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Characterization of the Arabidopsis thaliana E3 ubiquitin-ligase AtSINAL7 and identification of the ubiquitination sites.

Peralta DA, Araya A, Nardi CF, Busi MV, Gomez-Casati DF - PLoS ONE (2013)

Bottom Line: We also found that higher AtSINAL7 transcript levels are present in tissues undergoing active cell division during floral development.An interesting observation is the circadian expression pattern of AtSINAL7 mRNA in floral buds.Furthermore, UV-B irradiation induces the expression of this transcript indicating that AtSINAL7 may be involved in a wide range of different cell processes.

View Article: PubMed Central - PubMed

Affiliation: Centro de Estudios Fotosintéticos y Bioquímicos (CEFOBI-CONICET), Universidad Nacional de Rosario, Rosario, Argentina.

ABSTRACT
Protein ubiquitination leading to degradation by the proteasome is an important mechanism in regulating key cellular functions. Protein ubiquitination is carried out by a three step process involving ubiquitin (Ub) activation by a E1 enzyme, the transfer of Ub to a protein E2, finally an ubiquitin ligase E3 catalyzes the transfer of the Ub peptide to an acceptor protein. The E3 component is responsible for the specific recognition of the target, making the unveiling of E3 components essential to understand the mechanisms regulating fundamental cell processes through the protein degradation pathways. The Arabidopsis thaliana seven in absentia-like 7 (AtSINAL7) gene encodes for a protein with characteristics from a C3HC4-type E3 ubiquitin ligase. We demonstrate here that AtSINAL7 protein is indeed an E3 protein ligase based on the self-ubiquitination in vitro assay. This activity is dependent of the presence of a Lys residue in position 124. We also found that higher AtSINAL7 transcript levels are present in tissues undergoing active cell division during floral development. An interesting observation is the circadian expression pattern of AtSINAL7 mRNA in floral buds. Furthermore, UV-B irradiation induces the expression of this transcript indicating that AtSINAL7 may be involved in a wide range of different cell processes.

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Analysis of AtSINAL7 mRNA levels (A) AtSINAL7 transcript expression profile in different organs from A. thaliana.AtSINAL7 transcript levels relative to CBP control gene expression were determined by RT-qPCR in several A. thaliana organs. Data shown represent at least three independent experiments (significant statistical difference was determined using t test, P <0.05). (B) Time-course expression of AtSINAL7 transcripts. Determination of the transcription levels of AtSINAL7 at 0, 4, 8, 12, 16, 20, and 24 hours under long day growth conditions. Transcripts levels are plotted as relative values using the cap binding protein (At5g44200) mRNA as an internal control. Data shown represent at least three independent experiments (significant statistical difference was determined using t test, P <0.05). (C) Effect of the UV–B treatment on the AtSINAL7 transcript levels in wild type A. thaliana inflorescences. The AtSINAL7 transcript expression in A. thaliana inflorescences (stage 12) relative to cpk3 (calcium-dependent protein kinase 3, At4g23650) was determined after 4 h of UV–B exposure. Control plants were protected from UV–B irradiation using polyester filters (see Methods). Data shown represent at least three independent experiments (significant statistical differences determined using t test, P <0.05).
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pone-0073104-g002: Analysis of AtSINAL7 mRNA levels (A) AtSINAL7 transcript expression profile in different organs from A. thaliana.AtSINAL7 transcript levels relative to CBP control gene expression were determined by RT-qPCR in several A. thaliana organs. Data shown represent at least three independent experiments (significant statistical difference was determined using t test, P <0.05). (B) Time-course expression of AtSINAL7 transcripts. Determination of the transcription levels of AtSINAL7 at 0, 4, 8, 12, 16, 20, and 24 hours under long day growth conditions. Transcripts levels are plotted as relative values using the cap binding protein (At5g44200) mRNA as an internal control. Data shown represent at least three independent experiments (significant statistical difference was determined using t test, P <0.05). (C) Effect of the UV–B treatment on the AtSINAL7 transcript levels in wild type A. thaliana inflorescences. The AtSINAL7 transcript expression in A. thaliana inflorescences (stage 12) relative to cpk3 (calcium-dependent protein kinase 3, At4g23650) was determined after 4 h of UV–B exposure. Control plants were protected from UV–B irradiation using polyester filters (see Methods). Data shown represent at least three independent experiments (significant statistical differences determined using t test, P <0.05).

Mentions: The expression of AtSINAL7 was determined in several tissues from wild-type Arabidopsis by qRT-PCR (Figure 2A). RNA from Root, Rosette leaves, Inflorescence stage 6, Inflorescence stage 12, and Silique were isolated from 28 days old A. thaliana Col 0 plants grown under long-day condition greenhouse (see Methods). cDNA was synthesized from total RNA and was quantified spectrophotometrically. Identical amounts of different cDNA samples were used for qPCR amplifications using Arabidopsis sina like 7 with specific primers (Table 1). The data presented concern relative values obtained from the average of three biological and technical replicates using the cbp gene (cap binding protein At5g44200) as a house-keeping control [24] (Figure 2A). AtSINAL7 transcript levels are highly expressed in siliques (2.3770) and stage 12 inflorescences (0.7053), and roots (0.1524), being lower at Stage 6 inflorescences (0.0807) and rosette leaves (0.0600). Thus, AtSINAL7 seems to be expressed in a tissue-specific manner with a strong induction of 12 and 40 times in stage 12 flowers and silique respectively, compared to rosette leaves.


