Interaction of the Escherichia coli DEAD box protein DbpA with 23 S ribosomal RNA.

The Escherichia coli DEAD box protein DbpA is unique among the DEAD box family in that its ATPase activity is specifically stimulated by bacterial 23 S ribosomal RNA. We have analysed the interaction between DbpA and a specific region within 23 S rRNA (namely nucleotides 2508-2580) which stimulates full ATPase activity. Using electrophoretic mobility shift assays we show that DbpA binds to this "specific" region with greater efficiency than to other regions of 23 S rRNA, and is not competed off by a non-specific RNA or a mutant RNA in which one of the stem-loops has been disrupted. These data suggest that the secondary structure within this region of 23 S rRNA is important for its recognition and binding by DbpA. We have also examined the ability of DbpA to unwind RNA and show that the purified protein does not behave as an RNA helicase in vitro with the substrates tested.     

The Escherichia coli DEAD protein DbpA recognizes a small RNA hairpin in 23S rRNA

The Escherichia coli DEAD protein DbpA is an RNA-specific ATPase that is activated by a 153-nt fragment within domain V of 23S rRNA. A series of RNA subfragments and sequence changes were used to identify the recognition elements of this RNA-protein interaction. Reducing the size of the fully active 153-nt RNA yields compromised substrates in which both RNA and ATP binding are weakened considerably without affecting the maximal rate of ATP hydrolysis. All RNAs that stimulate ATPase activity contain hairpin 92 of 23S rRNA, which is known to interact with the 3' end of tRNAs in the ribosomal A-site. RNAs with base mutations within this hairpin fail to activate ATP hydrolysis, suggesting that it is a critical recognition element for DbpA. Although the isolated hairpin fails to activate DbpA, RNAs with an extension of approximately 15 nt on either the 5' or 3' side of hairpin 92 elicit full ATPase activity. These results suggest that the binding of DbpA to RNA requires sequence-specific interactions with hairpin 92 as well as nonspecific interactions with the RNA extension. A model relating the RNA binding and ATPase activities of DbpA is presented.     

Cloning and biochemical characterization of Bacillus subtilis YxiN, a DEAD protein specifically activated by 23S rRNA: delineation of a novel sub-family of bacterial DEAD proteins.

DEAD, DEAH and DExH proteins are involved in almost every facet of RNA biochemistry. Members of these protein families exhibit an RNA-dependent ATPase activity and some possess an ATP-dependent RNA helicase activity. Although genetic studies have identified specific functions for certain DEx(D)/(H)proteins from which an RNA substrate can be reasonably inferred, only DbpA from Escherichia coli has been shown to exhibit significant RNA specificity in vitro. Here we describe the characterization of YxiN from Bacillus subtilis, the second DEx(D)/(H)protein to show significant RNA specificity as an isolated, homogenous protein. The ATPase activity of YxiN, like that of DbpA, is stimulated by a 154 nt fragment of 23S rRNA. YxiN has a 2 nM apparent binding constant for this fragment, yet its ATPase activity shows 1800-fold RNA specificity. Along with the conserved motifs shared among all DEAD proteins, YxiN and DbpA have a conserved C-terminal extension. This extension is highly conserved in several additional DEAD proteins. We propose that the C-terminus identifies a protein sub-family whose members bind 23S rRNA and that proteins of this family are likely to function in rRNA maturation/ribosome biogenesis or an unappreciated aspect of translation.     

Characterization of DbpA, an Escherichia coli DEAD box protein with ATP independent RNA unwinding activity.

DbpA is a putative Escherichia coli ATP dependent RNA helicase belonging to the family of DEAD box proteins. It hydrolyzes ATP in the presence of 23S ribosomal RNA and 93 bases in the peptidyl transferase center of 23S rRNA are sufficient to trigger 100% of the ATPase activity of DbpA. In the present study we characterized the ATPase and RNA unwinding activities of DbpA in more detail. We report that-in contrast to eIF-4A, the prototype of the DEAD box protein family-the ATPase and the helicase activities of DbpA are not coupled. Moreover, the RNA unwinding activity of DbpA is not specific for 23S rRNA, since DbpA is also able to unwind 16S rRNA hybrids. Furthermore, we determined that the ATPase activity of DbpA is triggered to a significant extent not only by the 93 bases of the 23S rRNA previously reported but also by other regions of the 23S rRNA molecule. Since all these regions of 23S rRNA are either part of the 'functional core' of the 50S ribosomal subunit or involved in the 50S assembly, DbpA may play an important role in the ribosomal assembly process.     

p72: a human nuclear DEAD box protein highly related to p68.

