RHAU associates with RNA through its N terminus. A, CLIP
method to identify interactions of RHAU with nucleic acids (NA). HeLa
cells were transiently transfected with small interfering RNAs against RHAU
(siRHAU) or luciferase (siControl). Cells were UV irradiated
72 h later and harvested. Immunoprecipitation was performed with a RHAU
antibody. Bound nucleic acids were radiolabeled and detected as a band of
∼120 kDa corresponding to RHAU (lane siControl). However, this
band was strongly reduced when endogenous RHAU was depleted (lane
siRHAU). Western blot (INPUT) shows efficient RHAU depletion by
small interfering RNAs against RHAU. Actin was used as a loading control.
B, identification of associated nucleic acids as RNA. After
immunoprecipitation, the nucleic acids bound to RHAU were isolated,
radiolabeled, and treated with increasing concentrations (0.015, 0.15, 1.5,
and 15 units) of RNase A and 1 unit of RQ1DNase. Lane1, isolated and
non-treated nucleic acids(NA); lanes 2-5, isolated nucleic
acids treated with increasing concentrations of RNase; lane 6,
nucleic acids treated with DNase. -Fold changes in intensity ratio
(NA/digest NA) are depicted relative to the intensity of
non-treated nucleic acids in lane 1, set as 1. Note that nucleic
acids are RNase-sensitive but DNase-insensitive. C, RHAU binds to RNA
in vitro. 0, 2.5, 5, 10, 20, and 40 nm GST or GST-RHAU
(RHAU) was incubated with 5,000 cpm 5′-end-labeled RNA for 30
min at 37 °C and filtered through nitrocellulose and nylon membranes. The
percentage of bound RNA was plotted as a function of increasing concentration
of GST-RHAU. Note that dose-dependent RNA binding was seen only with GST-RHAU.
D, effect of stress on RHAU association with RNA in vivo.
HeLa cells were transfected with EGFP alone or EGFP-WT (a full-length RHAU).
Half were treated 24 h later with arsenite (0.5 mm for 45 min)
followed by UV irradiation and radiolabeling of RHAU-associated nucleic acids.
RHAU and associated RNA were analyzed by Western blotting and a
phosphorimaging system to detect levels of protein expression and the amount
of radiolabeled RNA, respectively. Note that the arsenite treatment (+) did
not abolish or dramatically change the RNA binding activity of RHAU.
ARS, arsenite. E, comparison of RNA binding activities of
wild-type RHAU and its N-terminal deletion mutants. HeLa cells were
transfected with EGFP, EGFP-WT, EGFP-(50-1008), or EGFP-(105-1008). Protein
and associated RNA were analyzed as in A. Radioactivity of bound RNA
was normalized to the expression levels of corresponding proteins. In sharp
contrast to constant RNA binding of endogenous RHAU in each lane, N-terminal
deletion mutants showed stepwise reduction of RNA interaction compared with
WT. ▸, overexpressed EGFP-RHAU or its N-terminal deletion mutants;
▸▸, endogenous RHAU.