Tài liệu Báo cáo khoa học: Functional interaction between RNA helicase II⁄Gua and ribosomal protein

Functional interaction between RNA helicase II⁄Gua and

ribosomal protein L4

Hushan Yang, Dale Henning and Benigno C. Valdez

Department of Pharmacology, Baylor College of Medicine, Houston, Texas, USA

Ribosome biogenesis is a complicated cellular process

which occurs in the nucleolus [1]. The entire scenario

begins at the end of mitosis and includes ribosomal

DNA transcription, pre-ribosomal RNA (pre-rRNA)

modifications and processing as well as assembly of

rRNAs and ribosomal proteins into preribosome sub￾units which are then exported to the cytoplasm to

form the mature ribosomes [2]. Errors in this process

are reported to be associated with several diseases

[3–8]. The availability of genetic manipulations makes

ribosome biogenesis much better studied in yeast than

in higher eukaryotes such as mammalian and frog sys￾tems, resulting in the identification of more than 80

yeast ribosomal proteins and numerous trans-acting

elements including small nucleolar RNAs (snoRNAs)

as well as nonribosomal proteins. However, in mam￾malian systems, ribosome biogenesis is far from being

thoroughly understood due to the increased complex￾ity. To date, only a few nucleolus-localized nonribo￾somal proteins have been implicated in pre-rRNA

processing in mammalian cells and include B23 ⁄

NO38 ⁄ NPM [9], C23 ⁄ nucleolin [3], fibrillarin [10,11],

p120 [12], EBP1 [13], Bop1 [14] and p19Arf [15]. No

bona fide RNA helicase has been implicated in this

process in higher eukaryotes except RNA helicase

II⁄ Gua [16,17].

RNA helicase II⁄ Gua is a multifunctional nucleolar

protein with in vitro RNA-dependent ATPase activity,

ATP-dependent RNA helicase activity and GTP-stimu￾lated RNA foldase activity [17–19]. The presence of

both RNA unwinding and RNA folding activities in

two distinct domains of the same protein highly sug￾gests a role of Gua in rRNA biogenesis [19]. Using

antisense oligodeoxynucleotide and siRNA to down￾regulate Gua expression in Xenopus oocytes [16] and

mammalian cells [17], respectively, we demonstrated

that Gua is important for 18S and 28S rRNA produc￾tion in both systems. In addition, Gua was also showed

to participate in other major cellular activities such as

cell growth and differentiation [19,20], regulation of

Keywords

ribosomal protein; ribosomal RNA

biogenesis; RNA helicase; nucleolus

Correspondence

B. C. Valdez, Department of Pharmacology,

Baylor College of Medicine, Houston,

TX 77030, USA

Fax: +1 713 798 3145

Tel: +1 713 798 7908

E-mail: [email protected]

(Received 11 April 05, revised 19 May 05,

accepted 9 June 05)

doi:10.1111/j.1742-4658.2005.04811.x

RNA helicase II⁄ Gua is a multifunctional nucleolar protein involved in

ribosomal RNA processing in Xenopus laevis oocytes and mammalian cells.

Downregulation of Gua using small interfering RNA (siRNA) in HeLa

cells resulted in 80% inhibition of both 18S and 28S rRNA production.

The mechanisms underlying this effect remain unclear. Here we show that

in mammalian cells, Gua physically interacts with ribosomal protein L4

(RPL4), a component of 60S ribosome large subunit. The ATPase activity

of Gua is important for this interaction and is also necessary for the func￾tion of Gua in the production of both 18S and 28S rRNAs. Knocking

down RPL4 expression using siRNA in mouse LAP3 cells inhibits the pro￾duction of 47 ⁄ 45S, 32S, 28S, and 18S rRNAs. This inhibition is reversed

by exogenous expression of wild-type human RPL4 protein but not the

mutant form lacking Gua-interacting motif. These observations have sug￾gested that the function of Gua in rRNA processing is at least partially

dependent on its ability to interact with RPL4.

Abbreviations

aa, amino acid; GST, glutathione S-transferase; Gua, RNA helicase II ⁄ Gua; HA, hemagglutinin; IPTG, isopropylthio-b-D-galactoside; NLS,

nuclear localization signal; NoLS, nucleolar localization signal; RPL4, Ribosomal Protein L4; rDNA, ribosomal DNA; rRNA, ribosomal RNA;

RNP, ribonucleoprotein; siRNA, small interfering RNA; snRNA, small nuclear RNA; snoRNA, small nucleolar RNA.

3788 FEBS Journal 272 (2005) 3788–3802 ª 2005 FEBS