P72, a novel human member of the DEAD box family of putative RNA-dependent ATPases and ATP-dependent RNA helicases was isolated from a HeLa cDNA library. The predicted amino acid sequence of p72 is highly homologous to that of the prototypic DEAD box protein p68. In addition to the conserved core domains characteristic of DEAD box proteins, p72 contains several N-terminal RGG RNA-binding domains and a serine/glycine rich C-terminus likely involved in mediating protein-protein interactions. A p72-specific probe detects two mRNAs of approximately 5300 and 9300 bases which, although ubiquitously expressed, show variability in their expression levels in different tissues. Purified recombinant p72 exhibits ATPase activity in the presence of a range of RNA moieties. Immunocytochemical studies of p68 and p72 show that these proteins localise to similar locations in the nucleus of HeLa cells, suggesting their involvement in a nuclear process.     

Hepatitis C virus core protein binds to a DEAD box RNA helicase.

Approximately 4 million Americans are infected with the hepatitis C virus (HCV), making it a major cause of chronic liver disease. Because of the lack of an efficient cell culture system, little is known about the interaction between HCV and host cells. We performed a yeast two-hybrid screen of a human liver cell cDNA library with HCV core protein as bait and isolated the DEAD box protein DBX. DBX has significant amino acid sequence identity to mouse PL10, an ATP-dependent RNA helicase. The binding of DBX to HCV core protein occurred in an in vitro binding assay in the presence of 1 M NaCl or detergent. When expressed in mammalian cells, HCV core protein and DBX were co-localized at the endoplasmic reticulum. In a mutant strain of Saccharomyces cerevisiae, DBX complemented the function of Ded1p, an essential DEAD box RNA helicase. HCV core protein inhibited the growth of DBX-complemented mutant yeast but not Ded1p-expressing yeast. HCV core protein also inhibited the in vitro translation of capped but not uncapped RNA. These findings demonstrate an interaction between HCV core protein and a host cell protein involved in RNA translation and suggest a mechanism by which HCV may inhibit host cell mRNA translation.     

Cooperative binding of ATP and RNA substrates to the DEAD/H protein DbpA

Unlike most DEAD/H proteins, the purified Escherichia coli protein DbpA demonstrates high specificity for its 23S rRNA substrate in vitro. Here we describe several assays designed to characterize the interaction of DbpA with its RNA and ATP substrates. Electrophoretic mobility shift assays reveal a sub-nanomolar binding affinity for a 153 nucleotide RNA substrate (R153) derived from the 23S rRNA. High affinity RNA binding requires both hairpin 92 and helix 90, as substrates lacking these structures bind DbpA with lower affinity. AMPPNP inhibition assays and ATP/ADP binding assays provide binding constants for ATP and ADP to DbpA with and without RNA substrates. These data have been used to describe a minimal thermodynamic scheme for the binding of the RNA and ATP substrates to DbpA, which reveals cooperative binding between larger RNAs and ATP with cooperative energies of approximately 1.3 kcal mol(-1). This cooperativity is lost upon removal of helix 89 from R153, suggesting this helix is either the preferred target for DbpA's helicase activity or is a necessary structural element for organization of the target site within R153.     

mRNA export: travelling with DEAD box proteins.

Recent studies have shown that the putative RNA helicase protein UAP56 and its yeast homologue Sub2p are not only involved in pre-mRNA splicing but also required for the export of mRNA out of the nucleus, even if the mRNA is encoded by an intron-less gene.     

Identification of a novel human member of the DEAD box protein family

The cDNA library of human pancreatic islets was screened with sera from patients with insulin-dependent diabetes mellitus (IDDM). From the library screening, we isolated a novel cDNA, RNA helicase-like protein (RHELP), which exhibited strong sequence homology to p68 RNA helicase, a prototypic member of the DEAD (Asp-Glu-Ala-Asp) box protein family. Sequence analysis of the cDNA revealed that RHELP contained DEAD sequence motif and other conserved motifs of the DEAD box protein family, indicating that RHELP is a new member of this family. DEAD box-containing proteins are involved in the RNA processing, ribosome assembly, spermatogenesis, embryogenesis, and cell growth and division. RHELP showed 42% and 44% amino acid sequence identity to human p68 RNA helicase and yeast DBP2 RNA helicase, respectively, among the DEAD box protein family. Northern blot analysis revealed that RHELP is expressed in most tissues including the liver, lung, tonsil, thymus, and muscle in addition to the pancreatic islets. In vivo or in vitro functions of RHELP as a putative RNA helicase and its potential role as a diabetic autoantigen need to be further investigated.     

Escherichia coli DbpA is an RNA helicase that requires hairpin 92 of 23S rRNA

Escherichia coli DbpA is a member of the DEAD/H family of proteins which has been shown to have robust ATPase activity only in the presence of a specific region of 23S rRNA. A series of bimolecular RNA substrates were designed based on this activating region of rRNA and used to demonstrate that DbpA is also a non-processive, sequence-specific RNA helicase. The high affinity of DbpA for the RNA substrates allowed both single and multiple turnover helicase assays to be performed. Helicase activity of DbpA is dependent on the presence of ATP or dATP, the sequence of the loop of hairpin 92 of 23S rRNA and the position of the substrate helix with respect to hairpin 92. This work indicates that certain RNA helicases require particular RNA structures in order for optimal unwinding activity to be observed.     

