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

Abstract

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 single-stranded poly(A) overhangs. 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 single-stranded poly(A) overhangs. The hnRNP K 1The abbreviations used are: hnRNP K, heterogeneous nuclear ribonucleoprotein K; GFP, green fluorescent protein; GST, glutathione S-transferase; RT, reverse transcription; DTT, dithiothreitol; PMSF, phenylmethylsulfonyl fluoride; LC/MS/MS, liquid chromatography/mass spectrometry/mass spectrometry. 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 (1Dreyfuss G. Matunis G.M. Pinol-Roma S. Burd C.G. Annu. Rev. Biochem. 1993; 62: 289-321Google Scholar, 2Weighardt F. Biamonti G. Riva S. BioEssays. 1996; 18: 747-756Google Scholar, 3Krecic A.M. Swanson M.S. Curr. Opin. Cell Biol. 1999; 11: 363-371Google Scholar). 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 (4Siomi H. Matunis M.J. Michael W.M. Dreyfuss G. Nucleic Acids Res. 1993; 21: 1193-1198Google Scholar). It contains both classical nuclear localization signal and the K nuclear shuttling domain that allow the protein to shuttle between nucleus and cytoplasm (5Michael W.M. Eder P.S. Dreyfuss G. EMBO J. 1997; 16: 3587-3598Google Scholar). 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-mycpromoter and subsequent activation of c-myc expression (6Takimoto M. Tomonaga T. Matunis M. Avigan M. Krutzsch H. Dreyfuss G. Levens D. J. Biol. Chem. 1993; 268: 18249-18258Google Scholar, 7Tomonaga T. Levens D. J. Biol. Chem. 1995; 270: 4875-4881Google Scholar, 8Michelotti E.F. Michelotti G.A. Aronsohn A.I. Levens D. Mol. Cell. Biol. 1996; 16: 2350-2360Google Scholar). Additionally, its association with CCAAT/enhancer-binding protein β results in the repression of CCAAT/enhancer-binding protein β target genes (9Miau L.H. Chang C.J. Shen B.J. Tsai W.H. Lee S.C. J. Biol. Chem. 1998; 273: 10784-10791Google Scholar). hnRNP K was also reported to bind 3′ end of 15-lipoxygenase, and HPV 16 L2 mRNA was reported to trigger translation inhibition (10Ostareck D.H. Ostareck-Lederer A. Wilm M. Thiele B.J. Mann M. Hentze M.W. Cell. 1997; 89: 597-606Google Scholar, 11Collier B. Goobar-Larsson L. Sokolowski M. Schwartz S. J. Biol. Chem. 1998; 273: 22648-22656Google Scholar). 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 (12Bustelo X.R. Suen K.L. Michael W.M. Dreyfuss G. Barbacid M. Mol. Cell. Biol. 1995; 15: 1324-1332Google Scholar, 13Hobert O. Jallal B. Schlessinger J. Ullrich A. J. Biol. Chem. 1994; 269: 20225-20228Google Scholar, 14Richards S., Yu, D. Blumer K.J. Hauslanden D. Olszowy M.W. Cennelly P.A. Shaw A.S. Mol. Cell. Biol. 1995; 15: 186-197Google Scholar, 15Van Seuningen I. Ostrowski J. Bustelo X.R. Bomsztyk K. J. Biol. Chem. 1995; 270: 26976-26985Google Scholar, 16Schullery D.S. Ostrowski J. Denisenko O.N. Stempka L. Shnyreva M. Suzuki H. Gschwendt M. Bomsztyk K. J. Biol. Chem. 1999; 274: 15101-15109Google Scholar). 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 (17Fuller-Pace F.V. Trends Cell Biol. 1994; 4: 271-274Google Scholar, 18Wassarman D.A. Stetz J.A. Nature. 1991; 340: 463-464Google Scholar). DEAD box proteins are a family of putative RNA helicases that are characterized by a core region of 300–360 amino acids that possesses eight conserved amino acid motifs, one of which is the ATP hydrolysis motif containing the core tetrapeptide DEX(D/H) (19Tanner N.K. Linder P. Mol. Cell. 2001; 8: 251-262Google Scholar). The eighth domain, HRIGRXXR, is part of a basic region and is involved in RNA binding. DEAD box proteins are assumed to be ATP-dependent RNA helicases, based on the observed in vitro RNA helicase activities of the mouse translation initiation factor eIF4A (20Rozen F. Edery I. Meerovitch K. Dever T.E. Merrick W.C. Sonenberg N. Mol. Cell. Biol. 1990; 10: 1134-1144Google Scholar), the human nuclear protein p68 (21Hirling H. Scheffner M. Restle T. Stahl H. Nature. 1989; 339: 562-564Google Scholar), the Xenopus An3 (22Gururajan R. Mathew L. Longo F. Weeks D.L. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 2056-2060Google Scholar), and the Drosophila vasa (23Liang L. Diehl-Jones W. Lasko P. Development. 