are the proposed targets for the helicases involved in nuclear mRNA splicing (Staley and Guthrie,... more are the proposed targets for the helicases involved in nuclear mRNA splicing (Staley and Guthrie, 1998). For this reason, RNA helicases are increasingly thought of as ATP-dependent translocases or as RNA chaperones that are used to ensure the correct folding, or refolding, Switzerland
We have identified a highly conserved phenylalanine in motif IV of the DEAD-box helicases that is... more We have identified a highly conserved phenylalanine in motif IV of the DEAD-box helicases that is important for their enzymatic activities. In vivo analyses of essential proteins in yeast showed that mutants of this residue had severe growth phenotypes. Most of the mutants also were temperature sensitive, which suggested that the mutations altered the conformational stability. Intragenic suppressors of the F405L mutation in yeast Ded1 mapped close to regions of the protein involved in ATP or RNA binding in DEAD-box crystal structures, which implicated a defect at this level. In vitro experiments showed that these mutations affected ATP binding and hydrolysis as well as strand displacement activity. However, the most pronounced effect was the loss of the ATP-dependent cooperative binding of the RNA substrates. Sequence analyses and an examination of the Protein Data Bank showed that the motif IV phenylalanine is conserved among superfamily 2 helicases. The phenylalanine appears to be...
RNA helicases are essential for virtually all cellular processes, however, their regulation is po... more RNA helicases are essential for virtually all cellular processes, however, their regulation is poorly understood. The activities of eight RNA helicases are required for pre-mRNA splicing. Amongst these, Brr2p is unusual in having two helicase modules, of which only the amino-terminal helicase domain appears to be catalytically active. Using genetic and biochemical approaches, we investigated interaction of the carboxy-terminal helicase module, in particular the carboxy-terminal Sec63-2 domain, with the splicing RNA helicase Prp16p. Combining mutations in BRR2 and PRP16 suppresses or enhances physical interaction and growth defects in an allele-specific manner, signifying functional interactions. Notably, we show that Brr2p Sec63-2 domain can modulate the ATPase activity of Prp16p in vitro by interfering with its ability to bind RNA. We therefore propose that the carboxy-terminal helicase module of Brr2p acquired a regulatory function that allows Brr2p to modulate the ATPase activity...
DEAD-box proteins are the most common RNA helicases, and they are associated with virtually all p... more DEAD-box proteins are the most common RNA helicases, and they are associated with virtually all processes involving RNA. They have nine conserved motifs that are required for ATP and RNA binding, and for linking phosphoanhydride cleavage of ATP with helicase activity. The Q motif is the most recently identified conserved element, and it occurs B17 amino acids upstream of motif I. There is a highly conserved, but isolated, aromatic group B17 amino acids upstream of the Q motif. These two elements are involved in adenine recognition and in ATPase activity of DEAD-box proteins. We made extensive analyses of the Q motif and upstream aromatic residue in the yeast translation-initiation factor Ded1. We made site-specific mutations and tested them for viability in yeast. Moreover, we purified various mutant proteins and obtained the Michaelis-Menten parameters for the ATPase activities. We also measured RNA affinities and strand-displacement activities. We find that the Q motif not only regulates ATP binding and hydrolysis but also regulates the affinity of the protein for RNA substrates and ultimately the helicase activity.
Proceedings of the National Academy of Sciences, 2004
Pre-mRNA splicing requires the function of a number of RNAdependent ATPases͞helicases, yet no thr... more Pre-mRNA splicing requires the function of a number of RNAdependent ATPases͞helicases, yet no three-dimensional structure of any spliceosomal ATPases͞helicases is known. The highly conserved DECD-box protein UAP56͞Sub2 is an essential splicing factor that is also important for mRNA export. The expected ATPase͞helicase activity appears to be essential for the UAP56͞ Sub2 functions. Here, we show that purified human UAP56 is an active RNA-dependent ATPase, and we also report the crystal structures of UAP56 alone and in complex with ADP, as well as a DECD to DEAD mutant. The structures reveal a unique spatial arrangement of the two conserved helicase domains, and ADPbinding induces significant conformational changes of key residues in the ATP-binding pocket. Our structural analyses suggest a specific protein-RNA displacement model of UAP56͞Sub2. The detailed structural information provides important mechanistic insights into the splicing function of UAP56͞Sub2. The structures also will be useful for the analysis of other spliceosomal DExD-box ATPases͞helicases.
