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. 2019 Feb;31(2):486-501.
doi: 10.1105/tpc.18.00556. Epub 2019 Jan 23.

The PROTEIN PHOSPHATASE4 Complex Promotes Transcription and Processing of Primary microRNAs in Arabidopsis

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Free PMC article

The PROTEIN PHOSPHATASE4 Complex Promotes Transcription and Processing of Primary microRNAs in Arabidopsis

Suikang Wang et al. Plant Cell. .
Free PMC article

Abstract

PROTEIN PHOSPHATASE4 (PP4) is a highly conserved Ser/Thr protein phosphatase found in yeast, plants, and animals. The composition and functions of PP4 in plants are poorly understood. Here, we uncovered the complexity of PP4 composition and function in Arabidopsis (Arabidopsis thaliana) and identified the composition of one form of PP4 containing the regulatory subunit PP4R3A. We show that PP4R3A, together with one of two redundant catalytic subunit genes, PROTEIN PHOSPHATASE X (PPX)1 and PPX2, promotes the biogenesis of microRNAs (miRNAs). PP4R3A is a chromatin-associated protein that interacts with RNA polymerase II and recruits it to the promoters of miRNA-encoding (MIR) genes to promote their transcription. PP4R3A likely also promotes the cotranscriptional processing of miRNA precursors, because it recruits the microprocessor component HYPONASTIC LEAVES1 to MIR genes and to nuclear dicing bodies. Finally, we show that hundreds of introns exhibit splicing defects in pp4r3a mutants. Together, this study reveals roles for Arabidopsis PP4 in transcription and nuclear RNA metabolism.

