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Reciprocal regulation of glycine-rich RNA-binding proteins via an interlocked feedback loop coupling alternative splicing to nonsense-mediated decay in Arabidopsis.

Schöning JC, Streitner C, Meyer IM, Gao Y, Staiger D - Nucleic Acids Res. (2008)

Bottom Line: In addition to its own pre-mRNA, AtGRP8 negatively regulates the AtGRP7 transcript through promoting the formation of the equivalent alternatively spliced as_AtGRP7 transcript, leading to a decrease in AtGRP7 abundance.AtGRP7 itself is part of a negative autoregulatory circuit that influences circadian oscillations of its own and the AtGRP8 transcript through alternative splicing linked to NMD.A high degree of evolutionary sequence conservation in the introns retained in as_AtGRP8 or as_AtGRP7 points to an important function of these sequences.

View Article: PubMed Central - PubMed

Affiliation: Molecular Cell Physiology, Bielefeld University, Bielefeld, Germany.

ABSTRACT
The Arabidopsis RNA-binding protein AtGRP8 undergoes negative autoregulation at the post-transcriptional level. An elevated AtGRP8 protein level promotes the use of a cryptic 5' splice site to generate an alternatively spliced transcript, as_AtGRP8, retaining the 5' half of the intron with a premature termination codon. In mutants defective in nonsense-mediated decay (NMD) abundance of as_AtGRP8 but not its pre-mRNA is elevated, indicating that as_AtGRP8 is a direct NMD target, thus limiting the production of functional AtGRP8 protein. In addition to its own pre-mRNA, AtGRP8 negatively regulates the AtGRP7 transcript through promoting the formation of the equivalent alternatively spliced as_AtGRP7 transcript, leading to a decrease in AtGRP7 abundance. Recombinant AtGRP8 binds to its own and the AtGRP7 pre-mRNA, suggesting that this interaction is relevant for the splicing decision in vivo. AtGRP7 itself is part of a negative autoregulatory circuit that influences circadian oscillations of its own and the AtGRP8 transcript through alternative splicing linked to NMD. Thus, we identify an interlocked feedback loop through which two RNA-binding proteins autoregulate and reciprocally crossregulate by coupling unproductive splicing to NMD. A high degree of evolutionary sequence conservation in the introns retained in as_AtGRP8 or as_AtGRP7 points to an important function of these sequences.

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Influence of ectopic AtGRP8 overexpression on the endogenous AtGRP7 and AtGRP8. (A) The immunoblot with total protein of WT plants, the AtGRP8-ox lines 5 and 21 and an AtGRP7-ox line, harvested at zt11, was probed with the AtGRP8 antibody (top) and an antibody against LHCP (light harvesting chlorophyll-binding protein) as loading control (bottom). The absence of crossreaction with overexpressed AtGRP7 protein in AtGRP7-ox plants demonstrates the specificity of the antibody. (B) A duplicate blot was probed with the AtGRP7 antibody (top) and the LHCP antibody (bottom). (C) WT and AtGRP8-ox plants were harvested at zt3 and zt11. The RNA gel blot was hybridized with the AtGRP8 cDNA to determine the total AtGRP8 transcript level. The stripped blot was rehybridized with the gene-specific probe to monitor the endogenous AtGRP8 transcript and subsequently with the gene-specific AtGRP7 probe. The position of the pre-mRNA, as_AtGRP8 and as_AtGRP7 retaining the first half of the intron and the mature mRNA are indicated. Boxes represent exons, lines represent the first and second half of the intron, respectively. The ethidium-bromide stained gel shows equal loading.
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Figure 1: Influence of ectopic AtGRP8 overexpression on the endogenous AtGRP7 and AtGRP8. (A) The immunoblot with total protein of WT plants, the AtGRP8-ox lines 5 and 21 and an AtGRP7-ox line, harvested at zt11, was probed with the AtGRP8 antibody (top) and an antibody against LHCP (light harvesting chlorophyll-binding protein) as loading control (bottom). The absence of crossreaction with overexpressed AtGRP7 protein in AtGRP7-ox plants demonstrates the specificity of the antibody. (B) A duplicate blot was probed with the AtGRP7 antibody (top) and the LHCP antibody (bottom). (C) WT and AtGRP8-ox plants were harvested at zt3 and zt11. The RNA gel blot was hybridized with the AtGRP8 cDNA to determine the total AtGRP8 transcript level. The stripped blot was rehybridized with the gene-specific probe to monitor the endogenous AtGRP8 transcript and subsequently with the gene-specific AtGRP7 probe. The position of the pre-mRNA, as_AtGRP8 and as_AtGRP7 retaining the first half of the intron and the mature mRNA are indicated. Boxes represent exons, lines represent the first and second half of the intron, respectively. The ethidium-bromide stained gel shows equal loading.

