Dbp5p, a cytosolic RNA helicase, is required for poly(A)+ RNA export

Tseng, Stephanie S I; Weaver, P.; Liu, Yan; Hitomi, Midori; Tartakoff, Alan M.; Chang, Tien Hsien

doi:10.1093/emboj/17.9.2651

Cited by 244 publications

(196 citation statements)

References 68 publications

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“…Our data indicate that purified recombinant DDX1 does not possess RNA unwinding activity, but the immunoprecipitated DDX1 complexes exhibit such activity to unwind a substrate with poly(A) overhang. This finding implies that, just like eIF-4A (20) and Dbp5p (27), DDX1 requires co-factors to unwind RNA duplexes. It was also demonstrated that DDX1 exhibits its RNA unwinding activity only for a specific RNA substrate, as previously observed in the case of An3 (37).…”

Section: Discussionmentioning

confidence: 94%

“…Two sets of RNA fragments, A and B or C and D, were annealed in buffer containing 10 mM Tris-HCl, pH 7.5, and 100 mM NaCl after boiling for 5 min and cooling down gradually to 30°C. RNA unwinding assay was performed as described (27). 40 fmol of duplex RNA in a 10-l reaction containing 17 mM HEPES, pH 7.5, 150 mM KCl, 2 mM DTT, 1 mM MgCl 2 , 5% glycerol, 0.3% PEG 8,000, 1 mM ATP, 40 units of RNasin, 1 g of tRNA, and the indicated proteins were incubated at 37°C for 20 min.…”

Section: Amino Acid Sequence Analysis By Lc/ms/ms-followingmentioning

confidence: 99%

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An RNA Helicase, DDX1, Interacting with Poly(A) RNA and Heterogeneous Nuclear Ribonucleoprotein K

Chen

Lin

Tsay

et al. 2002

Journal of Biological Chemistry

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Heterogeneous nuclear ribonucleoprotein K (hnRNP K) is a multifunctional protein known to be involved in the regulation of transcription, translation, nuclear transport, and signal transduction. To systematically obtain insight into mechanisms of hnRNP K activities, we set out to identify protein factors that interact with hnRNP K by using glutathione S-transferase-hnRNP K affinity chromatography followed by liquid chromatography/mass spectrometry/mass spectrometry analysis. Several partner proteins in the K562 cell lysates were identified through this method. One of them is a DEAD box-containing putative RNA helicase, DDX1. In vitro binding and co-immunoprecipitation studies confirmed the protein-protein interaction between hnRNP K with DDX1, and the region spanning amino acids 1-276 of hnRNP K is apparently responsible for its physical interaction with DDX1. Interestingly, their interaction was disrupted by the addition of poly(C), poly(A), and poly(U) RNA substrates. We found that DDX1 was a homopolymeric poly(A) RNA-binding protein. On the other hand, the ATPase activity of the purified recombinant DDX1 protein was stimulated by these homopolymeric RNAs and yeast total RNA but not by DNA. Moreover, the immunoprecipitated DDX1 complex but not purified DDX1 can unwind double-stranded RNA having singlestranded poly(A) overhangs.The hnRNP K 1 protein has been identified as a component of the heterogeneous nuclear ribonucleoprotein complexes. These hnRNP proteins bind pre-mRNAs directly and appear to facilitate various stages of mRNA biogenesis (1-3). hnRNP K can bind to RNA and single-stranded or double-stranded DNA. The nucleic acid binding activity of hnRNP K is mediated by three repeats of motifs termed the K homology domain, which consists of 65-70 residues (4). It contains both classical nuclear localization signal and the K nuclear shuttling domain that allow the protein to shuttle between nucleus and cytoplasm (5). hnRNP K appears to be involved in transcriptional regulation as exemplified by its binding to a C-rich sequence (the CT element) within the human c-myc promoter and subsequent activation of c-myc expression (6 -8). Additionally, its association with CCAAT/enhancer-binding protein ␤ results in the repression of CCAAT/enhancer-binding protein ␤ target genes (9). hnRNP K was also reported to bind 3Ј end of 15-lipoxygenase, and HPV 16 L2 mRNA was reported to trigger translation inhibition (10, 11). Moreover, direct protein-protein interactions between hnRNP K and some proto-oncogene products serve as clear evidence that hnRNP K acts as a docking platform to facilitate molecular interactions within signal transduction cascades (12-16).Modulation of RNA structure is an essential step in many fundamental cellular processes, including RNA synthesis, splicing, export, turnover, and translation. Recently, an increasing number of RNA helicases from different organisms, ranging from bacteria to yeast, human, and virus, has been identified (17, 18). DEAD box proteins are a family of putative RNA helicases t...