Mutational analysis of the Escherichia coli DEAD box protein CsdA

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 degrees C. In contrast to wild-type CsdA, a DQAD variant of CsdA (Glu-157-->Gln) had no detectible helicase or ATPase activity at 15 degrees C in vitro. A plasmid encoding the DQAD variant was also unable to suppress the impaired growth of the csdA null mutant at 15 degrees C. Plasmid-encoded CsdADelta444, which lacks most of the carboxy-terminal extension, enhanced the growth of a csdA null mutant at 25 degrees C but not at 15 degrees C; this truncated protein also has limited in vitro activity at 15 degrees C. These results support the physiological function of CsdA as a DEAD box ATP-dependent RNA helicase at low temperature.     

Interaction of Escherichia coli DbpA with 23S rRNA in different functional states of the enzyme

DEx^D/_H proteins catalyze structural rearrangements in RNA by coupling ATP hydrolysis to the destabilization of RNA helices or RNP complexes. The Escherichia coli DEx^D/_H protein DbpA specifically recognizes a region within the catalytic core of 23S rRNA. To better characterize the interaction of DbpA with this region and to identify changes in the complex between different nucleotide-bound states of the enzyme, RNase T1, RNase T2, kethoxal and DMS footprinting of DbpA on a 172 nt fragment of 23S rRNA were performed. A number of protections identified in helices 90 and 92 were consistent with biochemical experiments measuring the RNA binding and ATPase activity of DbpA with truncated RNAs. When DbpA was bound with AMPPNP, but not ADP, several additional footprints were detected in helix 93 and the single-stranded region 5′ of helix 90, suggesting binding of the helicase domains of DbpA at these sites. These results propose that DbpA can act at multiple sites and hint at the targets of its biological activity on rRNA.     

Ddx42p--a human DEAD box protein with RNA chaperone activities

The human gene ddx42 encodes a human DEAD box protein highly homologous to the p68 subfamily of RNA helicases. In HeLa cells, two ddx42 poly(A)⁺ RNA species were detected both encoding the nuclear localized 938 amino acid Ddx42p polypeptide. Ddx42p has been heterologously expressed and its biochemical properties characterized. It is an RNA binding protein, and ATP and ADP modulate its RNA binding affinity. Ddx42p is an NTPase with a preference for ATP, the hydrolysis of which is enhanced by various RNA substrates. It acts as a non-processive RNA helicase. Interestingly, RNA unwinding by Ddx42p is promoted in the presence of a single-strand (ss) binding protein (T4gp32). Ddx42p, particularly in the ADP-bound form (the state after ATP hydrolysis), also mediates efficient annealing of complementary RNA strands thereby displacing the ss binding protein. Ddx42p therefore represents the first example of a human DEAD box protein possessing RNA helicase, protein displacement and RNA annealing activities. The adenosine nucleotide cofactor bound to Ddx42p apparently acts as a switch that controls the two opposing activities: ATP triggers RNA strand separation, whereas ADP triggers annealing of complementary RNA strands.     

A conserved role of a DEAD box helicase in mRNA masking.

Clam p82 is a member of the cytoplasmic polyadenylation element-binding protein (CPEB) family of RNA-binding proteins and serves dual functions in regulating gene expression in early development. In the oocyte, p82/CPEB is a translational repressor, whereas in the activated egg, it acts as a polyadenylation factor. Coimmunoprecipitations were performed with p82 antibodies in clam oocyte and egg lysates to identify stage-regulated accessory factors. p47 coprecipitates with p82 from oocyte lysates in an RNA-dependent manner and is absent from egg lysate p92-bound material. Clam p47 is a member of the RCK/p54 family of DEAD box RNA helicases. Xp54, the Xenopus homolog, with bona fide helicase activity, is an abundant and integral component of stored mRNP in oocytes (Ladomery et al., 1997). In oocytes, clam p47 and p82/CPEB are found in large cytoplasmic mRNP complexes. Whereas the helicase level is constant during embryogenesis, in contrast to CPEB, clam p47 translocates to nuclei at the two-cell stage. To address the role of this class of helicase in masking, Xp54 was tethered via 3' UTR MS2-binding sites to firefly luciferase, following microinjection of fusion protein and nonadenylated reporter mRNAs into Xenopus oocytes. Tethered helicase repressed luciferase translation three- to fivefold and, strikingly, mutations in two helicase motifs (DEAD--> DQAD and HRIGR-->HRIGQ), activated translation three- to fourfold, relative to MS2. These data suggest that this helicase family represses translation of maternal mRNA in early development, and that its activity may be attenuated during meiotic maturation, prior to cytoplasmic polyadenylation.