Auto-regulation of the circadian slave oscillator component AtGRP7 and regulation of its targets is impaired by a single RNA recognition motif point mutation

Jan C Schöning, Corinna Streitner, Damian R Page, Sven Hennig, Kenko Uchida, Eva Wolf, Masaki Furuya, Dorothee Staiger

Research output: Contribution to JournalArticleAcademicpeer-review

Abstract

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.

Original languageEnglish
Pages (from-to)1119-30
Number of pages12
JournalPlant Journal
Volume52
Issue number6
DOIs
Publication statusPublished - Dec 2007

Fingerprint

autoregulation
RNA-Binding Proteins
alternative splicing
point mutation
Point Mutation
RNA-binding proteins
oxen
deterioration
RNA
oscillation
Nonsense Codon
Genetically Modified Plants
RNA Precursors
Alternative Splicing
Glutamine
Arabidopsis
Glycine
Arginine
mutants
stop codon

Keywords

  • Alternative Splicing
  • Amino Acid Motifs
  • Arabidopsis
  • Arabidopsis Proteins
  • Blotting, Northern
  • Blotting, Western
  • Circadian Rhythm
  • Circular Dichroism
  • Gene Expression Regulation, Plant
  • Mutagenesis, Site-Directed
  • Plants, Genetically Modified
  • Point Mutation
  • Protein Binding
  • RNA Helicases
  • RNA Stability
  • RNA, Plant
  • RNA-Binding Proteins
  • Recombinant Proteins
  • Journal Article
  • Research Support, Non-U.S. Gov't

Cite this

Schöning, Jan C ; Streitner, Corinna ; Page, Damian R ; Hennig, Sven ; Uchida, Kenko ; Wolf, Eva ; Furuya, Masaki ; Staiger, Dorothee. / Auto-regulation of the circadian slave oscillator component AtGRP7 and regulation of its targets is impaired by a single RNA recognition motif point mutation. In: Plant Journal. 2007 ; Vol. 52, No. 6. pp. 1119-30.
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abstract = "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.",
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author = "Sch{\"o}ning, {Jan C} and Corinna Streitner and Page, {Damian R} and Sven Hennig and Kenko Uchida and Eva Wolf and Masaki Furuya and Dorothee Staiger",
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Auto-regulation of the circadian slave oscillator component AtGRP7 and regulation of its targets is impaired by a single RNA recognition motif point mutation. / Schöning, Jan C; Streitner, Corinna; Page, Damian R; Hennig, Sven; Uchida, Kenko; Wolf, Eva; Furuya, Masaki; Staiger, Dorothee.

In: Plant Journal, Vol. 52, No. 6, 12.2007, p. 1119-30.

Research output: Contribution to JournalArticleAcademicpeer-review

TY - JOUR

T1 - Auto-regulation of the circadian slave oscillator component AtGRP7 and regulation of its targets is impaired by a single RNA recognition motif point mutation

AU - Schöning, Jan C

AU - Streitner, Corinna

AU - Page, Damian R

AU - Hennig, Sven

AU - Uchida, Kenko

AU - Wolf, Eva

AU - Furuya, Masaki

AU - Staiger, Dorothee

PY - 2007/12

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N2 - 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.

AB - 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.

KW - Alternative Splicing

KW - Amino Acid Motifs

KW - Arabidopsis

KW - Arabidopsis Proteins

KW - Blotting, Northern

KW - Blotting, Western

KW - Circadian Rhythm

KW - Circular Dichroism

KW - Gene Expression Regulation, Plant

KW - Mutagenesis, Site-Directed

KW - Plants, Genetically Modified

KW - Point Mutation

KW - Protein Binding

KW - RNA Helicases

KW - RNA Stability

KW - RNA, Plant

KW - RNA-Binding Proteins

KW - Recombinant Proteins

KW - Journal Article

KW - Research Support, Non-U.S. Gov't

U2 - 10.1111/j.1365-313X.2007.03302.x

DO - 10.1111/j.1365-313X.2007.03302.x

M3 - Article

VL - 52

SP - 1119

EP - 1130

JO - Plant Journal

JF - Plant Journal

SN - 0960-7412

IS - 6

ER -