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Linking key steps of microRNA biogenesis by TREX-2 and the nuclear pore complex in Arabidopsis

Nature Plants volume 6, pages 957–969 (2020)Cite this article

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Abstract

Unlike in metazoans, the stepwise biogenesis of microRNAs (miRNAs) in plants occurs within the nucleus. Whether or how the major steps in miRNA biogenesis are coordinated is largely unknown. Here we show that the plant TREX-2 complex promotes multiple steps in miRNA biogenesis, including transcription, processing and nuclear export. THP1 and SAC3A—the core subunits of TREX-2—interact and colocalize with RNA polymerase II to promote the transcription of MIR genes in the nucleoplasm. TREX-2 interacts with the microprocessor component SERRATE and promotes the formation of dicing bodies in the nucleoplasm. THP1 also interacts and colocalizes with the nucleoporin protein NUP1 at the nuclear envelope. NUP1 and THP1 promote the nuclear export of miRNAs and ARGONAUTE1. These results suggest that TREX-2 coordinates the transcription, processing and export steps in miRNA biogenesis to ensure efficient miRNA production.

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Fig. 1: The thp1-5 mutant exhibits pleiotropic developmental defects and reduced miRNA levels.
Fig. 2: Arabidopsis TREX-2 is required for miRNA production.
Fig. 3: TREX-2 promotes MIR gene transcription and associates with the transcription machinery.
Fig. 4: TREX-2 associates with and affects the microprocessor.
Fig. 5: Nucleocytoplasmic partitioning of miRNAs and AGO1, and TREX-2 protein localization.
Fig. 6: NUP1 promotes the nuclear export of miRNAs and AGO1.
Fig. 7: mTREX-2 in miRNA biogenesis.

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Data availability

Raw sequence data generated during this study were deposited into the NCBI GEO database under the accession code GSE150473. The authors declare that any other supporting data is available from the corresponding author upon request. Source data are provided with this paper.

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Acknowledgements

We thank D. Weigel (Max Planck Institute for Developmental Biology, Tubingen, Germany) for sharing the pSUC2:amiR-SUL line; Y. Cui (Agriculture and Agri-Food Canada, London, Ontario, Canada) for sharing the thp1-3 and sac3 mutants and TREX-2 Y2H plasmids; M. Chen (University of California, Riverside, CA, USA) for sharing the fluorescence protein-containing vectors and stimulating discussions; and B. Le, H. Hu and N. Flynn for comments on the manuscript. This work was funded by NIH GM129373 to X.C.

Author information

Authors and Affiliations

  1. Department of Botany and Plant Sciences, Institute of Integrative Genome Biology, University of California, Riverside, CA, USA

    Bailong Zhang, Chenjiang You, Yong Zhang, Liping Zeng, Jun Hu, Minglei Zhao & Xuemei Chen

  2. Guangdong Provincial Key Laboratory for Plant Epigenetics, College of Life Sciences and Oceanography, Shenzhen University, Shenzhen, China

    Chenjiang You

  3. State Key Laboratory of Hybrid Rice, College of Life Sciences, Wuhan University, Wuhan, China

    Jun Hu

  4. College of Horticulture, South China Agricultural University, Guangzhou, China

    Minglei Zhao

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Contributions

X.C. and B.Z. designed the research. B.Z. performed the majority of experiments. C.Y. and L.Z. conducted data analyses. Y.Z. performed nuclear AGO1 IP–MS. M.Z., J.H. and B.Z. performed genotyping for double mutant identification. B.Z. and X.C. wrote the paper.

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Correspondence to Xuemei Chen.

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Peer review information Nature Plants thanks Hiro-oki Iwakawa, Pablo Manavella, Rodrigo Reis and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 Small-RNA sequencing of thp1-5 amS.

a, Heatmaps to show reproducibility among amS and thp1-5 amS biological replicates. The whole genome was divided into 100-bp bins, and small-RNA reads whose 5' ends located in a bin were assigned to this bin. Color density indicates distance calculated by log-transformed normalized read counts assigned to each bin. b, Distribution of fold changes for all detected miRNAs (n = 232) and 21 nt (n=59742 windows of 100bp) and 24 nt (n=201374 windows of 100bp) small RNAs in thp1-5 amS relative to amS. The lower extreme, the lower hinge, the white dot, the upper hinge, and the upper extreme of the black box represent the minimum, the first quartile, the median, the third quartile, and the maximum of the data. The violin shape corresponds to the density of data. ***, P-value < 2.2e-16. P-values were determined by a paired two-sided Wilcoxon test.