Figures

Figure 1.
Figure 1.
The sup-e33 Mutant Is Defective in miRNA Biogenesis. (A) Morphological phenotypes of 3-week-old SUC2:amiR-SUL (amiR-SUL), sup-e33, and rescued sup-e33 (sup-e33 C1) plants. Scale bars = 1 cm. (B) RNA gel blot analysis to detect various miRNAs and tasiRNAs (siR155 and siR1511) in amiR-SUL, sup-e33, and rescued sup-e33 plants. U6 served as a loading control. The numbers represent relative abundance of small RNAs in the three genotypes. (C) Transcript levels of miRNA targets, as determined by RT-qPCR. UBIQUITIN5 (UBQ5) served as an internal control. Error bars indicate sd from three independent experiments; asterisks indicate significant difference (t test, P < 0.05). (D) Protein levels of miRNA targets (SUL/amiR-SUL, SE/miR863-3p, AGO2/miR403) in sup-e33, as determined by protein gel blot analysis. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and heat shock cognate (HSC)70 served as loading controls. (E) Global abundance of miRNAs in Col and pp4r3a alleles, as determined by small RNA-seq. The small RNAs were normalized against total reads, abundance is expressed as RPM (reads per million mapped reads), and log2 fold changes (mutant/wild type) were plotted. Asterisk indicates that the mean log2 fold change is significantly below 0 (Wilcox. test, P < 2.3e-10).
Figure 2.
Figure 2.
Gene Cloning and Genetic Characterization of PP4R3A and PP4R3B. (A) Gene structures of PP4R3A and PP4R3B (gray block, untranslated region; black blocks, exons; black lines, introns). The arrow indicates the point mutation site in sup-e33 (designated as pp4r3a-1), and the black triangles indicate the T-DNA insertion sites. (B) Both PP4R3A and PP4R3B possess a pleckstrin homology (PH) domain, a suppressor of MEK-1 (SMK-1) domain, and an Armadillo fold. (C) Morphological phenotypes of 3-week-old Col, pp4r3a-1 (sup-e33 in Col background), pp4r3a-2, pp4r3b-2, pp4r3a-1 pp4r3b-2, and pp44r3a-2 pp4r3b-2 plants. Scale bars = 1 cm. (D) Determination of miRNA levels in pp4r3a, pp4r3b and pp4r3a pp4r3b by RNA gel blot analysis of small RNAs. The numbers represent relative abundance of small RNAs.
Figure 3.
Figure 3.
Phenotypes of PP4 Catalytic Mutants. (A) Morphologic phenotypes of the ppx1 and ppx2 single mutants and the ppx1 ppx2 double mutant. Scale bars = 1 cm. (B) Accumulation of miRNAs and an siRNA in Col, ppx1, ppx2, and ppx1 ppx2 plants as detected by RNA gel blot analysis of small RNAs. U6 served as a loading control. The numbers indicate relative abundance of the small RNAs in the four genotypes.
Figure 4.
Figure 4.
Genetic Interactions between sup-e33 and Pri-miRNA Processing Mutants. (A) to (D) Morphological phenotypes of 3-week-old plants of the indicated genotypes. Scale bars = 1 cm. (E) RNA gel blot analysis of small RNAs to determine the levels of amiR-SUL in various genotypes. U6 served as a loading control. The numbers represent relative abundance.
Figure 5.
Figure 5.
Requirement of PP4R3A for MIR Transcription. (A) Determination of pri-miRNA levels by RT-qPCR. (B) GUS staining of representative samples harboring pMIR167a:GUS and pMIR172b:GUS in the Col and pp4r3a-1 backgrounds. Scale bars = 2 mm. (C) GUS transcript levels in samples harboring pMIR167a:GUS and pMIR172b:GUS in the Col and pp4r3a-1 backgrounds, as determined by RT-qPCR. (D) and (E) Genetic interactions between mutations in PP4R3A, NRPB2 and CDC5. (D) Additive phenotypes of nrpb2-3 pp4r3a-1 and cdc5-2 pp4r3a-1 double mutants. Scale bars = 1 cm. Note that the phenotypes of the double mutants were much stronger than the corresponding single mutants, but similar to that of pp4r3a-2. (E) The abundance of miRNAs in Col, pp4r3a-1, pp4r3a-2, nrpb2-3, cdc5-1, nrpb2-3 pp4r3a-1, and cdc5-1 pp4r3a-1, as detected by RNA gel blot analysis of small RNAs. In (A) and (C), UBQ5 served as the internal control; error bars indicate sd from three independent experiments; asterisks indicate significant difference (t test, P < 0.05).
Figure 6.
Figure 6.
Analysis of PP4R3A Localization and Pol II Occupancy at MIR Promoters. (A) YFP signals in the root of a 5-d-old PP4R3A:PP4R3A-YFP seedling. Scale bar = 200 μm. (B) Protein gel blot analysis showing the cytoplasmic, nuclear, and chromatin distribution of the PP4R3A-HA protein from a PP4R3A:PP4R3A-HA/pp4r3a-1 transgenic line. GAPDH and histone H3 were used as protein markers for the cytoplasmic and nuclear/chromatin fractions, respectively. (C) ChIP-qPCR analysis to determine the occupancy of PP4R3A at MIR promoters. ChIP was performed with no antibody (no Ab) or anti-HA antibody in Col and PP4R3A:PP4R3A-HA transgenic plants. (D) Co-immunoprecipitation of PP4R3A with Pol II. F1 plants from a cross between PP4R3A:PP4R3A-YFP and RPB1:RPB1-4xMYC homozygous transgenic lines were used to perform IP, using either anti-MYC or GFP-Trap antibodies. Protein gel blot analysis to detect PP4R3A–YFP and RPB1-MYC was performed with anti-GFP and anti-MYC antibodies, respectively. The protein ladder is labeled on the left. (E) ChIP-qPCR analysis to determine the occupancy of Pol II at MIR promoters using plants with a homozygous RPB1:RPB1-4xMYC transgene in the Col and pp4r3a-1 backgrounds. The intergenic region DEG15do3k between AT1G28310 and AT1G28320 was used as a negative control. The ChIP signal was normalized against input. In (C) and (E), error bars indicate sd from three independent experiments; asterisks indicate significant difference (t test, P < 0.05).
Figure 7.
Figure 7.
HYL1 Localization and Chromatin Association are Impaired in pp4r3a-1. (A) Representative confocal images of HYL1-YFP fluorescence in the meristematic zones of Col and pp4r3a-1 roots. Bars = 10 μm. (B) Quantification of HYL1-YFP D-bodies. The x axis represents the number of HYL1-YFP D-bodies (speckles) per cell, and the y axis indicates the percentage of cells with the corresponding HYL1-YFP D-body numbers. The number of HYL1-YFP D-bodies was calculated from 146 root cells of Col and 135 root cells of pp4r3a-1. (C) ChIP-qPCR assay to determine the association of HYL1 with MIR gene bodies in Col and pp4r3a-1. Error bars indicate sd from three independent experiments; asterisk indicates significant difference (t test, P < 0.05).
Figure 8.
Figure 8.
Defects in Gene Expression and Splicing of Pre-mRNAs in pp4r3a Mutants. (A) Venn diagrams showing the number of differentially expressed genes (DEGs) in each pp4r3a allele and the overlap of DEGs in the two alleles. Hyper- and hypo-DEGs are genes with increased and decreased expression, respectively. (B) Gene Ontology enrichment analysis of common DEGs in the two pp4r3a alleles. In the pp4r3a mutants, upregulated genes are enriched in various stress responses Gene Ontology terms, whereas downregulated genes are involved in oxidation-reduction processes, cellular ion metabolism, and signaling. (C) Scatterplots showing increased PI (percent of intron reads) in each pp4r3a mutant vs. wild type. Each dot represents an annotated intron in Araport11. The introns with a statistically significant increase in PI are shown in green. (D) Venn diagram showing the number of introns with splicing defects in pp4r3a-1 and pp4r3a-2 and the overlap between the two alleles.

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