1994; 120: 1201-1211Google Scholar). The family is growing steadily, and each member differs in its size. The specificity and properties of each protein, including subcellular localization, RNA binding specificity, and regions required for the interaction with accessory proteins, are conferred by the additional N- and C-terminal sequences (17Fuller-Pace F.V. Trends Cell Biol. 1994; 4: 271-274Google Scholar). hnRNP K is involved in multiple regulatory processes of gene expression. To gain more insight into the mechanisms of hnRNP K action, we set out to identify its associated proteins through the hnRNP K affinity column. One of them was DDX1. DDX1 (originally named HuDBP-RB for human DEAD boxprotein identified inretinoblastoma cells) was isolated from a screen for cDNA clones of transcripts preferentially expressed in the retinoblastoma cell lines (24Godbout R. Squire J. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 7578-7582Google Scholar). Co-amplification of DDX1and the proto-oncogene MYCN has been demonstrated in both retinoblastoma and neuroblastoma cell lines (24Godbout R. Squire J. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 7578-7582Google Scholar, 25Squire J.A. Thorner P.S. Weitzman S. Maggi J.D. Dirks P. Doyle J. Hale M. Godbout R. Oncogene. 1995; 10: 1417-1422Google Scholar). DDX1 protein levels in MYCN/DDX1-amplified neuroblastoma and retinoblastoma cell lines correlate well with DDX1 gene copy number and its transcript levels (26Godbout R. Packer M. Bie W. J. Biol. Chem. 1998; 273: 21161-21168Google Scholar). Although there have been extensive research efforts directed toward DDX1 and MYCN, the enzyme activity of DDX1 protein remains to be investigated. Here, we found that DDX1 could interact with hnRNP K and poly(A) RNA. The ATPase and RNA unwinding activities of DDX1 were also further examined and characterized. The full-length hnRNP K cDNA was cloned as described (9Miau L.H. Chang C.J. Shen B.J. Tsai W.H. Lee S.C. J. Biol. Chem. 1998; 273: 10784-10791Google Scholar). The EcoRI fragment of hnRNP K was subcloned into pEGFP-C (Clontech) for eukaryotic GFP-hnRNP K protein expression and also subcloned into pGEX (Amersham Biosciences) for GST fusion protein production. To construct truncated mutants, the EcoRI-HindIII (amino acids 1–180), XbaI-EcoRI (amino acids 277–464),HindIII-EcoRI (amino acids 179–464), andIndIII-XbaI fragments (amino acids 179–276) were individually subcloned into both pEGFP-C (Clontech) and pGEX (Amersham Biosciences) vectors. DDX1s were cloned by RT-PCR using the specific primers (sequence shown in the following method) and K562 cDNA. After being cloned into pCRII-TOPO vector (Invitrogen) and confirmed by nucleotide sequencing, the DDX1 cDNA was further subcloned into pCMV-Tag2 vector (Stratagene) for mammalian cell expression and pVL1392 (Pharmingen) for baculovirus/insect cell expression. Several deletion fragments including theBamHI-ScaI (amino acids 1–524),ScaI-XhoI (amino acids 525–740),BamHI-SmaI (amino acids 1–295), andEcoRI-ScaI fragments (amino acids 185–524) were subcloned into the pCMV-Tag2 vector. RT-PCR was undertaken to clone the DDX1 cDNA. This is done by using total RNA from K562 cells, ready-to-go RT-PCR (Amersham Biosciences), and specific primers for DDX1: primer A, 5′-ATGGGRGRAATGCCTGAG-3′; and primer B, 5′-GTAAATGTAAAAA- TCAGAAGG-3′ Whole cell extracts of K562 cells are prepared in buffer containing 25 mm HEPES, pH 7.7, 0.4 m NaCl, 1.5 mm MgCl2, 0.2 mm EDTA, 0.1% Triton X-100, 0.5 mm DTT, 20 mm β-glycerophosphate, 0.1 mmNa3VO4, 2 μg/ml leupeptine, and 100 μg/ml PMSF. For GST fusion protein columns, 0.5 ml of glutathione-Sepharose beads (Amersham Biosciences) carrying 5 mg of immobilized GST-hnRNP K or GST proteins are used. 100 mg of whole cell extracts are preincubated with GST-bound beads for 3 h at 4 °C, and then the flow-through was mixed with the GST-hnRNP K beads in binding buffer (20 mm HEPES, pH 7.6, mm NaCl, 0.2 mm EDTA, 0.1% Triton X-100, 0.5 mm DTT, 20 mm β-glycerophosphate, 0.1 mm 2 μg/ml leupeptine, and 