Throwing a monkey wrench in the motor: Targeting DExH/D box proteins with small molecule inhibitors

DExH/D box proteins are molecular motors that utilize the energy derived from NTP hydrolysis to perform work — from helicases that remodel RNA to RNPases that alter RNA–protein complexes. Members of this class of proteins are uniquely placed along the RNA information highway to regulate the flow of genetic information. They have been implicated in a number of nodal points encompassing nuclear, cytoplasmic, and organellar RNA-based processes. The identification and characterization of three unique natural products that selectively inhibit the activity of eukaryotic initiation factor (eIF)4A (DDX2) has provided proof-of-principle that the activity of DExH/D box family members can be selectively targeted. Extending these achievements to other DExH/D box proteins is an important future challenge for drugging this family of proteins. This article is part of a Special Issue entitled: The Biology of RNA helicases — Modulation for life.     

eIF4AIII binds spliced mRNA in the exon junction complex and is essential for nonsense-mediated decay

The exon junction complex (EJC), a set of proteins deposited on mRNAs as a consequence of pre-mRNA splicing, is a key effector of downstream mRNA metabolism. We have identified eIF4AIII, a member of the eukaryotic translation initiation factor 4A family of RNA helicases (also known as DExH/D box proteins), as a novel EJC core component. Crosslinking and antibody inhibition studies suggest that eIF4AIII constitutes at least part of the platform anchoring other EJC components to spliced mRNAs. A nucleocytoplasmic shuttling protein, eIF4AIII associates in vitro and in vivo with two other EJC core factors, Y14 and Magoh. In mammalian cells, eIF4AIII is essential for nonsense-mediated mRNA decay (NMD). Finally, a model is proposed by which eIF4AIII represents a new functional class of DExH/D box proteins that act as RNA clamps or 'place holders' for the sequence-independent attachment of additional factors to RNAs.     

Remodeling of ribonucleoprotein complexes with DExH/D RNA helicases

The DExH/D protein family is the largest group of enzymes in eukaryotic RNA metabolism. DExH/D proteins are mainly known for their ability to unwind RNA duplexes in an ATP-dependent fashion. However, it has become clear in recent years that these DExH/D RNA helicases are also involved in the ATP-dependent remodeling of RNA–protein complexes. Here we review recent studies that highlight physiological roles of DExH/D proteins in the displacement of proteins from RNA. We further discuss work with simple RNA–protein complexes in vitro, which illuminates mechanisms by which DExH/D proteins remove proteins from RNA. Although we are only beginning to understand how DExH/D proteins remodel RNA–protein complexes, these studies have shown that an ‘RNA helicase’ does not per se require cofactors to displace proteins from RNA, that protein displacement does not necessarily involve RNA duplex unwinding, and that not all DExH/D proteins are able to disassemble the same range of ribonucleoproteins.     

Discriminatory RNP remodeling by the DEAD-box protein DED1

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.     

The DExH/D protein family database

DExH/D proteins are essential for all aspects of cellular RNA metabolism and processing, in the replication of many viruses and in DNA replication. DExH/D proteins are subject to current biological, biochemical and biophysical research which provides a continuous wealth of data. The DExH/D protein family database compiles this information and makes it available over the WWW (http://www.columbia.edu/ ej67/dbhome.htm ). The database can be fully searched by text based queries, facilitating fast access to specific information about this important class of enzymes.     

Mutational analysis of vaccinia virus nucleoside triphosphate phosphohydrolase II, a DExH box RNA helicase.

Vaccinia virus nucleoside triphosphate phosphohydrolase II (NPH-II), a 3'-to-5' RNA helicase, displays sequence similarity to members of the DExH family of nucleic acid-dependent nucleoside triphosphatases (NTPases). The contributions of the conserved GxGKT and DExH motifs to enzyme activity were assessed by alanine scanning mutagenesis. Histidine-tagged versions of NPH-II were expressed in vaccinia virus-infected BSC40 cells and purified by nickel affinity and conventional fractionation steps. Wild-type His-NPH-II was indistinguishable from native NPH-II with respect to RNA helicase, RNA binding, and nucleic acid-stimulated NTPase activities. The K-191-->A (K191A), D296A, and E297A mutant proteins bound RNA as well as wild-type His-NPH-II did, but they were severely defective in NTPase and helicase functions. The H299A mutant was active in RNA binding and NTP hydrolysis but was defective in duplex unwinding. Whereas the NTPase of wild-type NPH-II was stimulated > 10-fold by polynucleotide cofactors, the NTPase of the H299A mutant was nucleic acid independent. Because the specific NTPase activity of the H299A mutant in the absence of nucleic acid was near that of wild-type enzyme in the presence of DNA or RNA and because the Km for ATP was unaltered by the H299A substitution, we regard this mutation as a "gain-of-function" mutation and suggest that the histidine residue in the DExH box is required to couple the NTPase and helicase activities.     