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Section: Discussionmentioning

confidence: 94%

Section: Amino Acid Sequence Analysis By Lc/ms/ms-followingmentioning

confidence: 99%

An RNA Helicase, DDX1, Interacting with Poly(A) RNA and Heterogeneous Nuclear Ribonucleoprotein K

Chen

Lin

Tsay

et al. 2002

Journal of Biological Chemistry

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“…Our demonstration that NOH61 exhibits the capacity to hydrolyze ATP is not surprising, because many proteins that possess the superfamily II helicase domain have been shown previously to be ATPases. However, NOH61 ATPase activity displayed only a modest stimulation (1.6-to 3.9-fold) in the presence of polynucleotides, in apparent contrast to other DEAD-box proteins (for example, see proteins Dbp5p [Tseng et al, 1998] and Ded1p [Iost et al, 1999]). Nevertheless, it should be noted that there are several viral proteins whose ATPase activity is stimulated only weakly by nucleic acids (Kadare and Haenni, 1997).…”

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confidence: 88%

A Novel Helicase-Type Protein in the Nucleolus: Protein NOH61

Zirwes

Eilbracht

Kneissel

et al. 2000

MBoC

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We report the identification, cDNA cloning, and molecular characterization of a novel, constitutive nucleolar protein. The cDNA-deduced amino acid sequence of the human protein defines a polypeptide of a calculated mass of 61.5 kDa and an isoelectric point of 9.9. Inspection of the primary sequence disclosed that the protein is a member of the family of "DEAD-box" proteins, representing a subgroup of putative ATP-dependent RNA helicases. ATPase activity of the recombinant protein is evident and stimulated by a variety of polynucleotides tested. Immunolocalization studies revealed that protein NOH61 (nucleolar helicase of 61 kDa) is highly conserved during evolution and shows a strong accumulation in nucleoli. Biochemical experiments have shown that protein NOH61 synthesized in vitro sediments with ϳ11.5 S, i.e., apparently as homo-oligomeric structures. By contrast, sucrose gradient centrifugation analysis of cellular extracts obtained with buffers of elevated ionic strength (600 mM NaCl) revealed that the solubilized native protein sediments with ϳ4 S, suggestive of the monomeric form. Interestingly, protein NOH61 has also been identified as a specific constituent of free nucleoplasmic 65S preribosomal particles but is absent from cytoplasmic ribosomes. Treatment of cultured cells with 1) the transcription inhibitor actinomycin D and 2) RNase A results in a complete dissociation of NOH61 from nucleolar structures. The specific intracellular localization and its striking sequence homology to other known RNA helicases lead to the hypothesis that protein NOH61 might be involved in ribosome synthesis, most likely during the assembly process of the large (60S) ribosomal subunit. INTRODUCTIONNucleoli, the most conspicuous intranuclear structures in eukaryotic cells, are known to be the main site of ribosome biosynthesis (Hadjiolov, 1985;Scheer and Weisenberger, 1994;Scheer and Hock, 1999). More recently, however, it has also become clear that the nucleolus has additional functions and may be involved in the transport or assembly of various kinds of ribonucleoprotein particles (reviewed by Pederson, 1998).The production of preribosomal particles is a complex process, involving the transcription of the rRNA genes, the processing of the primary transcripts, and the addition of proteins to the nascent preribosomes as well as the incorporation of the 5S rRNA, which is synthesized outside of the nucleolus. Besides the rRNA gene clusters and flanking sequences, nucleoli contain a large number of proteins and RNA components that are not part of mature cytoplasmic ribosomes but are involved in the transcriptional (Reeder, 1990;Paule, 1993) and post-transcriptional regulation of ribosome synthesis (Olson, 1990). Among these are small nucleolar RNAs (snoRNAs), which play a major role in the endo-and exonucleolytic processing of the large preribosomal precursor as well as in the specific modification of rRNA molecules by remodeling certain uridine residues into pseudouridines and by tagging ribose moieties with methyl groups (To...