Extended Data Fig. 2 Defects of thp1 mutants.

a-b, Flowers (a) and siliques (b) of amS and thp1-5 amS plants. Note that the shoot terminates in thp1-5 amS but not in wild type (amS). Scale bar in (a) = 1 mm. Scale bar in (b) = 10 mm. c, 21-day-old Col, thp1-1 and thp1-3 plants with differences in leaf shape. Scale bar = 10 mm. d, RNA gel blot analysis of 6 miRNAs in Col, thp1-1 and thp1-3. The miRNA signals were quantified using U6 as a loading control, and values were relative to Col. These experiments were repeated three times with similar results.

Source data

Extended Data Fig. 3 Genetic interactions between mutants in THP1 and miRNA biogenesis genes.

a, Morphological phenotypes of 25-day-old amS, thp1-5 amS, se-1 amS, hyl1-2 amS, hst-6 amS, hen1-8 amS, ago1-27 amS, and thp1-5 ago1-27 amS plants. Scale bar = 10 mm. All plants with the same genotype exhibit the same phenotype and one representative for each genotype is shown. b, Genetic segregation analyses. F2 plants of the indicated genotypes were selved and ~48 F3 plants were genotyped for the thp1-5 mutation. The ratios of thp1-5/+ : +/+ are around 2:1, indicating that thp1-5 homozygosity leads to embryo lethality.

Source data

Extended Data Fig. 4 Arabidopsis TREX-2 components and sac3 mutant alleles.

a, Yeast two-hybrid assays to test interactions among TREX-2 components (THP1, SAC3A, SAC3B, DSSI-(I), DSSI-(V), CEN1, and CEN2). The empty vectors AD and BD were included as negative controls. After mating, diploid yeast cells containing both the bait and prey plasmids grew on SD-Trp/-Leu (DDO) medium, and diploid yeast cells in which the bait and prey proteins interacted grew into blue colonies on SD-Trp/-Leu/-His/-Ade+X-α-gal Aureobasidin A (QDO/X/A) medium. The experiment was repeated two times with similar results. b, Diagrams of SAC3A, B, and C genes showing the different mutant alleles. Triangles indicate positions of T-DNA insertions. Black arrows indicate primers used for RT-PCR. Lines below the gene models indicate conserved protein domains in homologous proteins. c, RT-PCR analyses of SAC3 gene expression in different sac3 mutants. The primers used are shown as black arrows in (b). The experiment was repeated two times with similar results.

Source data

Extended Data Fig. 5 TREX-2 associates with the transcription machinery.

a, Yeast two-hybrid assays to test interactions between TREX-2 components (THP1, SAC3A, SAC3B, DSS1-(I), DSS1-(V), and CEN1) and subunits of Mediator (MED20A, MED20B, MED20C and MED18). The empty vectors AD and BD were included as negative controls. After mating, diploid yeast cells containing both the bait and prey plasmids grew on SD-Trp/-Leu (DDO) medium, and diploid yeast cells in which the bait and prey proteins interacted grew into blue colonies on SD-Trp/-Leu/-His/-Ade+X-α-gal Aureobasidin A (QDO/X/A) medium. The experiment was repeated two times with similar results. b, Yeast two-hybrid assays to test interactions between TREX-2 components (THP1, SAC3A, SAC3B, DSS1-(I), DSS1-(V), and CEN2) and subunits of Elongator (ELP4 and ELP5) or subunits of MAC (PRL1 and MAC8). The empty vectors AD and BD were included as negative controls. After mating, diploid yeast cells containing both the bait and prey plasmids grew on SD-Trp/-Leu (DDO) medium, and diploid yeast cells in which the bait and prey proteins interacted grew into blue colonies on SD-Trp/-Leu/-His/-Ade+X-α-gal Aureobasidin A (QDO/X/A) medium. The experiment was repeated two times with similar results. c, BiFC analysis to test interactions between SAC3A and Pol II CTD (C-terminal domain). Scale bar = 10 µm. Three independent experiments yielded similar results. d, RT-qPCR to determine pri-miR-SUL and Bar transcript levels in amS and thp1-5 amS. IPP2 was used as an internal control. More than 30 plants from each genotype were used in the experiment. e, ChIP-qPCR analyses to determine the occupancy of four MIR loci by Pol II in 12-day-old seedlings of amS and thp1-5 amS. IgG amS was served as a negative control. More than 200 plants from each genotype were used in the experiment.