The DEAD-box family of putative RNA helicases is composed of ubiquitous proteins that are found i... more The DEAD-box family of putative RNA helicases is composed of ubiquitous proteins that are found in nearly all organisms and that are involved in virtually all processes involving RNA. They are characterized by two tandemly linked, RecA-like domains that contain 11 conserved motifs and highly variable amino-and carboxy-terminal flanking sequences. For this reason, they are often considered to be modular multi-domain proteins. We tested this by making extensive BLASTs and sequence alignments to elucidate the minimal functional unit in nature. We then used this information to construct chimeras and deletions of six essential yeast proteins that were assayed in vivo. We purified many of the different constructs and characterized their biochemical properties in vitro. We found that sequence elements can only be switched between closely related proteins and that the carboxy-terminal sequences are important for high ATPase and strand displacement activities and for high RNA binding affinity. The amino-terminal elements were often toxic when overexpressed in vivo, and they may play regulatory roles. Both the amino and the carboxyl regions have a high frequency of sequences that are predicted to be intrinsically disordered, indicating that the flanking regions do not form distinct modular domains but probably assume an ordered structure with ligand binding. Finally, the minimal functional unit of the DEAD-box core starts two amino acids before the isolated phenylalanine of the Q motif and extends to about 35 residues beyond motif VI. These experiments provide evidence for how a highly conserved structural domain can be adapted to different cellular needs.
RNA helicases of the DEAD-box protein family have been shown to participate in every aspect of RN... more 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.
Pre-mRNA splicing requires the activities of several ATPases from the DEAH-box, DEAD-box and Ski2... more Pre-mRNA splicing requires the activities of several ATPases from the DEAH-box, DEAD-box and Ski2-like helicase families to control conformational rearrangements within the spliceosome. Recent findings indicate that several spliceosomal helicases can act at multiple stages of the splicing reaction, and information on how those multiple actions are controlled are emerging. The recently solved crystal structure of the DEAH-box helicase Prp43 provides novel insights into the similarities and differences between the three helicase families. Here we discuss the potential family-specific mechanisms of spliceosomal RNA helicases and their regulation.
SF1 and SF2 helicases have structurally conserved cores containing seven to eight distinctive mot... more SF1 and SF2 helicases have structurally conserved cores containing seven to eight distinctive motifs and variable amino- and carboxyl-terminal flanking sequences. We have discovered a motif upstream of motif I that is unique to and characteristic of the DEAD box family of RNA helicases. It consists of a 9 amino acid sequence containing an invariant glutamine. A conserved phenylalanine occurs 17 aa further upstream. Sequence alignments, site-specific mutagenesis, and ATPase assays show that this motif and the upstream phenylalanine are highly conserved, that they are essential for viability in the yeast Saccharomyces cerevisiae, and that they control ATP binding and hydrolysis in the yeast translation-initiation factor eIF4A. These results are consistent with computer studies of the solved crystal structures.
are the proposed targets for the helicases involved in nuclear mRNA splicing (Staley and Guthrie,... more are the proposed targets for the helicases involved in nuclear mRNA splicing (Staley and Guthrie, 1998). For this reason, RNA helicases are increasingly thought of as ATP-dependent translocases or as RNA chaperones that are used to ensure the correct folding, or refolding, Switzerland
We have identified a highly conserved phenylalanine in motif IV of the DEAD-box helicases that is... more We have identified a highly conserved phenylalanine in motif IV of the DEAD-box helicases that is important for their enzymatic activities. In vivo analyses of essential proteins in yeast showed that mutants of this residue had severe growth phenotypes. Most of the mutants also were temperature sensitive, which suggested that the mutations altered the conformational stability. Intragenic suppressors of the F405L mutation in yeast Ded1 mapped close to regions of the protein involved in ATP or RNA binding in DEAD-box crystal structures, which implicated a defect at this level. In vitro experiments showed that these mutations affected ATP binding and hydrolysis as well as strand displacement activity. However, the most pronounced effect was the loss of the ATP-dependent cooperative binding of the RNA substrates. Sequence analyses and an examination of the Protein Data Bank showed that the motif IV phenylalanine is conserved among superfamily 2 helicases. The phenylalanine appears to be...
RNA helicases are essential for virtually all cellular processes, however, their regulation is po... more RNA helicases are essential for virtually all cellular processes, however, their regulation is poorly understood. The activities of eight RNA helicases are required for pre-mRNA splicing. Amongst these, Brr2p is unusual in having two helicase modules, of which only the amino-terminal helicase domain appears to be catalytically active. Using genetic and biochemical approaches, we investigated interaction of the carboxy-terminal helicase module, in particular the carboxy-terminal Sec63-2 domain, with the splicing RNA helicase Prp16p. Combining mutations in BRR2 and PRP16 suppresses or enhances physical interaction and growth defects in an allele-specific manner, signifying functional interactions. Notably, we show that Brr2p Sec63-2 domain can modulate the ATPase activity of Prp16p in vitro by interfering with its ability to bind RNA. We therefore propose that the carboxy-terminal helicase module of Brr2p acquired a regulatory function that allows Brr2p to modulate the ATPase activity...
