An RNA helicase, DDX1, interacting with poly(A) RNA and heterogeneous nuclear ribonucleoprotein K.
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.
Institute of Molecular Medicine, College of Medicine, National Taiwan University, Academia Sinica, No. 1 Sec. 4, Roosevelt Road, Taipei 106, Taiwan.
Hui-Chin
HC
Chen
Wei-Chin
WC
Lin
Yeou-Guang
YG
Tsay
Sheng-Chung
SC
Lee
Ching-Jin
CJ
Chang
D000251:Adenosine Triphosphatases / Q000378:metabolism; D000595:Amino Acid Sequence; D001483:Base Sequence; D002846:Chromatography, Affinity; D053487:DEAD-box RNA Helicases; D017931:DNA Primers; D034641:Heterogeneous-Nuclear Ribonucleoprotein K / Q000378:metabolism*; D006801:Humans; D020014:K562 Cells; D008969:Molecular Sequence Data; D011233:Precipitin Tests; D011485:Protein Binding; D020365:RNA Helicases / Q000737:chemistry / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction
D017931:DNA Primers; D034641:Heterogeneous-Nuclear Ribonucleoprotein K; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; D000251:Adenosine Triphosphatases; C086252:DDX1 protein, human; D053487:DEAD-box RNA Helicases; D020365:RNA Helicases