Source data

Extended Data Fig. 6 mRNA sequencing of wild type (Col), thp1-5, and sac3b-1/sac3b-2 sac3c-1/sac3c-1.

a, Correlation analysis across WT, thp1-5 and sac3b-1/sac3b-2 sac3c-1/sac3c-1 samples. The three samples from each genotype are biological replicates. Color density indicates Euclidean distance calculated by log-transformed normalized read counts assigned to each gene. b, Venn diagrams showing the overlap in down-regulated (left) and up-regulated (right) genes between thp1-5 and sac3b-1/sac3b-2 sac3c-1/sac3c-1. c, GO enrichment analysis of 852 commonly up-regulated genes in thp1-5 and sac3b-1/sac3b-2 sac3c-1/sac3c-1. d, GO enrichment analysis of 819 commonly down-regulated genes in thp1-5 and sac3b-1/sac3b-2 sac3c-1/sac3c-1.

Extended Data Fig. 7 TREX-2 associates with the microprocessor.

a, Yeast two-hybrid assays to test interactions between TREX-2 components (THP1, SAC3A, SAC3B, DSS1-(I), DSS1-(V), and CEN2) and the microprocessor components DCL1 and HYL1. The empty vectors AD and BD were included as negative controls. After mating, diploid yeast cells containing both the bait and prey plasmids grew on SD-Trp/-Leu (DDO) medium, and diploid yeast cells in which the bait and prey proteins interacted were expected to grow into blue colonies on SD-Trp/-Leu/-His/-Ade+X-α-gal Aureobasidin A (QDO/X/A) medium. DCL1-N, amino acids 1 to 831 of DCL1; DCL1-C, amino acids of 832 to 1909 of DCL1. The experiment was repeated two times with similar results. b, BiFC assay to test interactions between SAC3A and SE. Scale bar = 10 µm. Two independent experiments yielded similar results. c, Determination of HYL1 phosphorylation status. The same blot as that of the upper panel in Fig. 4c with different exposure time. The different forms of HYL1 are as indicated (P+ = phosphorylated HYL1; P− = non-phosphorylated HYL1). The experiment was repeated three times with similar results. d, Another replicate for Fig. 4c. e, A western blot to show the levels of HYL1-YFP and endogenous HYL1 in 35S:HYL1-YFP and 35S:HYL1-YFP thp1-5. RPN6 is a loading control. Two independent experiments yielded similar results.

Source data

Extended Data Fig. 8 DCL1 D-body formation is compromised in thp1-5.

a, A western blot to detect DCL1 with the anti-DCL1 antibody in WT and dcl1-9 (a truncation mutant). This shows that the antibody was able to specifically detect DCL1. The experiment was conducted one time. b, Detection of DCL1 D-body by immunofluorescence of isolated nuclei from inflorescence tissue of Col and thp1-5. DCL1 D-body signals are in green and DAPI stained nuclei are in blue. One representative image is shown for Col and thp1-5 each. Scale bar =1µm. The experiment was repeated independently two times with similar results. c, Quantification of DCL1 D-body numbers in Col (n=78) and thp1-5 (n=101) nuclei. The numbers 0-4 represent the numbers of D-bodies in each nucleus. d, ChIP-qPCR analyses to determine the occupancy of four MIR loci by DCL1 in 12-day-old seedlings of amS and thp1-5 amS. amS and thp1-5 amS were immunoprecipitated by DCL1 antibody. SN1 (located between At3g44000 and At3g44005 at nucleotides 15,805,617–15,805,773 of chromosome 3) was used as a negative control. IgG amS served as a negative control.

