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Regulation of pri-miRNA processing by the hnRNP-like protein AtGRP7 in Arabidopsis.

Köster T, Meyer K, Weinholdt C, Smith LM, Lummer M, Speth C, Grosse I, Weigel D, Staiger D - Nucleic Acids Res. (2014)

Bottom Line: AtGRP7 overexpression caused a significant reduction in the level of 30 miRNAs and an increase for 14 miRNAs with a minimum log2 fold change of ± 0.5.Mutation of an arginine in the RNA recognition motif abrogated in vivo binding and the effect on miRNA and pri-miRNA levels, indicating that AtGRP7 inhibits processing of these pri-miRNAs by direct binding.Thus, AtGRP7 is an hnRNP-like protein with a role in processing of pri-miRNAs in addition to its role in pre-mRNA splicing.

View Article: PubMed Central - PubMed

Affiliation: Molecular Cell Physiology, Bielefeld University.

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Related in: MedlinePlus

MiR398 and miR398 targets are affected in AtGRP7-ox but not AtGRP7-R49Q-ox plants. (A) Stem–loop RT-PCR of miR398. Shown are the mean ± SD of three biological replicates. Asterisks denote statistically significant differences according to Student's t-test (P < 0.05). (B) Relative transcript levels of the miR398 targets CSD1, CSD2, CCS and COX5b-1. (C) CSD1, CSD2 and CCS protein levels. AB, Amidoblack staining of the membrane to show equal loading. (D) AtGRP7 and AtGRP8 protein levels.
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Figure 2: MiR398 and miR398 targets are affected in AtGRP7-ox but not AtGRP7-R49Q-ox plants. (A) Stem–loop RT-PCR of miR398. Shown are the mean ± SD of three biological replicates. Asterisks denote statistically significant differences according to Student's t-test (P < 0.05). (B) Relative transcript levels of the miR398 targets CSD1, CSD2, CCS and COX5b-1. (C) CSD1, CSD2 and CCS protein levels. AB, Amidoblack staining of the membrane to show equal loading. (D) AtGRP7 and AtGRP8 protein levels.

Mentions: To validate the RNA-seq data, steady-state abundance of selected miRNAs was monitored by stem–loop RT-PCR. In independent AtGRP7-ox lines grown on half-strength MS medium, miR398 levels reached only 25–50% of the wt level (Figure 2A, Supplementary Figure S3). In contrast, in plants overexpressing AtGRP7 with a single arginine in the RRM mutated (AtGRP7-R49Q-ox) (32) miR398 levels were similar to wt levels. This suggests that AtGRP7 contributes to reduced miR398 steady-state abundance through a mechanism requiring RNA binding. Transcript levels of the miR398 targets CSD1 localized in the cytosol, CSD2 localized in the chloroplast and CCS (COPPER CHAPERONE FOR CSD) that delivers the copper cofactor to the CSDs (50) were elevated in AtGRP7-ox but not AtGRP7-R49Q-ox plants (Figure 2B). The abundance of the predicted target COX5b-1 was not altered in AtGRP7-ox plants, in agreement with a previous observation that COX5b-1 is not substantially regulated by miR398 (51). An immunoblot analysis demonstrated elevated levels of CSD2, CSD1 and CCS in AtGRP7-ox but not AtGRP7-R49Q-ox plants (Figure 2C). Taken together, the reduced miR398 level in AtGRP7-ox plants correlates with elevated CSD1, CSD2 and CCS transcript and protein levels.


Regulation of pri-miRNA processing by the hnRNP-like protein AtGRP7 in Arabidopsis.

Köster T, Meyer K, Weinholdt C, Smith LM, Lummer M, Speth C, Grosse I, Weigel D, Staiger D - Nucleic Acids Res. (2014)

MiR398 and miR398 targets are affected in AtGRP7-ox but not AtGRP7-R49Q-ox plants. (A) Stem–loop RT-PCR of miR398. Shown are the mean ± SD of three biological replicates. Asterisks denote statistically significant differences according to Student's t-test (P < 0.05). (B) Relative transcript levels of the miR398 targets CSD1, CSD2, CCS and COX5b-1. (C) CSD1, CSD2 and CCS protein levels. AB, Amidoblack staining of the membrane to show equal loading. (D) AtGRP7 and AtGRP8 protein levels.
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Figure 2: MiR398 and miR398 targets are affected in AtGRP7-ox but not AtGRP7-R49Q-ox plants. (A) Stem–loop RT-PCR of miR398. Shown are the mean ± SD of three biological replicates. Asterisks denote statistically significant differences according to Student's t-test (P < 0.05). (B) Relative transcript levels of the miR398 targets CSD1, CSD2, CCS and COX5b-1. (C) CSD1, CSD2 and CCS protein levels. AB, Amidoblack staining of the membrane to show equal loading. (D) AtGRP7 and AtGRP8 protein levels.
Mentions: To validate the RNA-seq data, steady-state abundance of selected miRNAs was monitored by stem–loop RT-PCR. In independent AtGRP7-ox lines grown on half-strength MS medium, miR398 levels reached only 25–50% of the wt level (Figure 2A, Supplementary Figure S3). In contrast, in plants overexpressing AtGRP7 with a single arginine in the RRM mutated (AtGRP7-R49Q-ox) (32) miR398 levels were similar to wt levels. This suggests that AtGRP7 contributes to reduced miR398 steady-state abundance through a mechanism requiring RNA binding. Transcript levels of the miR398 targets CSD1 localized in the cytosol, CSD2 localized in the chloroplast and CCS (COPPER CHAPERONE FOR CSD) that delivers the copper cofactor to the CSDs (50) were elevated in AtGRP7-ox but not AtGRP7-R49Q-ox plants (Figure 2B). The abundance of the predicted target COX5b-1 was not altered in AtGRP7-ox plants, in agreement with a previous observation that COX5b-1 is not substantially regulated by miR398 (51). An immunoblot analysis demonstrated elevated levels of CSD2, CSD1 and CCS in AtGRP7-ox but not AtGRP7-R49Q-ox plants (Figure 2C). Taken together, the reduced miR398 level in AtGRP7-ox plants correlates with elevated CSD1, CSD2 and CCS transcript and protein levels.

Bottom Line: AtGRP7 overexpression caused a significant reduction in the level of 30 miRNAs and an increase for 14 miRNAs with a minimum log2 fold change of ± 0.5.Mutation of an arginine in the RNA recognition motif abrogated in vivo binding and the effect on miRNA and pri-miRNA levels, indicating that AtGRP7 inhibits processing of these pri-miRNAs by direct binding.Thus, AtGRP7 is an hnRNP-like protein with a role in processing of pri-miRNAs in addition to its role in pre-mRNA splicing.

View Article: PubMed Central - PubMed

Affiliation: Molecular Cell Physiology, Bielefeld University.

Show MeSH
Related in: MedlinePlus