Functional requirement for symmetric assembly of archaeal box C/D small ribonucleoprotein particles

Box C/D small ribonucleoprotein particles (sRNPs) are archaeal homologs of small nucleolar ribonucleoprotein particles (snoRNPs) in eukaryotes that are responsible for site specific 2'-O-methylation of ribosomal and transfer RNAs. The function of box C/D sRNPs is characterized by step-wise assembly of three core proteins around a box C/D RNA that include fibrillarin, Nop5p, and L7Ae. The most distinct structural feature in all box C/D RNAs is the presence of two conserved box C/D motifs accompanied by often a single, and sometimes two, antisense elements located immediately upstream of either the D or D' box. Despite this asymmetric distribution of antisense elements, the bipartite feature of the box C/D motifs appears to be in pleasing agreement with a recently reported three-dimensional structure of the core protein complex between fibrillarin and Nop5p. This investigates functional implications of the symmetric features both in box C/D RNAs and in the fibrillarin-Nop5p complex. Site-directed mutagenesis was employed to generate box C/D RNAs lacking one of the two box C/D motifs and a mutant fibrillarin-Nop5p complex deficient in self-association. The ability of the mutated components to assemble and to direct methyl transfer reactions was assessed by gel mobility-shift, analytical ultracentrifugation, and in vitro catalysis studies. The results presented here suggest that, while a box C/D sRNP is capable of asymmetrical assembly, the symmetries in both the box C/D RNA and in the fibrillarin-Nop5p complex are required for efficient catalysis. These findings underscore the importance of functional assembly in methyl transfer reactions.     

A DExH/D-box protein coordinates the two steps of splicing in a group I intron

Proteins of the DExH/D family are ATPases that can unwind duplex RNA in vitro. Individual members of this family coordinate many steps in ribonucleoprotein enzyme assembly and catalysis in vivo, but it is largely unknown how the action of these co-factors is specified and precisely timed. As a first step to address this question biochemically, we describe the development of a new protein-dependent group I intron splicing system that requires such an ATPase for coordinating successive steps in splicing. While genetic analysis in yeast has shown that at least five nuclear-encoded proteins are required for splicing of the mitochondrial aI5β group I intron, we show that efficient in vitro splicing of aI5β occurs with only two of these co-factors and, furthermore, they fulfill distinct functions in vitro. The Mrs1p protein stabilizes RNA structure and promotes the first step in splicing. In contrast, a DExH/D protein, Mss116p, acts after the first step and, utilizing ATP hydrolysis, specifically enhances the efficiency of exon ligation. An analysis of Mss116p variants with mutations that impair its RNA-stimulated ATP hydrolysis activity or reduce its ability to unwind duplexes show that the efficiency of ATP hydrolysis is a major determinant in promoting exon ligation. These observations suggest that Mss116p acts in aI5β splicing by catalyzing changes in the structure of the RNA/protein splicing intermediate that promote the second step. More broadly, these observations are consistent with a model in which the “functional-timing” of DExH/D-box protein action can be specified by a specific conformation of its substrate due to the “upstream” activity of other co-factors.     

Nuclear retention elements of U3 small nucleolar RNA

The processing and methylation of precursor rRNA is mediated by the box C/D small nucleolar RNAs (snoRNAs). These snoRNAs differ from most cellular RNAs in that they are not exported to the cytoplasm. Instead, these RNAs are actively retained in the nucleus where they assemble with proteins into mature small nucleolar ribonucleoprotein particles and are targeted to their intranuclear site of action, the nucleolus. In this study, we have identified the cis-acting sequences responsible for the nuclear retention of U3 box C/D snoRNA by analyzing the nucleocytoplasmic distributions of an extensive panel of U3 RNA variants after injection of the RNAs into Xenopus oocyte nuclei. Our data indicate the importance of two conserved sequence motifs in retaining U3 RNA in the nucleus. The first motif is comprised of the conserved box C' and box D sequences that characterize the box C/D family. The second motif contains conserved box sequences B and C. Either motif is sufficient for nuclear retention, but disruption of both motifs leads to mislocalization of the RNAs to the cytoplasm. Variant RNAs that are not retained also lack 5' cap hypermethylation and fail to associate with fibrillarin. Furthermore, our results indicate that nuclear retention of U3 RNA does not simply reflect its nucleolar localization. A fragment of U3 containing the box B/C motif is not localized to nucleoli but retained in coiled bodies. Thus, nuclear retention and nucleolar localization are distinct processes with differing sequence requirements.     