DEAD-box proteins are the most common RNA helicases, and they are associated with virtually all p... more DEAD-box proteins are the most common RNA helicases, and they are associated with virtually all processes involving RNA. They have nine conserved motifs that are required for ATP and RNA binding, and for linking phosphoanhydride cleavage of ATP with helicase activity. The Q motif is the most recently identified conserved element, and it occurs B17 amino acids upstream of motif I. There is a highly conserved, but isolated, aromatic group B17 amino acids upstream of the Q motif. These two elements are involved in adenine recognition and in ATPase activity of DEAD-box proteins. We made extensive analyses of the Q motif and upstream aromatic residue in the yeast translation-initiation factor Ded1. We made site-specific mutations and tested them for viability in yeast. Moreover, we purified various mutant proteins and obtained the Michaelis-Menten parameters for the ATPase activities. We also measured RNA affinities and strand-displacement activities. We find that the Q motif not only regulates ATP binding and hydrolysis but also regulates the affinity of the protein for RNA substrates and ultimately the helicase activity.
Proceedings of the National Academy of Sciences, 2004
Pre-mRNA splicing requires the function of a number of RNAdependent ATPases͞helicases, yet no thr... more Pre-mRNA splicing requires the function of a number of RNAdependent ATPases͞helicases, yet no three-dimensional structure of any spliceosomal ATPases͞helicases is known. The highly conserved DECD-box protein UAP56͞Sub2 is an essential splicing factor that is also important for mRNA export. The expected ATPase͞helicase activity appears to be essential for the UAP56͞ Sub2 functions. Here, we show that purified human UAP56 is an active RNA-dependent ATPase, and we also report the crystal structures of UAP56 alone and in complex with ADP, as well as a DECD to DEAD mutant. The structures reveal a unique spatial arrangement of the two conserved helicase domains, and ADPbinding induces significant conformational changes of key residues in the ATP-binding pocket. Our structural analyses suggest a specific protein-RNA displacement model of UAP56͞Sub2. The detailed structural information provides important mechanistic insights into the splicing function of UAP56͞Sub2. The structures also will be useful for the analysis of other spliceosomal DExD-box ATPases͞helicases.
The DEAD-box family of putative RNA helicases is composed of ubiquitous proteins that are found i... more The DEAD-box family of putative RNA helicases is composed of ubiquitous proteins that are found in nearly all organisms and that are involved in virtually all processes involving RNA. They are characterized by two tandemly linked, RecA-like domains that contain 11 conserved motifs and highly variable amino-and carboxy-terminal flanking sequences. For this reason, they are often considered to be modular multi-domain proteins. We tested this by making extensive BLASTs and sequence alignments to elucidate the minimal functional unit in nature. We then used this information to construct chimeras and deletions of six essential yeast proteins that were assayed in vivo. We purified many of the different constructs and characterized their biochemical properties in vitro. We found that sequence elements can only be switched between closely related proteins and that the carboxy-terminal sequences are important for high ATPase and strand displacement activities and for high RNA binding affinity. The amino-terminal elements were often toxic when overexpressed in vivo, and they may play regulatory roles. Both the amino and the carboxyl regions have a high frequency of sequences that are predicted to be intrinsically disordered, indicating that the flanking regions do not form distinct modular domains but probably assume an ordered structure with ligand binding. Finally, the minimal functional unit of the DEAD-box core starts two amino acids before the isolated phenylalanine of the Q motif and extends to about 35 residues beyond motif VI. These experiments provide evidence for how a highly conserved structural domain can be adapted to different cellular needs.
RNA helicases of the DEAD-box protein family have been shown to participate in every aspect of RN... more 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.
Pre-mRNA splicing requires the activities of several ATPases from the DEAH-box, DEAD-box and Ski2... more Pre-mRNA splicing requires the activities of several ATPases from the DEAH-box, DEAD-box and Ski2-like helicase families to control conformational rearrangements within the spliceosome. Recent findings indicate that several spliceosomal helicases can act at multiple stages of the splicing reaction, and information on how those multiple actions are controlled are emerging. The recently solved crystal structure of the DEAH-box helicase Prp43 provides novel insights into the similarities and differences between the three helicase families. Here we discuss the potential family-specific mechanisms of spliceosomal RNA helicases and their regulation.
SF1 and SF2 helicases have structurally conserved cores containing seven to eight distinctive mot... more SF1 and SF2 helicases have structurally conserved cores containing seven to eight distinctive motifs and variable amino- and carboxyl-terminal flanking sequences. We have discovered a motif upstream of motif I that is unique to and characteristic of the DEAD box family of RNA helicases. It consists of a 9 amino acid sequence containing an invariant glutamine. A conserved phenylalanine occurs 17 aa further upstream. Sequence alignments, site-specific mutagenesis, and ATPase assays show that this motif and the upstream phenylalanine are highly conserved, that they are essential for viability in the yeast Saccharomyces cerevisiae, and that they control ATP binding and hydrolysis in the yeast translation-initiation factor eIF4A. These results are consistent with computer studies of the solved crystal structures.
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Papers by Olivier Cordin