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

Abstract

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.

Figure 1

The core regions of Ddx42p and p68 are highly homologous. The sequences of p68 (amino acids 54–475), Ddx42p (amino acids 213–632) and eIF4A (amino acids 1–406) were aligned. Residues that are homologous (i.e. identical or similar) in Ddx42p and either p68 or eIF4A are highlighted in gray.

Figure 2

Characterization of ddx42 mRNA and protein. (A) Schematic representation of the structure of the ddx42 mRNA. The ddx42 cds is represented as a bar starting with ATG and ending with TAG. ATG_n denotes the true translation initiation codon of Ddx42p (this study), while ATG_o is the previously reported one, and the shaded bar area shows the 5′ ward extended cds. Thin lines left and right of the ddx42 cds symbolize the 5′- and 3′-UTRs, respectively, and gray parts are genomic sequences not found in ddx42 poly(A)⁺ RNA (i1, i2, i3 are introns 1, 2 and 3). Exon 2 (e2) is absent in the shorter ddx42 poly(A)⁺ RNA. PCR primers used for analysis are indicated as arrows, and those not leading to a PCR product are highlighted in gray. (B) ddx42 mRNA is present in similar low levels in different cell lines. A northern blot of poly(A)⁺ RNA (100 ng each) was probed with a ddx42-specific antisense RNA probe. β-Actin RNA served as a loading control. The size of RNA marker bands (Fermentas RNA ladder, high range) is indicated on the right. (C) The protein Ddx42p is expressed differentially in different cell lines. Ddx42p was detected by western blotting in whole cell extracts (15 µg total protein each) with antiserum α-D42N. Actin was detected as a loading control. The molecular weight of marker bands (RPN800, Amersham Biosciences) is indicated on the right. (D) Ddx42p is found exclusively in nuclear extracts. The western blot was performed as above using nuclear extracts (ne) from HeLa and COS cells (lanes 1 and 2). Soluble cellular extracts (ce; lanes 3 and 5) and ne (lanes 4 and 6) from COS cells transfected with full-length ddx42 cDNA (pCddx42; lanes 5 and 6) or a C-terminally shortened form (pCddx42Δ; lanes 3 and 4), respectively, show that even overexpressed protein resides in the nuclei. The overexpressed full-length form is probably subject to proteolytic degradation.

Figure 3

Purification and characterization of recombinant Ddx42p. (A) Ddx42p is purified to apparent homogeneity after expression in E.coli. A Coomassie-stained SDS–PAGE of the purification course is shown. sup, soluble fraction after sonication of E.coli; ft, IMAC flow-through; LMW, low molecular weight calibration kit (Amersham Biosciences); w1 (2), IMAC washes 1 (2); IMAC, Ddx42p-containing IMAC eluate; GF, Ddx42p eluate of gel filtration. Marker band sizes are indicated on the right. (B) Ddx42p is heterologously expressed and in vitro translated as a full-length protein. Lanes 1 and 2 show a western blot with antiserum α-D42C of a HeLa nuclear extract and Ddx42p expressed in E.coli. Lane 3 is an autoradiograph of ³⁵S-labeled Ddx42p in vitro transcribed/translated ddx42 cDNA. The molecular weight of the protein marker bands is indicated. (C) Ddx42p forms complexes of high molecular weight. Purified Ddx42p was subjected to native PAGE and silver staining (lane 1). The molecular weight of the high molecular weight calibration kit proteins (Amersham Biosciences, lane 2) is indicated. Lane 3 shows a nuclear extract of COS cells transfected with full-length ddx42 cDNA (pCddx42) separated on a native PAGE and detected by western blotting with α-D42C.

Figure 4

Ddx42p is an RNA binding protein and RNA-stimulated ATPase. (A) RNA binding by Ddx42p. Purified Ddx42p was incubated with an in vitro transcript (lane 2) and a partial RNA duplex (lane 4), respectively. Lower electrophoretic mobility proves binding of Ddx42p to both RNAs. (B) NTPase activity of Ddx42p. Ddx42p shows a basal NTPase activity with all four NTPs, which is lower than that of p68. Only the Ddx42p ATPase is stimulated in the presence of heteropolymeric RNA. (C) Ddx42p ATPase as a function of Ddx42p concentration. Formation of orthophosphate owing to ATP hydrolysis was monitored at increasing Ddx42p concentrations in the absence and in the presence of poly(A)⁺ RNA. Values depicted represent the average of at least three measurements per point.

Figure 5

Ddx42p is a non-processive RNA helicase stimulated in the presence of a ss binding protein. RNA helicase assay samples were separated by SDS–PAGE and autoradiographed. M, AK, BK and PK denote the partially dsRNA substrates depicted above the respective autoradiograph. An asterisk marks the respective ³²P-labeled RNA strand. In lane 1 of each panel, the expected product of the helicase reaction, the released ss, is shown. The ss binding protein, T4gp32, does not unwind any of the dsRNA substrates used (lanes 2, 6, 10 and 14). Limited unwinding of substrates M and AK by Ddx42p was observed (lanes 3, 4, 7 and 8). For the less stable substrates BK and PK, respectively, the weak Ddx42p-mediated strand-separating activity (lanes 11 and 15, respectively) is promoted by T4gp32 (lanes 12 and 16, respectively). The positions of the respective dsRNA and ssRNA species are indicated.

Figure 6

RNA duplex formation by Ddx42p. (A) Ddx42p mediates annealing of complementary regions in RNA. RNA strands containing complementary regions of differing length were used as substrates for Ddx42p-catalyzed annealing reactions. The ss and ds control samples were incubated under identical conditions, but in the absence of Ddx42p. RNA annealing samples were subjected to SDS–PAGE and autoradiographed. D, M and P7 denote the partially ds product of the respective annealing reaction. The efficiency of RNA annealing by Ddx42p under helicase conditions increases with increasing length of the base pairing region: M (14 bp) < D (46 bp) < P7 (123 bp). The positions of the respective dsRNA and ssRNA species are indicated on the right. (B) Ddx42p-mediated RNA annealing is most efficient with ADP as the nucleotide cofactor. Annealing reactions with 50 nM Ddx42p were carried out with ssRNAs DT7 and DSP6 (complementary region of 46 bases) to exclude a possible counteracting of Ddx42p helicase activity. The reaction is most efficient with ADP, indicating that ATP must be hydrolyzed before RNA annealing. The weak RNA duplex band in lane 3 is attributed to 10% ADP content in the ATPγS preparation as declared by the manufacturer. (C) RNA annealing as a function of Ddx42p concentration. Formation of duplex D (46 bp) was monitored at increasing Ddx42p concentrations. Each point is the average of at least three measurements. In the presence of ADP (after ATP hydrolysis), significant annealing activity was observed even at a Ddx42p concentration used in helicase assays. (D) Ddx42p removes bound protein from RNA. Ddx42p-mediated RNA annealing reactions were carried out with the indicated nucleotide cofactors in the presence of RNA coated with saturating concentrations of the ss binding protein T4gp32. T4gp32 affects the Ddx42p-mediated RNA annealing reaction only when a 50-fold molar excess of T4gp32 over Ddx42p is used in the presence of ATP (lane 6). ADP-bound Ddx42p is even more effective as essentially no inhibition of annealing is seen under these conditions (lane 9).