RNomics: an experimental approach that identifies 201 candidates for novel, small, non-messenger RNAs in mouse

In mouse brain cDNA libraries generated from small RNA molecules we have identified a total of 201 different expressed RNA sequences potentially encoding novel small non-messenger RNA species (snmRNAs). Based on sequence and structural motifs, 113 of these RNAs can be assigned to the C/D box or H/ACA box subclass of small nucleolar RNAs (snoRNAs), known as guide RNAs for rRNA. While 30 RNAs represent mouse homologues of previously identified human C/D or H/ACA snoRNAs, 83 correspond to entirely novel snoRNAS: Among these, for the first time, we identified four C/D box snoRNAs and four H/ACA box snoRNAs predicted to direct modifications within U2, U4 or U6 small nuclear RNAs (snRNAs). Furthermore, 25 snoRNAs from either class lacked antisense elements for rRNAs or snRNAS: Therefore, additional snoRNA targets have to be considered. Surprisingly, six C/D box snoRNAs and one H/ACA box snoRNA were expressed exclusively in brain. Of the 88 RNAs not belonging to either snoRNA subclass, at least 26 are probably derived from truncated heterogeneous nuclear RNAs (hnRNAs) or mRNAS: Short interspersed repetitive elements (SINEs) are located on five RNA sequences and may represent rare examples of transcribed SINES: The remaining RNA species could not as yet be assigned either to any snmRNA class or to a part of a larger hnRNA/mRNA. It is likely that at least some of the latter will represent novel, unclassified snmRNAS:     

The DExH/D box protein HEL/UAP56 is essential for mRNA nuclear export in Drosophila.

Dbp5 is the only member of the DExH/D box family of RNA helicases that is directly implicated in the export of messenger RNAs from the nucleus of yeast and vertebrate cells. Dbp5 localizes in the cytoplasm and at the cytoplasmic face of the nuclear pore complex (NPC). In an attempt to identify proteins present in a highly enriched NPC fraction, two other helicases were detected: RNA helicase A (RHA) and UAP56. This suggested a role for these proteins in nuclear transport. Contrary to expectation, we show that the Drosophila homolog of Dbp5 is not essential for mRNA export in cultured Schneider cells. In contrast, depletion of HEL, the Drosophila homolog of UAP56, inhibits growth and results in a robust accumulation of polyadenylated RNAs within the nucleus. Consequently, incorporation of [35S]methionine into newly synthesized proteins is inhibited. This inhibition affects the expression of both heat-shock and non-heat-shock mRNAs, as well as intron-containing and intronless mRNAs. In HeLa nuclear extracts, UAP56 preferentially, but not exclusively, associates with spliced mRNAs carrying the exon junction complex (EJC). We conclude that HEL is essential for the export of bulk mRNA in Drosophila. The association of human UAP56 with spliced mRNAs suggests that this protein might provide a functional link between splicing and export.     

A role for the Dicer helicase domain in the processing of thermodynamically unstable hairpin RNAs

In humans a single species of the RNAseIII enzyme Dicer processes both microRNA precursors into miRNAs and long double-stranded RNAs into small interfering RNAs (siRNAs). An interesting but poorly understood domain of the mammalian Dicer protein is the N-terminal helicase-like domain that possesses a signature DExH motif. Cummins et al. created a human Dicer mutant cell line by inserting an AAV targeting cassette into the helicase domain of both Dicer alleles in HCT116 cells generating an in-frame 43-amino-acid insertion immediately adjacent to the DExH box. This insertion creates a Dicer mutant protein with defects in the processing of most, but not all, endogenous pre-miRNAs into mature miRNA. Using both biochemical and computational approaches, we provide evidence that the Dicer helicase mutant is sensitive to the thermodynamic properties of the stems in microRNAs and short-hairpin RNAs, with thermodynamically unstable stems resulting in poor processing and a reduction in the levels of functional mi/siRNAs. Paradoxically, this mutant exhibits enhanced processing efficiency and concomitant RNA interference when thermodynamically stable, long-hairpin RNAs are used. These results suggest an important function for the Dicer helicase domain in the processing of thermodynamically unstable hairpin structures.     

