Auto-regulation of the circadian slave oscillator component AtGRP7 and regulation of its targets is impaired by a single RNA recognition motif point mutation.
The clock-regulated RNA-binding protein AtGRP7 (Arabidopsis thaliana glycine-rich RNA-binding protein) influences circadian oscillations of its transcript by negative feedback at the post-transcriptional level. Here we show that site-specific mutation of one conserved arginine to glutamine within the RNA recognition motif impairs binding of recombinant AtGRP7 to its pre-mRNA in vitro. This correlates with the loss of the negative auto-regulation in vivo: in transgenic plants constitutively overexpressing AtGRP7 (AtGRP7-ox), a shift occurs to an alternatively spliced AtGRP7 transcript that decays rapidly, and thus does not accumulate to high levels. In contrast, constitutive ectopic overexpression of the AtGRP7-RQ mutant does not lead to alternative splicing of the endogenous AtGRP7 transcript and concomitant damping of the oscillations. This highlights the importance of AtGRP7 binding to its own transcript for the negative auto-regulatory circuit. Moreover, regulation of AtGRP7 downstream targets also depends on its RNA-binding activity, as AtGRP8 and other targets identified by transcript profiling of wild-type and AtGRP7-ox plants using fluorescent differential display are negatively affected by AtGRP7 but not by AtGRP7-RQ. In mutants impaired in the nonsense-mediated decay (NMD) components UPF1 or UPF3, levels of the alternatively spliced AtGRP7 and AtGRP8 transcripts that contain premature termination codons are strongly elevated, implicating UPF1 and UPF3 in the decay of these clock-regulated transcripts.
Schöning
Jan C
JC
Department of Molecular Cell Physiology, University of Bielefeld, D-33615 Bielefeld, Germany.
Streitner
Corinna
C
Page
Damian R
DR
Hennig
Sven
S
Uchida
Kenko
K
Wolf
Eva
E
Furuya
Masaki
M
Staiger
Dorothee
D
D017398:Alternative Splicing; D020816:Amino Acid Motifs / Q000235:genetics; D017360:Arabidopsis / Q000235:genetics* / Q000378:metabolism; D029681:Arabidopsis Proteins / Q000235:genetics* / Q000378:metabolism; D015152:Blotting, Northern; D015153:Blotting, Western; D002940:Circadian Rhythm / Q000235:genetics; D002942:Circular Dichroism; D018506:Gene Expression Regulation, Plant; D016297:Mutagenesis, Site-Directed; D030821:Plants, Genetically Modified; D017354:Point Mutation; D011485:Protein Binding; D020365:RNA Helicases / Q000235:genetics / Q000378:metabolism; D020871:RNA Stability; D018749:RNA, Plant / Q000235:genetics / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism
D029681:Arabidopsis Proteins; D020365:RNA Helicases; D018749:RNA, Plant; D016601:RNA-Binding Proteins; D011994:Recombinant Proteins; C118496:GRP7 protein, Arabidopsis; C504799:UPF3 protein, Arabidopsis; C513125:UPF1 protein, Arabidopsis