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 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...

A Role for DEAD Box 1 at DNA Double-Strand Breaks

“…In a previous study, recombinant DDX1 was unable to unwind RNA-RNA duplexes containing either 10 or 14 bp of complementary sequence (16). Here we have shown that DDX1 can unwind both RNA-RNA and RNA-DNA duplexes containing 29 bp of double-stranded structure in an ADP-dependent manner.…”

Identification of Genes Differentially Expressed in Mouse Fetuses from Streptozotocin-induced Diabetic Pregnancy by cDNA Subtraction

“…Brcc3 is a subunit included in BRCA complexes that are involved in DNA repair [19]. Ddx1 is a putative RNA helicase that binds hnRNP [20] and Commd3 is assumed to block NF-kB function via inhibiting access of NF-kB to chromatin [17]. We also quantified the expression level of genes that have been reported to be involved in neural crest/tube defects, and found that 3 genes, EphrinA5 [21], Folbp1 [22], and Sox10 [23], were differentially expressed (Fig.…”

The hnRNA-Binding Proteins hnRNP L and PTB Are Required for Efficient Translation of the Cat-1 Arginine/Lysine Transporter mRNA during Amino Acid Starvation

“…We found that hnRNP K, YB-1, and DDX1 form complexes with the Cat-1 IRES (see Table S2 in the supplemental material). These proteins are known to interact with one another (7,60,75) and with hnRNP L and PTB (13,29,40). The recruitment of the ribosome may involve interactions of these proteins with ribosome-associated proteins such as RACK1 (59).…”