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Auto‐regulation of the circadian slave oscillator component At GRP7 and regulation of its targets is impaired by a single RNA recognition motif point mutation
Author(s) -
Schöning Jan C.,
Streitner Corinna,
Page Damian R.,
Hennig Sven,
Uchida Kenko,
Wolf Eva,
Furuya Masaki,
Staiger Dorothee
Publication year - 2007
Publication title -
the plant journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.058
H-Index - 269
eISSN - 1365-313X
pISSN - 0960-7412
DOI - 10.1111/j.1365-313x.2007.03302.x
Subject(s) - rna recognition motif , biology , rna binding protein , rna , rna splicing , nonsense mediated decay , mutant , microbiology and biotechnology , intron , ectopic expression , genetics , gene
Summary The clock‐regulated RNA‐binding protein At GRP7 ( 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 At GRP7 to its pre‐mRNA in vitro . This correlates with the loss of the negative auto‐regulation in vivo : in transgenic plants constitutively overexpressing At GRP7 ( At GRP7‐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 At GRP7‐RQ mutant does not lead to alternative splicing of the endogenous AtGRP7 transcript and concomitant damping of the oscillations. This highlights the importance of At GRP7 binding to its own transcript for the negative auto‐regulatory circuit. Moreover, regulation of At GRP7 downstream targets also depends on its RNA‐binding activity, as AtGRP8 and other targets identified by transcript profiling of wild‐type and At GRP7‐ox plants using fluorescent differential display are negatively affected by At GRP7 but not by At GRP7‐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.