100 μg/ml for further 3 h at 4 After extensive with binding was with 5 ml of binding buffer containing and m The were by the protein were from the The were with 25 mm containing at The was to liquid chromatography followed by spectrometry and deletion of DDX1 proteins were using the from of of pCMV-Tag2 as The proteins were GST fusion proteins were expressed in and then lysates were prepared to the To the DDX1, the truncated protein amino was The amino fusion protein was purified for The K was as described (9Miau L.H. Chang C.J. Shen B.J. Tsai W.H. Lee S.C. J. Biol. Chem. 1998; 273: 10784-10791Google Scholar). beads were in containing Triton and then mixed with GST fusion proteins on a for 20 at After three with the beads were with 5 of in in a of of (20 mm HEPES, pH 100 mm NaCl, mm MgCl2, 0.1 mm EDTA, and Triton mm DTT, and mm on a The at 4 for the beads were with containing m and with mm pH The proteins were by in buffer and by In some the nucleic acids were as poly(A), and poly(U) RNAs were from and were from The binding in vitro protein and of RNA in 0.5 ml of binding buffer mm HEPES, pH 100 mm NaCl, 2 mm MgCl2, Triton X-100, and 0.1 and was for with at 4 The beads were three with binding The proteins to the RNA beads were by in protein on and by cells were in (Invitrogen) with and 100 μg/ml at in containing K562 cells and retinoblastoma cells were in with cells were in containing and 100 μg/ml at The cells not was by a as described (9Miau L.H. Chang C.J. Shen B.J. Tsai W.H. Lee S.C. J. Biol. Chem. 1998; 273: 10784-10791Google Scholar). and GFP-hnRNP K were by the For recombinant protein multiple were with 2 and then at The cells were then with a of recombinant After at for 3 the cells were and at Whole cell extracts were To whole cell cells were in of buffer (20 mm HEPES, pH 7.6, 0.4 m NaCl, 1.5 mm MgCl2, mm EDTA, 0.1% Triton X-100, mm DTT, μg/ml μg/ml A, and 100 μg/ml The cell was at 4 for and by at The protein was by protein The cell lysates were with beads for 4 h in the binding buffer mm HEPES, pH 7.6, 100 mm NaCl, 1.5 mm MgCl2, 0.2 mm EDTA, 0.1% Triton X-100, 0.5 mm DTT, mm PMSF, μg/ml and μg/ml at 4 The were three with buffer (20 mm HEPES, pH 7.6, 0.2 or NaCl, 0.5 mm MgCl2, 0.1 mm EDTA, 0.1% or Triton X-100, 0.5 mm DTT, mm PMSF, μg/ml and μg/ml The were by or further The were by 0.5 in three of The of the were by and the were and at for further The proteins by were to (Amersham Biosciences), and was done using or The were out in a total of which mm pH mm MgCl2, of and various of protein The were for h at and by to a of 20 One of the was chromatography The chromatography was in m pH and by The of in the were from the using a The RNAs used in unwinding were prepared as A, vector was with EcoRI and with RNA B, vector was with and with RNA vector was and with RNA and vector was with and with RNA RNA and were with of RNA and or and were in buffer containing mm pH and 100 mm for 5 and to RNA unwinding was as described M. A.M. Chang EMBO J. 1998; Scholar). of RNA in a containing mm HEPES, pH mm 2 mm DTT, mm MgCl2, mm of of and the proteins were at for 20 One of buffer K, was then to the and further for The were by To the proteins that interact with hnRNP K, we GST-hnRNP K affinity chromatography using K562 whole cell The protein from GST and GST-hnRNP K chromatography are on followed by Several specific protein were identified through between the protein from GST and GST-hnRNP K These specific purified from the GST-hnRNP K were and by from the were directly by Several proteins were identified by this We were in one of the putative RNA helicase termed DDX1, of the of helicases in mRNA and translation and their to hnRNP K. eight conserved motifs of DEAD box proteins are in DDX1. To further the physical and interaction between hnRNP K and DDX1, we cloned the of DDX1 by To DDX1 directly with hnRNP K, and were using GST the in DDX1 protein was for binding to the beads with GST or GST-hnRNP K. The as shown in A, that the in vitro DDX1 could with GST-hnRNP K. The molecular of GST-hnRNP K and DDX1 are a of protein was on the the GST-hnRNP K was both hnRNP