Mentions: To begin to investigate the molecular properties of the predicted RNA-binding protein AtGRP8, we generated transgenic plants ectopically overexpressing the AtGRP8 coding region under control of the CaMV promoter with the duplicated enhancer (AtGRP8-ox plants). Immunoblot analysis using a specific AtGRP8 antibody identified transgenic lines with strongly elevated AtGRP8 protein levels (Figure 1A). Compared to WT plants, the total AtGRP8 transcript level was strongly elevated in AtGRP8-ox plants harvested at zt3 (zeitgeber time 3, that is 3 h after lights on), the circadian minimum of AtGRP8 oscillations, and zt11, the circadian maximum, due to the expression of the transgene (Figure 1C). The endogenous AtGRP8 transcript forms are selectively detected with a gene-specific probe derived from the 5′ UTR that is not contained in the overexpression construct (Figure 1C). In WT plants, the endogenous AtGRP8 mRNA and a small amount of its pre-mRNA containing the 283-nt intron were present. In the AtGRP8-ox plants, almost no mature endogenous AtGRP8 mRNA was detectable. Instead, an intermediate size transcript appears at a low level, corresponding to the alternatively spliced AtGRP8 transcript (as_AtGRP8) that is generated through the use of a cryptic 5′ splice site within the intron. These data indicate that AtGRP8 exerts negative autoregulation on its own pre-mRNA.Figure 1.


Reciprocal regulation of glycine-rich RNA-binding proteins via an interlocked feedback loop coupling alternative splicing to nonsense-mediated decay in Arabidopsis.

Schöning JC, Streitner C, Meyer IM, Gao Y, Staiger D - Nucleic Acids Res. (2008)