The three-dimensional arcitecture of the EJC core

The exon junction complex (EJC) is a macromolecular complex deposited at splice junctions on mRNAs as a consequence of splicing. At the core of the EJC are four proteins: eIF4AIII, a member of the DExH/D-box family of NTP-dependent RNA binding proteins, Y14, Magoh, and MLN51. These proteins form a stable heterotetramer that remains bound to the mRNA throughout many different cellular environments. We have determined the three-dimensional (3D) structure of this EJC core using negative-stain random-conical tilt electron microscopy. This structure represents the first structure of a DExH/D-box protein in complex with its binding partners. The EJC core is a four-lobed complex with a central channel and dimensions consistent with its known RNA footprint of about ten nucleotides. Using known X-ray crystallographic structures and a model of three of the four components, we propose a model for complex assembly on RNA and explain how Y14:Magoh may influence eIF4AIII's RNA binding.     

Structural features of the guide:target RNA duplex required for archaeal box C/D sRNA-guided nucleotide 2'-O-methylation

Archaeal box C/D sRNAs guide the 2'-O-methylation of target nucleotides using both terminal box C/D and internal C'/D' RNP complexes. In vitro assembly of a catalytically active Methanocaldococcus jannaschii sR8 box C/D RNP provides a model complex to determine those structural features of the guide:target RNA duplex important for sRNA-guided nucleotide methylation. Watson-Crick pairing of guide and target nucleotides was found to be essential for methylation, and mismatched bases within the guide:target RNA duplex also disrupted nucleotide modification. However, dependence upon Watson-Crick base-paired guide:target nucleotides for methylation was compromised in elevated Mg(2+) concentrations where mismatched target nucleotides were modified. Nucleotide methylation required that the guide:target duplex consist of an RNA:RNA duplex as a target ribonucleotide within a guide RNA:target DNA duplex that was not methylated. Interestingly, D and D' target RNAs exhibited different levels of methylation when deoxynucleotides were inserted into the target RNA or when target methylation was carried out in elevated Mg(2+) concentrations. These observations suggested that unique structural features of the box C/D and C'/D' RNPs differentially affect their respective methylation capabilities. The ability of the sR8 box C/D sRNP to methylate target nucleotides positioned within highly structured RNA hairpins suggested that the sRNP can facilitate unwinding of double-stranded target RNAs. Finally, increasing target RNA length to extend beyond those nucleotides that base pair with the sRNA guide sequence significantly increased sRNP turnover and thus nucleotide methylation. This suggests that target RNA interaction with the sRNP core proteins is also important for box C/D sRNP-guided nucleotide methylation.     

Dynamic guide-target interactions contribute to sequential 2'-O-methylation by a unique archaeal dual guide box C/D sRNP

Assembly and guide-target interaction of an archaeal box C/D-guide sRNP was investigated under various conditions by analyzing the lead (II)-induced cleavage of the guide RNA. Guide and target RNAs derived from Haloferax volcanii pre-tRNA(Trp) were used with recombinant Methanocaldococcus jannaschii core proteins in the reactions. Core protein L7Ae binds differentially to C/D and C'/D' motifs of the guide RNA, and interchanging the two motifs relative to the termini of the guide RNA did not affect L7Ae binding or sRNA function. L7Ae binding to the guide RNA exposes its D'-guide sequence first followed by the D guide. These exposures are reduced when aNop5p and aFib proteins are added. The exposed guide sequences did not pair with the target sequences in the presence of L7Ae alone. The D-guide sequence could pair with the target in the presence of L7Ae and aNop5p, suggesting a role of aNop5p in target recruitment and rearrangement of sRNA structure. aFib binding further stabilizes this pairing. After box C/D-guided modification, target-guide pairing at the D-guide sequence is disrupted, suggesting that each round of methylation may require some conformational change or reassembly of the RNP. Asymmetric RNPs containing only one L7Ae at either of the two box motifs can be assembled, but a functional RNP requires L7Ae at the box C/D motif. This arrangement resembles the asymmetric eukaryal snoRNP. Observations of initial D-guide-target pairing and the functional requirement for L7Ae at the box C/D motif are consistent with our previous report of the sequential 2'-O-methylations of the target RNA.     

Direct cloning of double-stranded RNAs from RNase protection analysis reveals processing patterns of C/D box snoRNAs and provides evidence for widespread antisense transcript expression

We describe a new method that allows cloning of double-stranded RNAs (dsRNAs) that are generated in RNase protection experiments. We demonstrate that the mouse C/D box snoRNA MBII-85 (SNORD116) is processed into at least five shorter RNAs using processing sites near known functional elements of C/D box snoRNAs. Surprisingly, the majority of cloned RNAs from RNase protection experiments were derived from endogenous cellular RNA, indicating widespread antisense expression. The cloned dsRNAs could be mapped to genome areas that show RNA expression on both DNA strands and partially overlapped with experimentally determined argonaute-binding sites. The data suggest a conserved processing pattern for some C/D box snoRNAs and abundant expression of longer, non-coding RNAs in the cell that can potentially form dsRNAs.