K and DDX1 have RNA-binding their interaction be mediated through RNA binding. we observed that the interaction between hnRNP K with DDX1 to be in the of not To further the of RNA on their the binding DDX1 to GST-hnRNP K was in the of homopolymeric poly(A), and poly(U) the interaction of DDX1 with hnRNP K, not this binding 2 To further the in interaction between DDX1 and co-immunoprecipitation was using cell lysates from cells and Whole cell extracts from cells were immunoprecipitated with beads and by with DDX1. DDX1 protein was found to with K 3 In cell were with recombinant DDX1 and hnRNP K. In this hnRNP K was also through as a component of the 3 results that DDX1 can interact with vitro and in To the region within hnRNP K that binding to DDX1, we a of deletion of the hnRNP K protein. Whole cell extracts of cells with various GFP-hnRNP K were with beads with The in vitro binding was then by shown in A, DDX1 was found to with the K K K and K but binding was observed with the K and protein. GST the results 4 These the DDX1 interaction region of hnRNP K to an between amino acids and a of DDX1 deletion mutants, we the region within DDX1 that is responsible for binding to hnRNP K. The results of the in vitro binding in that regions of DDX1, amino acids amino acids can bind to hnRNP K, their binding are that of full-length DDX1. 2 that the interaction of DDX1 with hnRNP K could be by the of poly(A), poly(C), and poly(U) homopolymeric poly(A) RNA has been to interaction between hnRNP K and other associated proteins, we DDX1 can bind to poly(A) RNA. RNA binding that DDX1 to poly(A) beads but not to beads A, The RNA-binding of DDX1 the RNA binding activity A, with a M.J. Michael W.M. Dreyfuss G. Mol. Cell. Biol. Scholar), hnRNP K a specific RNA binding A, that DDX1 could be by poly(A) beads from the cytoplasm Interestingly, we that hnRNP K was in this poly(A) RNA-binding protein and but not of the DEAD box proteins have ATPase DDX1 protein has an ATPase motif to other DEAD box the ATPase activity of the purified DDX1 protein has not been characterized. K was expressed in cells, and recombinant was expressed in cells To the ATPase activity in was with and ATP hydrolysis was based on the of from as observed on the shown in B, recombinant protein DDX1 was found to ATPase in the of various We the of a of RNAs and on the ATPase activity of DDX1 was stimulated by poly(A), poly(C), and total RNA from yeast from the in the of and and an on the ATPase activity of DDX1, We have demonstrated that hnRNP K can a complex with DDX1 in cell lysates We further examined the ATPase activity of hnRNP or truncated a as shown in from cell lysates was immunoprecipitated with After with or the protein were with and were to and ATPase The that the the full-length hnRNP K could the DDX1 but truncated hnRNP K could not and that the DDX1 ATPase the more hnRNP K could not interact with DDX1, and ATPase activity was from the Several DEAD box proteins RNA unwinding activities in To the RNA helicase activity of DDX1, we out the RNA unwinding activity by DDX1 or hnRNP K on protein by or K and subsequent with RNA. of RNA were prepared as described and the RNA has of overhangs. that recombinant DDX1 RNA unwinding activity and but protein by DDX1 were to the RNA unwinding activity on the RNA in ATP is required in this not with the activity of DDX1 that of immunoprecipitated hnRNP K protein was The fundamental processes of the eukaryotic as RNA and are by multiple protein of to many of that as well as regulatory To more the molecular mechanisms of these processes and the of the the of their and the of their interaction are hnRNP K is a component of the hnRNP and a of has in the regulation of gene expression on different Although many hnRNP proteins have been identified E.F. Michelotti G.A. Aronsohn A.I. Levens D. Mol. Cell. Biol. 1996; 16: 2350-2360Google Scholar, X.R. Suen K.L. Michael W.M. Dreyfuss G. Barbacid M. Mol. Cell. Biol. 1995; 15: 1324-1332Google Scholar, 15Van Seuningen I. Ostrowski J. Bustelo X.R. Bomsztyk K. J. Biol. Chem. 1995; 270: 26976-26985Google Scholar, 16Schullery D.S. Ostrowski