tailieunhanh - Báo cáo khoa học: Molecular characterization of Arabidopsis thaliana PUF proteins – binding specificity and target candidates

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. | Molecular characterization of Arabidopsis thaliana PUF proteins - binding specificity and target candidates Carlos W. Francischini and Ronaldo B. Quaggio Departamento de Bioquimica Institute de Quimica Universidade de Sao Paulo Brazil Keywords Arabidopsis PUF proteins RNA-binding protein three-hybrid system translational control Correspondence R. B. Quaggio Instituto de Quimica Departamento de Bioquimica Universidade de Scio Paulo Avenida Professor Lineu Prestes 748 Sao Paulo 05508-000 Brazil Fax Tel 55 11 3091 2171 E-mail rquaggio@ Received 24 March 2009 revised 15 July 2009 accepted 22 July 2009 doi PUF proteins regulate both stability and translation through sequence-specific binding to the 3 UTR of target mRNA transcripts. Binding is mediated 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 transcripts 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 .