Influence of ectopic AtGRP8 overexpression on the endogenous AtGRP7 and AtGRP8. (A) The immunoblot with total protein of WT plants, the AtGRP8-ox lines 5 and 21 and an AtGRP7-ox line, harvested at zt11, was probed with the AtGRP8 antibody (top) and an antibody against LHCP (light harvesting chlorophyll-binding protein) as loading control (bottom). The absence of crossreaction with overexpressed AtGRP7 protein in AtGRP7-ox plants demonstrates the specificity of the antibody. (B) A duplicate blot was probed with the AtGRP7 antibody (top) and the LHCP antibody (bottom). (C) WT and AtGRP8-ox plants were harvested at zt3 and zt11. The RNA gel blot was hybridized with the AtGRP8 cDNA to determine the total AtGRP8 transcript level. The stripped blot was rehybridized with the gene-specific probe to monitor the endogenous AtGRP8 transcript and subsequently with the gene-specific AtGRP7 probe. The position of the pre-mRNA, as_AtGRP8 and as_AtGRP7 retaining the first half of the intron and the mature mRNA are indicated. Boxes represent exons, lines represent the first and second half of the intron, respectively. The ethidium-bromide stained gel shows equal loading.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Show All Figures
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Figure 1: Influence of ectopic AtGRP8 overexpression on the endogenous AtGRP7 and AtGRP8. (A) The immunoblot with total protein of WT plants, the AtGRP8-ox lines 5 and 21 and an AtGRP7-ox line, harvested at zt11, was probed with the AtGRP8 antibody (top) and an antibody against LHCP (light harvesting chlorophyll-binding protein) as loading control (bottom). The absence of crossreaction with overexpressed AtGRP7 protein in AtGRP7-ox plants demonstrates the specificity of the antibody. (B) A duplicate blot was probed with the AtGRP7 antibody (top) and the LHCP antibody (bottom). (C) WT and AtGRP8-ox plants were harvested at zt3 and zt11. The RNA gel blot was hybridized with the AtGRP8 cDNA to determine the total AtGRP8 transcript level. The stripped blot was rehybridized with the gene-specific probe to monitor the endogenous AtGRP8 transcript and subsequently with the gene-specific AtGRP7 probe. The position of the pre-mRNA, as_AtGRP8 and as_AtGRP7 retaining the first half of the intron and the mature mRNA are indicated. Boxes represent exons, lines represent the first and second half of the intron, respectively. The ethidium-bromide stained gel shows equal loading.
Mentions: To begin to investigate the molecular properties of the predicted RNA-binding protein AtGRP8, we generated transgenic plants ectopically overexpressing the AtGRP8 coding region under control of the CaMV promoter with the duplicated enhancer (AtGRP8-ox plants). Immunoblot analysis using a specific AtGRP8 antibody identified transgenic lines with strongly elevated AtGRP8 protein levels (Figure 1A). Compared to WT plants, the total AtGRP8 transcript level was strongly elevated in AtGRP8-ox plants harvested at zt3 (zeitgeber time 3, that is 3 h after lights on), the circadian minimum of AtGRP8 oscillations, and zt11, the circadian maximum, due to the expression of the transgene (Figure 1C). The endogenous AtGRP8 transcript forms are selectively detected with a gene-specific probe derived from the 5′ UTR that is not contained in the overexpression construct (Figure 1C). In WT plants, the endogenous AtGRP8 mRNA and a small amount of its pre-mRNA containing the 283-nt intron were present. In the AtGRP8-ox plants, almost no mature endogenous AtGRP8 mRNA was detectable. Instead, an intermediate size transcript appears at a low level, corresponding to the alternatively spliced AtGRP8 transcript (as_AtGRP8) that is generated through the use of a cryptic 5′ splice site within the intron. These data indicate that AtGRP8 exerts negative autoregulation on its own pre-mRNA.Figure 1.

Bottom Line: In addition to its own pre-mRNA, AtGRP8 negatively regulates the AtGRP7 transcript through promoting the formation of the equivalent alternatively spliced as_AtGRP7 transcript, leading to a decrease in AtGRP7 abundance.AtGRP7 itself is part of a negative autoregulatory circuit that influences circadian oscillations of its own and the AtGRP8 transcript through alternative splicing linked to NMD.A high degree of evolutionary sequence conservation in the introns retained in as_AtGRP8 or as_AtGRP7 points to an important function of these sequences.

View Article: PubMed Central - PubMed

Affiliation: Molecular Cell Physiology, Bielefeld University, Bielefeld, Germany.

ABSTRACT
The Arabidopsis RNA-binding protein AtGRP8 undergoes negative autoregulation at the post-transcriptional level. An elevated AtGRP8 protein level promotes the use of a cryptic 5' splice site to generate an alternatively spliced transcript, as_AtGRP8, retaining the 5' half of the intron with a premature termination codon. In mutants defective in nonsense-mediated decay (NMD) abundance of as_AtGRP8 but not its pre-mRNA is elevated, indicating that as_AtGRP8 is a direct NMD target, thus limiting the production of functional AtGRP8 protein. In addition to its own pre-mRNA, AtGRP8 negatively regulates the AtGRP7 transcript through promoting the formation of the equivalent alternatively spliced as_AtGRP7 transcript, leading to a decrease in AtGRP7 abundance. Recombinant AtGRP8 binds to its own and the AtGRP7 pre-mRNA, suggesting that this interaction is relevant for the splicing decision in vivo. AtGRP7 itself is part of a negative autoregulatory circuit that influences circadian oscillations of its own and the AtGRP8 transcript through alternative splicing linked to NMD. Thus, we identify an interlocked feedback loop through which two RNA-binding proteins autoregulate and reciprocally crossregulate by coupling unproductive splicing to NMD. A high degree of evolutionary sequence conservation in the introns retained in as_AtGRP8 or as_AtGRP7 points to an important function of these sequences.

Show MeSH
Related in: MedlinePlus