J. Denisenko O.N. Stempka L. Shnyreva M. Suzuki H. Gschwendt M. Bomsztyk K. J. Biol. Chem. 1999; 274: 15101-15109Google Scholar, O.N. B. Ostrowski J. Seuningen K. J. Biol. Chem. 1996; Scholar, M. D.S. Suzuki H. Bomsztyk K. J. Biol. Chem. Scholar, M. R. Lee A.S. J. Biol. Chem. 1998; 273: Scholar, C.J. Lee S.C. Cell Biol. 2001; Scholar), the of hnRNP in gene expression regulation are In this we identified a putative RNA helicase DDX1 from GST-hnRNP K affinity chromatography as a hnRNP The GST binding and further confirmed the physical interaction between these RNA helicases have been to be for RNA structure or for P. N.K. J. Trends Biochem. Sci. 2001; Scholar). We that DDX1 hnRNP K RNA are the nucleic interaction between hnRNP K and associated which to hnRNP K, binding to hnRNP K, poly(A), which not bind to hnRNP K, not binding O.N. B. Ostrowski J. Seuningen K. J. Biol. Chem. 1996; Scholar). the interaction of with hnRNP K, the other M. D.S. Suzuki H. Bomsztyk K. J. Biol. Chem. Scholar). we also reported that the interaction between hnRNP K and the core protein was in the of and poly(U) C.J. Lee S.C. Cell Biol. 2001; Scholar). 4 that the region in hnRNP K contains the K homology or domain that is known to nucleic acid binding. We the binding in the of acids and found that the interaction of DDX1 and hnRNP K was in the of poly(C), poly(A), and poly(U) 2 We further demonstrated that DDX1 could bind to poly(A), sequence to facilitate its RNA unwinding that binding of DDX1 to poly(A) and hnRNP K or of hnRNP K to and DDX1 to be It that protein-protein interactions of DDX1 and hnRNP K could their RNA binding activity and their The of DDX1 is not Recently, S. M.J. Packer M. Godbout R. Mol. Biol. Cell. 2001; reported that DDX1 associated with a 3′ end protein that DDX1 is one of the poly(A) RNA binding proteins, can be that DDX1 is involved in the 3′ end of DEAD box family proteins have been reported to be associated with ATP hydrolysis and for many of them the activity is Although RNA binding that DDX1 to bind to poly(A), the ATPase activity of recombinant DDX1 was stimulated by RNAs as poly(A), poly(C), and yeast total to various the known DEAD box proteins, some have RNA specificity, and of them not have specificity or have that are to their assumed in For mammalian which is required for translation initiation of is stimulated more by poly(U) by mRNA (20Rozen F. Edery I. Meerovitch K. Dever T.E. Merrick W.C. Sonenberg N. Mol. Cell. Biol. 1990; 10: 1134-1144Google Scholar). For and yeast specific RNA can their ATP hydrolysis activity and nuclear F.V. EMBO J. 1993; Scholar, D. S. D. J.D. Nature. 1996; Scholar). the in of these proteins be more complex and be from the in vitro This that of RNA helicases is and is on other RNA This is also with helicase activity of recombinant DDX1. some of DEAD box-containing proteins and have been shown to have both ATP hydrolysis and RNA helicase activities in vitro (17Fuller-Pace F.V. Trends Cell Biol. 1994; 4: 271-274Google Scholar). RNA helicases unwind in a to 3′ or a 3′ to in The the double-stranded region with or 3′ single-stranded (19Tanner N.K. Linder P. Mol. Cell. 2001; 8: 251-262Google Scholar). These the helicase to the that purified recombinant DDX1 not RNA unwinding but the immunoprecipitated DDX1 activity to unwind a with poly(A) This (20Rozen F. Edery I. Meerovitch K. Dever T.E. Merrick W.C. Sonenberg N. Mol. Cell. Biol. 1990; 10: 1134-1144Google and M. A.M. Chang EMBO J. 1998; Scholar), DDX1 to unwind RNA It was also demonstrated that DDX1 its RNA unwinding activity for a specific RNA as observed in the of An3 R. Weeks D.L. 1997; Scholar). DDX1 as a RNA helicase in the of a specific RNA and other accessory The properties of DDX1 be further investigated. hnRNP K is in cells K. H. P. B. H. J. Mol. Biol. 1994; and in human and the of human cells M. R. D. L. R. J. R. J. Biol. Chem. 2001; Scholar). DDX1 is with MYCN in retinoblastoma and neuroblastoma cells and be involved in the or the of a of these the interaction of hnRNP K with DDX1 and the associated RNA unwinding activity in the of both and We for the of the