Interactions of the C-Terminal Truncated DEAD-Box Protein DDX3X With RNA and Nucleotide Substrates

Abstract

DDX3X is a human DEAD-box RNA helicase implicated in many important cellular processes. In addition to the RecA-like catalytic core, DDX3X contains N- and C-terminal domains. The ancillary domains of DEAD-box RNA helicases have been shown to modulate their interactions with RNA and nucleotide substrates. Here, with the goal of understanding the role of N- and C-terminal domains of DDX3X on the DDX3X catalytic activity, we examined the interactions of RNA substrates and nucleotides with a DDX3X construct possessing the entire N-terminal domain and the catalytic core but lacking 80 residues from its C-terminal domain. Next, we compared our results with previously investigated DDX3X constructs. Our data show that the C-terminal truncated DDX3X does not bind to a blunt-ended double-helix RNA. This conclusion agrees with the data obtained on the wild-type LAF-1 protein, the DDX3X ortholog in Caenorhabditis elegans , and disagrees with the data obtained on the minimally active DDX3X construct, which misses 131 residues from its N-terminal domain and 80 residues from its C-terminal domain. The minimally active DDX3X construct was able to bind to the blunt-ended RNA construct. Combined, the previous studies and our results indicate that the N-terminal of DDX3X modulates the choice of DDX3X–RNA substrates. Furthermore, a previous study showed that the wild-type DDX3X construct hydrolyzes all four nucleotides and deoxynucleotides, both in the presence and absence of RNA. The C-terminal truncated DDX3X investigated here hydrolyzes only cytidine triphosphate (CTP) in the absence of RNA and CTP, adenosine triphosphate (ATP), and deoxyribose adenosine triphosphate (dATP) in the presence of RNA. Hence, the C-terminal truncated DDX3X has a more stringent nucleotide specificity than wild-type DDX3X.

Substrate Specificities of DDX1: A Human DEAD-box protein

“…Single-molecule fluorescence experiments revealed that the Laf-1 DEAD-box protein from Caenorhabditis elegans ( C. elegans ) does not bind to blunt-ended dsRNA 56 . In addition, the human ortholog of Laf-1 - the DDX3X protein - which lacked 80 residues from its C-terminal domain was unable to bind to blunt-ended dsRNA molecules; thus, these helixes did not support the C-terminal truncated DDX1 protein’s ATP hydrolysis activity 37 . Moreover, the ATPase activity of a few DEAD-box proteins is stimulated by DNA molecules 26, 27 28 29 30 31 .…”

“…The TLC assay was employed to determine the RNA length required to support the DDX1 protein ATP hydrolysis activity. TLC has been used extensively to examine the stimulation of the DEAD-box proteins’ ATP hydrolysis activity by various nucleic acid substrates 37, 38, 41 . For the experiments outlined here, we employed single-stranded RNA (ssRNA) molecules of similar sequences and different lengths.…”

“…The Malachite Green-molybdate assay was employed to measure the DDX1 protein’s NTP and deoxy-NTP nucleotide hydrolysis activity 36, 37 . The Malachite Green–molybdate reagent was prepared by the mixing 0.034% of malachite green, 1.04% of ammonium molybdate, and 1 M HCl stirring constantly overnight at 4°C, filtering the solution, and storing it at 4°C.…”

“…Hydrolysis of ATP was monitored using TLC as previously described 37, 38, 41 . First, the RNA was annealed, if necessary, by incubating it with 50 mM HEPES-KOH pH 7.5, 50 mM KCl at 95 °C for 1 min, 65 °C for 3 min, and cooled down to 25 °C for 1 min, before adding MgCl 2 to a final concentration of 10 mM.…”

“…Here, taking advantage of Malachite Green 36, 37 and Thin Layer Chromatography (TLC) 37, 38 assays, we systematically examined whether the DDX1 protein hydrolyzes additional nucleotides in addition to ATP and deoxy-ATP. Moreover, we examined the RNA sequence and structural properties required to support the DDX1 protein ATP hydrolysis activity.…”