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. 2014 Sep;42(15):9925-36.
doi: 10.1093/nar/gku716. Epub 2014 Aug 7.

Regulation of pri-miRNA Processing by the hnRNP-like Protein AtGRP7 in Arabidopsis

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Free PMC article

Regulation of pri-miRNA Processing by the hnRNP-like Protein AtGRP7 in Arabidopsis

Tino Köster et al. Nucleic Acids Res. .
Free PMC article

Abstract

The hnRNP-like glycine-rich RNA-binding protein AtGRP7 regulates pre-mRNA splicing in Arabidopsis. Here we used small RNA-seq to show that AtGRP7 also affects the miRNA inventory. 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. Overaccumulation of several pri-miRNAs including pri-miR398b, pri-miR398c, pri-miR172b, pri-miR159a and pri-miR390 at the expense of the mature miRNAs suggested that AtGRP7 affects pri-miRNA processing. Indeed, RNA immunoprecipitation revealed that AtGRP7 interacts with these pri-miRNAs in vivo. 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. In contrast, pri-miRNAs of selected miRNAs that were elevated or not changed in response to high AtGRP7 levels were not bound in vivo. Reduced accumulation of miR390, an initiator of trans-acting small interfering RNA (ta-siRNA) formation, also led to lower TAS3 ta-siRNA levels and increased mRNA expression of the target AUXIN RESPONSE FACTOR4. Furthermore, AtGRP7 affected splicing of pri-miR172b and pri-miR162a. Thus, AtGRP7 is an hnRNP-like protein with a role in processing of pri-miRNAs in addition to its role in pre-mRNA splicing.

Figures

Figure 1.
Figure 1.
MiR398 levels are reduced in AtGRP7-ox plants. An RNA gel blot of the AtGRP7-ox lines D and G in Col-2, AtGRP7-ox lines in L er and C24 and the corresponding wt plants was hybridised with anti-miR398 (top) and a U6 control (bottom). Fold changes of miR398 normalized to U6 in AtGRP7-ox plants are expressed relative to wt.
Figure 2.
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.
Figure 3.
Figure 3.
AtGRP7 affects miR390-dependent TAS3 ta-siRNAs and ARF4 in AtGRP7-ox but not AtGRP7 R49Q-ox plants. (A) Stem–loop RT-PCR of miR390. (B) Stem–loop RT-PCR of TAS3 5′D7(+) RNA. (C) Levels of the miR390 target ARF4. Shown are the mean ± SD of three biological reps. Asterisks denote statistically significant differences according to Student's t-test (P < 0.05).
Figure 4.
Figure 4.
Pri-miRNA levels are elevated at the expense of mature miRNAs in AtGRP7-ox plants. The levels of the pri-miRNAs were analyzed in AtGRP7-ox, AtGRP7-R49Q-ox and C24 wt plants (A) and in the AtGRP7-ox lines D and G and Col-2 (B, C). Pri-miR398a, b and c, pri-miR390b, pri-miR172b, pri-miR159a and pri-miR399b correspond to miRNAs with reduced level in AtGRP7-ox plants. Pri-miR395e and pri-miR319b correspond to miRNAs with elevated levels in AtGRP7-ox plants, and pri-miR408a and pri-miR171c correspond to miRNAs that are not affected by AtGRP7 overexpression. Data are based on three biological reps. Asterisks denote a significant difference according to Student's t-test (P < 0.05).
Figure 5.
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.
Figure 6.
Figure 6.
AtGRP7 affects alternative splicing of pri-miRNAs. (A) Scheme of MIR172b. Black boxes = exons, grey box = position of the pri-miRNA, thin line = introns. The arrows denote the position of the primers used in (B). (B) RNA from the AtGRP7-ox lines D and G and Col-2 wt was analysed by RT-PCR. The amplification products corresponding to the intron-retained form and the fully spliced forms are indicated by arrowheads and asterisks, respectively. PP2A served as a control. DNA = genomic DNA. (C) The ratio of intron-retained versus spliced pri-miR172b was quantified using Bioanalyzer DNA1000 chips. Shown is the mean of two reps. (D) Scheme of the non-protein-coding RNA harbouring MIR162a. Black boxes = exons, grey box = position of the pri-miRNA, open boxes = annotated 5′and 3′UTRs, thin line = introns. The arrows denote the position of the primers used in (E). (E) RNA from the AtGRP7-ox lines D and G and Col-2 wt was analysed by RT-PCR. The transcript forms corresponding to the amplification products are indicated. The rhombus denotes an alternative version of the 168 nt band generated by an alternative 3′splice site 3 nt downstream of the authentic 3′splice site. PP2A served as a control. DNA = genomic DNA. (F) The ratio of the alternative splice forms versus the spliced form was quantified using Bioanalyzer DNA1000 chips. Shown is the mean of two reps.

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