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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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AtGRP7 binds to pri-miRNAs in vivo. Plants expressing AtGRP7::AtGRP7:GFP (A, D) or AtGRP7::AtGRP7-R49Q:GFP (B) in the atgrp7–1 background and AtGRP7:GFP in Col-2 (C) were subjected to RIP. The levels of the pri-miRNAs and PP2A were determined in the GFP-Trap® bead precipitate (IP+), the RFP-Trap® bead precipitate (IP−) and the input fraction (IN), respectively. Pri-miR398b and c, pri-miR172b and pri-miR159a correspond to miRNAs with reduced level in AtGRP7-ox plants, pri-miR319b corresponds to a miRNA with elevated levels in AtGRP7-ox plants, and pri-miR408a and pri-miR171 b and c correspond to miRNAs that are not affected. Data are based on three biological replicates. Asterisks denote a significant difference according to Student's t-test (P < 0.05). n.s., not significant; n.d., not detectable.
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Figure 5: AtGRP7 binds to pri-miRNAs in vivo. Plants expressing AtGRP7::AtGRP7:GFP (A, D) or AtGRP7::AtGRP7-R49Q:GFP (B) in the atgrp7–1 background and AtGRP7:GFP in Col-2 (C) were subjected to RIP. The levels of the pri-miRNAs and PP2A were determined in the GFP-Trap® bead precipitate (IP+), the RFP-Trap® bead precipitate (IP−) and the input fraction (IN), respectively. Pri-miR398b and c, pri-miR172b and pri-miR159a correspond to miRNAs with reduced level in AtGRP7-ox plants, pri-miR319b corresponds to a miRNA with elevated levels in AtGRP7-ox plants, and pri-miR408a and pri-miR171 b and c correspond to miRNAs that are not affected. Data are based on three biological replicates. Asterisks denote a significant difference according to Student's t-test (P < 0.05). n.s., not significant; n.d., not detectable.

Mentions: Because AtGRP7 did not appear to affect miRNA levels indirectly via a global effect on processing factors, we tested whether it directly interacts with pri-miRNAs in vivo. We performed RIP on transgenic plants expressing AtGRP7:GFP under control of its own promoter in the atgrp7–1 background. Pri-miR398b and pri-miR398c were enriched in RNP complexes precipitated with GFP-Trap® beads (IP+) from these AtGRP7::AtGRP7:GFP plants relative to mock precipitates with RFP-Trap® beads (IP−) (Figure 5A). PP2A which served as a negative control was not significantly enriched. Both pri-miR398b and pri-miR398c were not enriched in precipitates from plants expressing GFP alone. Notably, they were also not recovered from plants expressing AtGRP7::AtGRP7-R49Q:GFP (Figure 5B). The pri-miR398a level was too low to allow reliable quantification, in line with its weak expression (68). Furthermore, pri-miR172b and pri-miR159a were enriched in IP+ relative to IP− and not present in precipitates from plants expressing AtGRP7::AtGRP7-R49Q:GFP or GFP only. For pri-miR390b, the expression level was also too low to allow reliable quantification.


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)

AtGRP7 binds to pri-miRNAs in vivo. Plants expressing AtGRP7::AtGRP7:GFP (A, D) or AtGRP7::AtGRP7-R49Q:GFP (B) in the atgrp7–1 background and AtGRP7:GFP in Col-2 (C) were subjected to RIP. The levels of the pri-miRNAs and PP2A were determined in the GFP-Trap® bead precipitate (IP+), the RFP-Trap® bead precipitate (IP−) and the input fraction (IN), respectively. Pri-miR398b and c, pri-miR172b and pri-miR159a correspond to miRNAs with reduced level in AtGRP7-ox plants, pri-miR319b corresponds to a miRNA with elevated levels in AtGRP7-ox plants, and pri-miR408a and pri-miR171 b and c correspond to miRNAs that are not affected. Data are based on three biological replicates. Asterisks denote a significant difference according to Student's t-test (P < 0.05). n.s., not significant; n.d., not detectable.
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Figure 5: AtGRP7 binds to pri-miRNAs in vivo. Plants expressing AtGRP7::AtGRP7:GFP (A, D) or AtGRP7::AtGRP7-R49Q:GFP (B) in the atgrp7–1 background and AtGRP7:GFP in Col-2 (C) were subjected to RIP. The levels of the pri-miRNAs and PP2A were determined in the GFP-Trap® bead precipitate (IP+), the RFP-Trap® bead precipitate (IP−) and the input fraction (IN), respectively. Pri-miR398b and c, pri-miR172b and pri-miR159a correspond to miRNAs with reduced level in AtGRP7-ox plants, pri-miR319b corresponds to a miRNA with elevated levels in AtGRP7-ox plants, and pri-miR408a and pri-miR171 b and c correspond to miRNAs that are not affected. Data are based on three biological replicates. Asterisks denote a significant difference according to Student's t-test (P < 0.05). n.s., not significant; n.d., not detectable.
Mentions: Because AtGRP7 did not appear to affect miRNA levels indirectly via a global effect on processing factors, we tested whether it directly interacts with pri-miRNAs in vivo. We performed RIP on transgenic plants expressing AtGRP7:GFP under control of its own promoter in the atgrp7–1 background. Pri-miR398b and pri-miR398c were enriched in RNP complexes precipitated with GFP-Trap® beads (IP+) from these AtGRP7::AtGRP7:GFP plants relative to mock precipitates with RFP-Trap® beads (IP−) (Figure 5A). PP2A which served as a negative control was not significantly enriched. Both pri-miR398b and pri-miR398c were not enriched in precipitates from plants expressing GFP alone. Notably, they were also not recovered from plants expressing AtGRP7::AtGRP7-R49Q:GFP (Figure 5B). The pri-miR398a level was too low to allow reliable quantification, in line with its weak expression (68). Furthermore, pri-miR172b and pri-miR159a were enriched in IP+ relative to IP− and not present in precipitates from plants expressing AtGRP7::AtGRP7-R49Q:GFP or GFP only. For pri-miR390b, the expression level was also too low to allow reliable quantification.

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