Human DDX21 binds and unwinds RNA guanine quadruplexes - PubMed (original) (raw)

Human DDX21 binds and unwinds RNA guanine quadruplexes

Ewan K S McRae et al. Nucleic Acids Res. 2017.

Abstract

Guanine quadruplexes (G4s) are an important structure of nucleic acids (DNA and RNA) with roles in several cellular processes. RNA G4s require specialized unwinding enzymes, of which only two have been previously identified. We describe the results of a simple and specific mass spectrometry guided method used to screen HEK293T cell lysate for G4 binding proteins. From these results, we validated the RNA helicase protein DDX21. DDX21 is an established RNA helicase, but has not yet been validated as a G4 binding protein. Through biochemical techniques, we confirm that DDX21-quadruplex RNA interactions are direct and mediated via a site of interaction at the C-terminus of the protein. Furthermore, through monitoring changes in nuclease sensitivity we show that DDX21 can unwind RNA G4. Finally, as proof of principle, we demonstrate the ability of DDX21 to suppress the expression of a protein with G4s in the 3΄ UTR of its mRNA.

© The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

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Figures

Figure 1.

Figure 1.

DDX21 is an RNA helicase recovered in abundance from pull-down assays with an RNA G4. (A) Proteins identified from streptavidin pull-down assays (SPDA) with RNA G4 by mass spectrometry and sorted by the difference in unique peptide count between G4 and control samples (top 35 hits shown), error bars represent the standard error between four biological replicates. The black bars represent the number of unique peptides from the G4 SPDA and the grey bars represent the number of unique peptides recovered from the negative control samples. (B) Analysis of gene ontology shows a statistically relevant (P < 0.05) overrepresentation of RNA G4 binding proteins when compared to the expected composition of the cell lysate. (C) SPDA were performed with each of the PITX1-derived G4s, hTR(10–43), C9ORF72+/– as well as the non quadruplex Q1-mutant. Recovery of DDX21 and DHX36 was detected by western blot. DHX36 was enriched by all the G4 quadruplexes, while DDX21 was only enriched by Q2 and C9ORF72+.

Figure 2.

Figure 2.

The C terminus of DDX21 (DDX21(574–783)) is both necessary and sufficient for the interaction with the Q2 RNA. (A) Cartoon representation of N-terminally FLAG-tagged truncations of DDX21. (B) Overexpression levels of FLAG-tagged DDX21 truncations in HEK293T cell lysate compared by western blot with anti-FLAG and anti-alpha-tubulin antibodies (∼55 kDa). (C) Detection of co-precipitated DDX21 truncations from streptavidin pull-down assays with biotinylated Q2 RNA in HEK293T cell lysates. Constructs containing the C-terminal 209 amino acids were all enriched by pull-down with biotinylated Q2, whereas constructs missing the C-terminal 209 amino acids were undetectable.

Figure 3.

Figure 3.

DDX21(1-783) and DDX21(574-783) bind quadruplex with high affinity, mutation of four amino acids in a unique repeat region of DDX21 abrogates the interaction. Dose–response data from microscale thermophoresis experiments were fit to a quadratic binding formula to obtain apparent dissociation constants and Hill coefficients for interactions between Q2 (A–C), hTR(10–43) (D–F), Q2-mutant (G–I) and full length DDX21 (A, D, G), DDX21(574–783) (B, E, H) and DDX21 m4 (C, F, I). Nano-molar affinities are determined between both quadruplex and the wild type DDX21 and DDX21(574–783) constructs, while no, or weak, binding is observed with DDX21 m4. No significant binding was observed between the non-quadruplex Q2-mutant and any DDX21 construct. Error bars represent the standard error between three replicate experiments.

Figure 4.

Figure 4.

Both the top band and bottom band conformation of the S1 RNA are G4 and DDX21 can unwind these G4 in an ATP dependent manner. Analysis of the two conformations of the S1 RNA (as well as negative control ssRNA and positive control hTR(10-43)) by in (A) gel staining with Thioflavin T and Toluidine blue, fluorescence intensity of (B) Thioflavin T, (C) circular dichroism and (D) thermal difference spectra. (E) Unwinding of the S1 RNA by DDX21, DDX21 C-terminus and DHX36. S1 top band (left) or S1 bottom band (right) was incubated with each purified protein without and with 0.5 mM (+) or 5 mM (+++) ATP for 20 min at 30°C before resolution on a native TBE gel and detection by SYBR gold. (F) RNase A and RNase T digestion patterns of 3΄-Cy5 labelled S1 top and bottom band species indicate the use of different guanines but not cytosines or uracils in their secondary structure.

Figure 5.

Figure 5.

DDX21(1-783) and DDX21(574-783) significantly enhance the nuclease sensitivity of (A) Q2 and (B) hTR(10-43) RNA G4 while DDX21 m4 does not. Each RNA was incubated with each protein construct with and without RNase T1 and under different ATP conditions, as indicated at the top of the figure, before resolution on by TBE-UREA PAGE and detection by SYBR gold. Disappearance of the main band and appearance of faster migrating bands is indicative of increased accessibility of guanine residues to RNase T1 due to G4 destabilizing activity.

Figure 6.

Figure 6.

DDX21 wild type but not DDX21 m4 can supress the expression of a beta-galactosidase reporter gene with G4s in its 3΄UTR. (A) Expression of beta-galactosidase in HEK293T cells from a transfected plasmid with the PITX1 3΄UTR or with a mutated quadruplex region under conditions of DDX21 knock down and rescue with DDX21 wild type, DDX21 m4, DDX21 SAT-mutant or DDX21(574-783). Data is the mean of six biological replicates with standard error shown by error bars. Expression was quantified by spectrophotometric detection of ONPG hydrolysis and normalization to protein concentration and the expression level of cells transfected with only control siRNA. (B) Representative western blot of the levels of DDX21 and tubulin in the control, knock down and rescue samples. The DDX21 SAT-mutant and DDX21(574-783) samples were imaged on a separate western blot and cropped into the figure. DDX21(574–783) was visualized with anti-FLAG primary antibodies because it lacks the binding epitope for the anti-DDX21 antibody.

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