular processes. The Neurospora crassa DEAD-Box Protein CYT-19 promotes mitochondrial group I intron splicing and functions as a general RNA chaperone. CYT-19 includes a disordered, arginine-rich “C-tail” that binds RNA, positioning the helicase core to capture and unwind nearby RNA helices. Here we probed the C-tail further by varying the number and positions of arginines within it. We found that removing sets of as few as four of the 11 arginines reduced RNA unwinding activity (kcat/KM) to a degree equivalent to that seen upon removal of the C-tail, suggesting that a minimum or “threshold” number of arginines is required. In addition, a mutant with 16 arginines displayed RNA unwinding activity greater than that of wild-type CYT-19. The C-tail modifications impacted unwinding only of RNA helices within constructs that included an adjacent helix or structured RNA element that would allow C-tail binding, indicating that the hel...

  • DEAD-Box Protein CYT-19 is activated by exposed helices in a group I intron RNA
    Proceedings of the National Academy of Sciences of the United States of America, 2014
    Co-Authors: Inga Jarmoskaite, Hari Bhaskaran, Soenke Seifert, Rick Russell
    Abstract:

    DEAD-Box Proteins are nonprocessive RNA helicases and can function as RNA chaperones, but the mechanisms of their chaperone activity remain incompletely understood. The Neurospora crassa DEAD-Box Protein CYT-19 is a mitochondrial RNA chaperone that promotes group I intron splicing and has been shown to resolve misfolded group I intron structures, allowing them to refold. Building on previous results, here we use a series of tertiary contact mutants of the Tetrahymena group I intron ribozyme to demonstrate that the efficiency of CYT-19–mediated unfolding of the ribozyme is tightly linked to global RNA tertiary stability. Efficient unfolding of destabilized ribozyme variants is accompanied by increased ATPase activity of CYT-19, suggesting that destabilized ribozymes provide more productive interaction opportunities. The strongest ATPase stimulation occurs with a ribozyme that lacks all five tertiary contacts and does not form a compact structure, and small-angle X-ray scattering indicates that ATPase activity tracks with ribozyme compactness. Further, deletion of three helices that are prominently exposed in the folded structure decreases the ATPase stimulation by the folded ribozyme. Together, these results lead to a model in which CYT-19, and likely related DEAD-Box Proteins, rearranges complex RNA structures by preferentially interacting with and unwinding exposed RNA secondary structure. Importantly, this mechanism could bias DEAD-Box Proteins to act on misfolded RNAs and ribonucleoProteins, which are likely to be less compact and more dynamic than their native counterparts.

  • ATP-Dependent Roles of the DEAD-Box Protein Mss116p in Group II Intron Splicing In Vitro and In Vivo
    Journal of molecular biology, 2011
    Co-Authors: Jeffrey P. Potratz, Mark Del Campo, Alan M. Lambowitz, Rachel Z. Wolf, Rick Russell
    Abstract:

    The yeast DEAD-Box Protein Mss116p functions as a general RNA chaperone in splicing mitochondrial group I and group II introns. For most of its functions, Mss116p is thought to use ATP-dependent RNA unwinding to facilitate RNA structural transitions, but it has been suggested to assist in the folding of one group II intron (aI5γ) primarily by stabilizing a folding intermediate. Here we compare three aI5γ constructs: one with long exons, one with short exons, and a ribozyme construct lacking exons. The long exons result in slower splicing, suggesting that they misfold and/or stabilize nonnative intronic structures. Nevertheless, Mss116p acceleration of all three constructs depends on ATP and is inhibited by mutations that compromise RNA unwinding, suggesting similar mechanisms. Results of splicing assays and a new two-stage assay that separates ribozyme folding and catalysis indicate that maximal folding of all three constructs by Mss116p requires ATP-dependent RNA unwinding. ATP-independent activation is appreciable for only a subpopulation of the minimal ribozyme construct and not for constructs containing exons. As expected for a general RNA chaperone, Mss116p can also disrupt the native ribozyme, which can refold after Mss116p removal. Finally, using yeast strains with mitochondrial DNA containing only the single intron aI5γ, we show that Mss116p mutants promote splicing in vivo to degrees that correlate with their residual ATP-dependent RNA-unwinding activities. Together, our results indicate that, although DEAD-Box Proteins play multiple roles in RNA folding, the physiological function of Mss116p in aI5γ splicing includes a requirement for ATP-dependent local unfolding, allowing the conversion of nonfunctional RNA structure into functional RNA structure.

  • Do DEAD-Box Proteins Promote Group II Intron Splicing without Unwinding RNA?
    Molecular cell, 2007
    Co-Authors: Mark Del Campo, Sabine Mohr, Eckhard Jankowsky, Rick Russell, Quansheng Yang, Pilar Tijerina, Hari Bhaskaran, Alan M. Lambowitz
    Abstract:

    The DEAD-Box Protein Mss116p promotes group II intron splicing in vivo and in vitro. Here we explore two hypotheses for how Mss116p promotes group II intron splicing: by using its RNA unwinding activity to act as an RNA chaperone or by stabilizing RNA folding intermediates. We show that an Mss116p mutant in helicase motif III (SAT/AAA), which was reported to stimulate splicing without unwinding RNA, retains ATP-dependent unwinding activity and promotes unfolding of a structured RNA. Its unwinding activity increases sharply with decreasing duplex length and correlates with group II intron splicing activity in quantitative assays. Additionally, we show that Mss116p can promote ATP-independent RNA unwinding, presumably via single-strand capture, also potentially contributing to DEAD-Box Protein RNA chaperone activity. Our findings favor the hypothesis that DEAD-Box Proteins function in group II intron splicing as in other processes by using their unwinding activity to act as RNA chaperones.