Tài liệu Báo cáo khoa học: Molecular characterization of Arabidopsis thaliana PUF proteins – binding

Molecular characterization of Arabidopsis thaliana PUF

proteins – binding specificity and target candidates

Carlos W. Francischini and Ronaldo B. Quaggio

Departamento de Bioquı´mica, Instituto de Quı´mica, Universidade de Sa˜o Paulo, Brazil

Introduction

The translational control of RNA is an important reg￾ulatory process in animal development. This regulation

is accomplished by sequence-specific RNA-binding

proteins that recognize cis-acting elements usually

located in the 3¢ UTR. In recent years, and as a result

of great efforts aiming to understand the mechanism

of RNA control in animals, the function of a diverse

number of RNA-binding proteins has been elucidated

[1–4]. Despite this, translational control through the

binding of RNA-binding proteins to 3¢ UTR tran￾scripts has been poorly described in plants.

PUF proteins are a large family of RNA-binding

proteins found in all eukaryotes. These proteins reduce

the expression of mRNA targets by binding in 3¢ UTR

regulatory elements, thus controlling translation or

mRNA stability [5]. Members of the PUF family have

been implicated in diverse processes in development. In

Drosophila, Pumilio binds to the Nanos response ele￾ment (NRE) sequence within the 3¢ UTR of maternal

hunchback mRNA and reduces its expression in the

posterior pole of the embryo. This control is essential

for abdomen formation [6]. In Caenorhabditis elegans

hermaphrodites, the Pumilio homolog FBF binds to

the 3¢ UTR of fem-3 mRNA, repressing its translation

and controlling the sperm–oocyte switch [7]. Dictyoste￾lium PufA represses pkaC mRNA and inhibits the

Keywords

Arabidopsis; PUF proteins; RNA-binding

protein; three-hybrid system; translational

control

Correspondence

R. B. Quaggio, Instituto de Quı´mica,

Departamento de Bioquı´mica, Universidade

de Sa˜o Paulo, Avenida Professor Lineu

Prestes, 748, Sa˜o Paulo 05508-000, Brazil

Fax/Tel: +55 11 3091 2171

E-mail: [email protected]

(Received 24 March 2009, revised 15 July

2009, accepted 22 July 2009)

doi:10.1111/j.1742-4658.2009.07230.x

PUF proteins regulate both stability and translation through sequence-spe￾cific binding to the 3¢ UTR of target mRNA transcripts. Binding is medi￾ated by a conserved PUF domain, which contains eight repeats of

approximately 36 amino acids each. Found in all eukaryotes, they have

been related to several developmental processes. Analysis of the 25 Arabid￾opsis Pumilio (APUM) proteins presenting PUF repeats reveals that 12

(APUM-1 to APUM-12) have a PUF domain with 50–75% similarity to

the Drosophila PUF domain. Through three-hybrid assays, we show that

APUM-1 to APUM-6 can bind specifically to the Nanos response element

sequence recognized by Drosophila Pumilio. Using an Arabidopsis RNA

library in a three-hybrid screening, we were able to identify an APUM￾binding consensus sequence. Computational analysis allowed us to identify

the APUM-binding element within the 3¢ UTR in many Arabidopsis tran￾scripts, even in important mRNAs related to shoot stem cell maintenance.

We demonstrate that APUM-1 to APUM-6 are able to bind specifically to

APUM-binding elements in the 3¢ UTR of WUSCHEL, CLAVATA-1,

PINHEAD ⁄ ZWILLE and FASCIATA-2 transcripts. The results obtained

in the present study indicate that the APUM proteins may act as regulators

in Arabidopsis through an evolutionarily conserved mechanism, which may

open up a new approach for investigating mRNA regulation in plants.

Abbreviations

APBE, APUM-binding element; APUM, Arabidopsis Pumilio; IRP, iron regulatory protein; NRE, Nanos response element.

5456 FEBS Journal 276 (2009) 5456–5470 ª 2009 The Authors Journal compilation ª 2009 FEBS