Molecular characterization of Arabidopsis thaliana PUF proteins--binding specificity and target candidates.
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 Arabidopsis 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 regulation in plants.
Francischini
Carlos W
CW
Departamento de Bioquímica, Instituto de Química, Universidade de São Paulo, Brazil. rquaggio@iq.usp.br
Quaggio
Ronaldo B
RB
D020413:3' Untranslated Regions; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D017360:Arabidopsis / Q000235:genetics / Q000378:metabolism; D029681:Arabidopsis Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010802:Phylogeny; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D018749:RNA, Plant / Q000235:genetics / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D020798:Two-Hybrid System Techniques
D020413:3' Untranslated Regions; D029681:Arabidopsis Proteins; D012333:RNA, Messenger; D018749:RNA, Plant; D016601:RNA-Binding Proteins; D011994:Recombinant Proteins