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“…Dbp5 (DDX19 and DDX25 in vertebrates) represents a furthe r emerging paradigm of how DEAD box proteins are regulated: through local activation. It interacts with the transcription machinery and with nascent RNPs 74,75 to mediate mRNA export from the nucleus 110,111 , and it has also been implicated in the termination of translation 112,113 . Although it is not known whether Dbp5 remains bound to mRNAs from transcription to export, as eIF4AIII does, elegant studies have demonstrated that Dbp5 activity must be highly localized during mRNA export 114,115 .…”

Section: Nuclear Specklesmentioning

confidence: 99%

From unwinding to clamping — the DEAD box RNA helicase family

Linder

Jankowsky

2011

Nat Rev Mol Cell Biol

956

1,069

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Nearly all aspects of RNA metabolism, from transcription and translation to mRNA decay, involve RNA helicases, which are enzymes that use ATP to bind or remodel RNA and RNA-protein complexes (ribonucleoprotein (RNP) complexes) 1 . RNA helicases are found in all three domains of life, and many viruses also encode one or more of these proteins 2,3 . Together with the structurally related DNA helicases that function in replication, recombination and repair, the RNA helicases are classified into superfamilies and families, based on sequence and structural features 3,4 . DEAD box proteins form the largest helicase family, with 37 members in humans and 26 in Saccharomyces cerevisiae 3 , and are characterized by the presence of an Asp-Glu-Ala-Asp (DEAD) motif.DEAD box helicases have central and, in many cases, essential physiological roles in cellular RNA metabolism 5 (FIG. 1). The proteins generally function as part of larger multicomponent assemblies, such as the spliceosom e or the eukaryotic translation initiation machinery 6 . Mutations and deregulation of several DEAD box proteins have been linked to disease states, including cancer 7 . Although all DEAD box proteins contain a structurally highly conserved core with conserved ATP-binding and RNA-binding sites, different proteins have been associated with diverse and seemingly unrelated functions, including the disassembly of RNPs, chaperoning during RNA folding and even stabil ization of protein complexes on RNA 5,6 . How these highly conserved proteins fulfil such an array of different functions has been a long-standing question.Research has now started to illuminate the molecular basis for this functional diversity. In this Review, we summarize how structural data, together with biochemical and biophysical studies, have revealed unexpected modes by which these proteins function. We also discuss how novel molecular and cell biological approaches have better defined the cellular roles of DEAD box helicases. We outline our current view on common structural and mechan istic themes that have emerged, and on physical models of how these 'unusual' RNA helicases work.Structures of DEAD box RNA helicases A number of structural studies have universally shown that members of the DEAD box family contain a highly conserved helicase core that harbours the binding sites for ATP and RNA 3,4,8 . The core is surrounded by variable auxiliary domains, which are thought to be critical for the diverse functions of these enzymes.Structure of the helicase core. DEAD box proteins belong to helicase superfamily 2 (SF2) 2 . Similarly to all SF2 helicases, DEAD box proteins are built around a highly conserved helicase core of two virtually identical domains that resemble the bacterial recombination protein recombinase A (RecA) 3,4,8 (FIG. 2a,b). Within this helicase core, at least 12 characteristic sequence motifs are located at conserved positions (FIG. 2a,b). Some of these motifs are conserved across the entire SF2 family, whereas others are found only in the DEAD box family 3 ....

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Dbp5p, a cytosolic RNA helicase, is required for poly(A)+ RNA export

Cited by 244 publications

References 68 publications

An RNA Helicase, DDX1, Interacting with Poly(A) RNA and Heterogeneous Nuclear Ribonucleoprotein K

An RNA Helicase, DDX1, Interacting with Poly(A) RNA and Heterogeneous Nuclear Ribonucleoprotein K

A Novel Helicase-Type Protein in the Nucleolus: Protein NOH61

From unwinding to clamping — the DEAD box RNA helicase family

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