Characterization of the Arabidopsis thaliana E3 ubiquitin-ligase AtSINAL7 and identification of the ubiquitination sites.

Peralta DA, Araya A, Nardi CF, Busi MV, Gomez-Casati DF - PLoS ONE (2013)

Analysis of AtSINAL7 mRNA levels (A) AtSINAL7 transcript expression profile in different organs from A. thaliana.AtSINAL7 transcript levels relative to CBP control gene expression were determined by RT-qPCR in several A. thaliana organs. Data shown represent at least three independent experiments (significant statistical difference was determined using t test, P <0.05). (B) Time-course expression of AtSINAL7 transcripts. Determination of the transcription levels of AtSINAL7 at 0, 4, 8, 12, 16, 20, and 24 hours under long day growth conditions. Transcripts levels are plotted as relative values using the cap binding protein (At5g44200) mRNA as an internal control. Data shown represent at least three independent experiments (significant statistical difference was determined using t test, P <0.05). (C) Effect of the UV–B treatment on the AtSINAL7 transcript levels in wild type A. thaliana inflorescences. The AtSINAL7 transcript expression in A. thaliana inflorescences (stage 12) relative to cpk3 (calcium-dependent protein kinase 3, At4g23650) was determined after 4 h of UV–B exposure. Control plants were protected from UV–B irradiation using polyester filters (see Methods). Data shown represent at least three independent experiments (significant statistical differences determined using t test, P <0.05).
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Related In: Results  -  Collection

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pone-0073104-g002: Analysis of AtSINAL7 mRNA levels (A) AtSINAL7 transcript expression profile in different organs from A. thaliana.AtSINAL7 transcript levels relative to CBP control gene expression were determined by RT-qPCR in several A. thaliana organs. Data shown represent at least three independent experiments (significant statistical difference was determined using t test, P <0.05). (B) Time-course expression of AtSINAL7 transcripts. Determination of the transcription levels of AtSINAL7 at 0, 4, 8, 12, 16, 20, and 24 hours under long day growth conditions. Transcripts levels are plotted as relative values using the cap binding protein (At5g44200) mRNA as an internal control. Data shown represent at least three independent experiments (significant statistical difference was determined using t test, P <0.05). (C) Effect of the UV–B treatment on the AtSINAL7 transcript levels in wild type A. thaliana inflorescences. The AtSINAL7 transcript expression in A. thaliana inflorescences (stage 12) relative to cpk3 (calcium-dependent protein kinase 3, At4g23650) was determined after 4 h of UV–B exposure. Control plants were protected from UV–B irradiation using polyester filters (see Methods). Data shown represent at least three independent experiments (significant statistical differences determined using t test, P <0.05).
Mentions: The expression of AtSINAL7 was determined in several tissues from wild-type Arabidopsis by qRT-PCR (Figure 2A). RNA from Root, Rosette leaves, Inflorescence stage 6, Inflorescence stage 12, and Silique were isolated from 28 days old A. thaliana Col 0 plants grown under long-day condition greenhouse (see Methods). cDNA was synthesized from total RNA and was quantified spectrophotometrically. Identical amounts of different cDNA samples were used for qPCR amplifications using Arabidopsis sina like 7 with specific primers (Table 1). The data presented concern relative values obtained from the average of three biological and technical replicates using the cbp gene (cap binding protein At5g44200) as a house-keeping control [24] (Figure 2A). AtSINAL7 transcript levels are highly expressed in siliques (2.3770) and stage 12 inflorescences (0.7053), and roots (0.1524), being lower at Stage 6 inflorescences (0.0807) and rosette leaves (0.0600). Thus, AtSINAL7 seems to be expressed in a tissue-specific manner with a strong induction of 12 and 40 times in stage 12 flowers and silique respectively, compared to rosette leaves.

Bottom Line: We also found that higher AtSINAL7 transcript levels are present in tissues undergoing active cell division during floral development.An interesting observation is the circadian expression pattern of AtSINAL7 mRNA in floral buds.Furthermore, UV-B irradiation induces the expression of this transcript indicating that AtSINAL7 may be involved in a wide range of different cell processes.

View Article: PubMed Central - PubMed

Affiliation: Centro de Estudios Fotosintéticos y Bioquímicos (CEFOBI-CONICET), Universidad Nacional de Rosario, Rosario, Argentina.

ABSTRACT
Protein ubiquitination leading to degradation by the proteasome is an important mechanism in regulating key cellular functions. Protein ubiquitination is carried out by a three step process involving ubiquitin (Ub) activation by a E1 enzyme, the transfer of Ub to a protein E2, finally an ubiquitin ligase E3 catalyzes the transfer of the Ub peptide to an acceptor protein. The E3 component is responsible for the specific recognition of the target, making the unveiling of E3 components essential to understand the mechanisms regulating fundamental cell processes through the protein degradation pathways. The Arabidopsis thaliana seven in absentia-like 7 (AtSINAL7) gene encodes for a protein with characteristics from a C3HC4-type E3 ubiquitin ligase. We demonstrate here that AtSINAL7 protein is indeed an E3 protein ligase based on the self-ubiquitination in vitro assay. This activity is dependent of the presence of a Lys residue in position 124. We also found that higher AtSINAL7 transcript levels are present in tissues undergoing active cell division during floral development. An interesting observation is the circadian expression pattern of AtSINAL7 mRNA in floral buds. Furthermore, UV-B irradiation induces the expression of this transcript indicating that AtSINAL7 may be involved in a wide range of different cell processes.

Show MeSH
Related in: MedlinePlus