No abstract

Members of the DExH/D superfamily of nucleic acid-activated nucleotide triphosphatases are essential for virtually all aspects of RNA metabolism, including pre-messenger RNA splicing, RNA interference, translation, and nucleocytoplasmic trafficking. Physiological substrates for these enzymes are thought to be regions of double-stranded RNA, because several DExH/D proteins catalyze strand separation in vitro. These "RNA helicases" can also disrupt RNA-protein interactions, but it is unclear whether this activity is coupled to duplex unwinding. Here we demonstrate that two unrelated DExH/D proteins catalyze protein displacement independently of duplex unwinding. Therefore, the essential functions of DExH/D proteins are not confined to RNA duplexes but can be exerted on a wide range of ribonucleoprotein substrates.

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 al arge number of RNA and RNP complexes in ap hysiological context. It is unknown how this discrimination between potential substrates is achieved. Here we show one possible way by which anon-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 at ightly 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 ad iscriminatory fashion. In addition, our study illuminates possible mechanisms for protein displacement by DExH/D proteins.

RNAs in biological processes often interconvert between defined structures. These RNA structure conversions are assisted by proteins and are frequently coupled to ATP hydrolysis. It is not well understood how proteins coordinate RNA structure conversions and which role ATP hydrolysis has in these processes. Here, we have investigated in vitro how the DEAD-box ATPase Ded1 facilitates RNA structure conversions in a simple model system. We find that Ded1 assists RNA structure conversions via two distinct pathways. One pathway requires ATP hydrolysis and involves the complete disassembly of the RNA strands. This pathway represents a kinetically controlled steady state between the RNA structures, which allows formation of less stable from more stable RNA conformations and thus RNA structure conversion against thermodynamic equilibrium values. The other pathway is ATP-independent and proceeds via multipartite intermediates that are stabilized by Ded1. Our results provide a basic mechanistic framework for protein-assisted RNA structure conversions that illuminates the role of ATP hydrolysis and reveal an unexpected diversity of pathways.

Hepatitis C virus (HCV) infects over 170 million persons worldwide. It is the leading cause of liver disease in the U.S. and is responsible for most liver transplants. Current treatments for this infectious disease are inadequate; therefore, new therapies must be developed. Several labs have obtained evidence for a protein complex that involves many of the nonstructural (NS) proteins encoded by the virus. NS3, NS4A, NS4B, NS5A, and NS5B appear to interact structurally and functionally. In this study, we investigated the interaction between the helicase, NS3, and the RNA polymerase, NS5B. Pull-down experiments and surface plasmon resonance data indicate a direct interaction between NS3 and NS5B that is primarily mediated through the protease domain of NS3. This interaction reduces the basal ATPase activity of NS3. However, NS5B stimulates product formation in RNA unwinding experiments under conditions of excess nucleic acid substrate. When the concentrations of NS3 and NS5B are in excess of nucleic acid substrate, NS5B reduces the rate of NS3-catalyzed unwinding. Under pre-steady-state conditions, in which NS3 and substrate concentrations are similar, product formation increased in the presence of NS5B. The increase was consistent with 1:1 complex formed between the two proteins. A fluorescently labeled form of NS3 was used to investigate this interaction through fluorescence polarization binding assays. Results from this assay support interactions that include a 1:1 complex formed between NS3 and NS5B. The modulation of NS3 by NS5B suggests that these proteins may function together during replication of the HCV genome.

RNA helicases are a large family of molecular motors that utilize nucleoside triphosphates to unwind RNA duplexes and to remodel RNA protein complexes. In this review, we discuss the structure and function of RNA helicases with an emphasis on the potential application of these enzymes to control conformational changes in nanoassemblies that contain RNA.

RNA helicases are essential for the adenosine 5'-triphosphate (ATP)-driven rearrangement of many RNAs and RNA-protein complexes (ribonucleoproteins, RNPs) throughout RNA metabolism. We describe assays to measure RNA and RNP remodeling by RNA helicases in vitro. We show how to prepare substrates for these reactions and how to monitor unwinding of RNA duplexes and displacement of proteins from RNA using standard molecular biology techniques.