RNA helicase DDX19 stabilizes ribosomal elongation and termination complexes

Abstract

The human DEAD-box RNA-helicase DDX19 functions in mRNA export through the nuclear pore complex. The yeast homolog of this protein, Dbp5, has been reported to participate in translation termination. Using a reconstituted mammalian in vitro translation system, we show that the human protein DDX19 is also important for translation termination. It is associated with the fraction of translating ribosomes. We show that DDX19 interacts with pre-termination complexes (preTCs) in a nucleotide-dependent manner. Furthermore, DDX19 increases the efficiency of termination complex (TC) formation and the peptide release in the presence of eukaryotic release factors. Using the eRF1(AGQ) mutant protein or a non-hydrolysable analog of GTP to inhibit subsequent peptidyl-tRNA hydrolysis, we reveal that the activation of translation termination by DDX19 occurs during the stop codon recognition. This activation is a result of DDX19 binding to preTC and a concomitant stabilization of terminating ribosomes. Moreover, we show that DDX19 stabilizes ribosome complexes with translation elongation factors eEF1 and eEF2. Taken together, our findings reveal that the human RNA helicase DDX19 actively participates in protein biosynthesis.

Figure 1.

DDX19 is associated with polysomes during translation. Supernatant of HEK 293 cell lysate were loaded onto a 10–60% sucrose gradient, centrifuged, fractionated and subjected to Western blot analysis. Antibodies against ribosomal proteins L9 and S15 were used to detect ribosomal subunits. The absorbance at 260 nm (OD, optical density) shows the distribution of ribosomes. DDX19 and eRF1 are detected by specific antibodies.

Figure 2.

preTC binding experiments with DDX19 and release factors. (A) Western blot analyses of DDX19B and purified preTCs separated via SDG centrifugation using antibodies raised against DDX19B and ribosomal protein L9, respectively. (B) Western blot analysis of purified preTCs or TCs, with the complex of release factors eRF1-eRF3a-GTP, bound with DDX19B in the presence of ATP, ADP and AMPPNP and separated via SDG centrifugation. Antibodies raised against DDX19B were used for detection. (C) Western blot analysis of purified preTCs bound with DDX19B E243Q in the presence of ATP and separated via SDG centrifugation. Antibodies raised against DDX19B were used for detection.

Figure 3.

DDX19B increases TC formation. (A) A scheme of toe-print analysis of a ribosome–mRNA complex formation. Stable ribosomal complex on the mRNA stops reverse transcriptase (AMV) at a certain position generating cDNA products of specific lengths. cDNA molecules synthesized with fluorescently labeled primers are separated and detected using fragment analysis. (B) Examples of raw data from capillary electrophoresis of cDNA products obtained using fluorescently labeled primers for toe-print analysis. PreTCs are shown after SDG purification, TC formation is induced by addition of eRF1 and eRF3c to the preTCs. Positions of preTCs and TCs are labeled by white and black triangles, respectively. (C) Toe-print analysis of termination complexes formed by addition to the preTCs of eRF1, eRF1+eRF3c+GTP, eRF1+eRF3a+GTP and DDX19B+ATP. Rfu – relative fluorescence unit. Positions of preTCs and TCs are labeled by white and black triangles, respectively. (D) Relative quantitative analysis of the stop codon binding efficiency of eRFs in the presence of DDX19B. Stop codon binding efficiency of eRF1 alone was set as 100%. The error bars represent the standard deviation, stars (**) mark a significant difference from the respective control P < 0.01 (n = 3).

Figure 4.

DDX19B increases the efficiency of peptidyl-tRNA hydrolysis induced by release factors. (A) Hydrolysis of peptidyl-tRNA induced by the addition of eRF1 in the presence/absence of DDX19B with ATP or AMPPNP (n = 3). (B) Hydrolysis of peptidyl-tRNA induced by the addition of eRF1 and eRF3c in the presence/absence of DDX19B with ATP or AMPPNP (n = 3). The error bars represent the standard deviation, stars (**) mark a significant difference from the respective control P < 0.01.

Figure 5.

DDX19B activity in translation termination in the presence of eRFs variants. Relative quantitative analysis of the stop codon binding efficiency of (A) eRF1(AGQ), eRF1(AGQ)+eRF3c+GTP, eRF1+eRF3c+GMPPNP and (B) eRF1(K83N), hydroxylated eRF1 (eRF1-OH) in the presence of DDX19B. Stop codon binding efficiency of eRF1 alone was set as 100% (n = 3). (C) ATPase activity of DDX19B mutants E243Q, R372G and R429Q (n = 3). (D) Relative quantitative analysis of the stop codon binding efficiency of eRF1, eRF1+eRF3c+GTP and DDX19B mutants in the presence of ATP or AMPPNP. Stop codon binding efficiency of eRF1 alone was set as 100% (n = 3). The error bars represent the standard deviation.

Figure 6.

DDX19B stabilizes translation elongation complexes. (A) Toe-print analyses of eEF2-preTC complexes with GTP or GMPPNP in the presence of DDX19B. (C) Toe-print analyses of the ribosomal complexes obtained as a result of the decoding of the stop codon by eEF1-suptRNA^Ser-GTP following the ribosome translocation induced by eEF2-GTP in the presence of DDX19B. Rfu – relative fluorescence unit. Positions of preTC are labelled by white triangles, preTC-1 – by grey, preTC+3 – by black. (B) Relative quantitative analysis of the −1 shift efficiency of eEF2-GMPPNP in the presence of DDX19B. −1 shift efficiency of eEF2-GMPPNP was set as 100%. The error bars represent the standard deviation, stars (**) mark a significant difference from the respective control P < 0.01 (n = 3). (D) A quantitative analysis of the translocation efficiency in the presence of DDX19B.