Source data

Extended Data Fig. 9 The nucleo-cytoplasmic partitioning of miRNAs and AGO1 in thp1-5.

a, Comparison of an improved nucleo-cytoplasmic fractionation method and the traditional method. The fractionated samples were subjected to protein gel blot analysis using anti-HYL1, anti-GAPDH and anti-H3 antibodies, respectively. T = total extract; C = cytoplasm; N = nucleus. HYL1 and H3 are nuclear proteins; GAPDH is a cytoplasmic protein. The experiment was repeated two times with similar results. b, Optimization of nucleo-cytoplasmic fractionation in terms of the duration of paraformaldehyde crosslinking (8min, 15min and 20min). The fractionated samples were subjected to protein gel blot analysis using anti-AGO1, anti-cFBPase, anti-HYL1, anti-BIP and anti-H3 antibodies, respectively. T = total extract; C = cytoplasm; N = nucleus. H3 is a nuclear marker; cFBPase is a cytoplasmic marker. They were also used in the quantification of AGO1 levels (represented by the numbers) between T and N and between T and C, respectively. Three independent experiments gave similar results. c, Small RNA gel blot assays to determine the levels of various miRNAs in Col and thp1-5 following fractionation with the improved method. T = total extract; C = cytoplasm; N = nucleus. Signal intensity of T was arbitrarily set to 1.0; that of C and N was normalized to T against U6 and tRNA-Met, respectively, as nuclear and cytoplasmic RNA markers. The experiment was repeated two times with similar results. d, Cytoplasmic/nuclear ratios of various miRNAs as determined in (c).

Source data

Extended Data Fig. 10 Characterization of nup1-cs lines.

a, 45-day-old plants of wild type (Col) and three lines harboring the pNUP1:NUP1-mEGFP transgene (the three on the right). Two of the three are nup1-cs (nup1 co-suppression) lines. Scale bar = 10 mm. b, RT-PCR analysis of NUP1 transcripts in Col, pNUP1:NUP1-mEGFP and nup1-cs lines. Note that the signal represents transcripts from both the endogenous NUP1 gene and the pNUP1:NUP1-mEGFP transgene. The experiment was repeated two times with similar results. c, Protein gel blot analysis of the NUP1-mEGFP protein in Col, pNUP1:NUP1-mEGFP and nup1-cs lines. The experiment was conducted one time. d, RNA gel blot assays to determine the levels of miR166 and miR172 in Col and a nup1-cs-1 line. Inflorescences were used for RNA extraction. U6 served as an internal control. The experiment was repeated two times with similar results. e-f, Protein gel blot assays to determine the nucleo-cytoplasmic partitioning of AGO1 in Col and nup1-cs (e) and in Col, nup1-cs, and thp1-5 (f). Three independent experiments gave similar results. The fractions were subjected to protein gel blot analysis using anti-AGO1, anti-cFBPase, anti-H3 and anti-BIP antibodies, respectively. H3 is a nuclear marker; cFBPase is a cytoplasmic marker. They were also used in the quantification of AGO1 levels (represented by the numbers) between T and N and between T and C, respectively. BIP is an ER marker to indicate the level of ER contamination in the nuclear fractions.

Source data

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Unprocessed plant images, northern blots and RT–qPCR source.

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Unprocessed plant images.

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Unprocessed plant images, yeast two-hybrid images and western blots.

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Unprocessed confocal microscope images, yeast two-hybrid images, western blots and RT–qPCR source.

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Unprocessed northern blots, western blots and confocal microscope images.

Source Data Fig. 6

Unprocessed plant images, northern blots, western blots and RT–qPCR source.

Source Data Extended Data Fig. 2

Unprocessed plant images and northern blots.

Source Data Extended Data Fig. 3

Unprocessed plant images.

Source Data Extended Data Fig. 4

Unprocessed yeast two-hybrid images and gels.

Source Data Extended Data Fig. 5

Unprocessed yeast two-hybrid images, confocal microscope images and RT–qPCR source.

Source Data Extended Data Fig. 7

Unprocessed yeast two-hybrid images, confocal microscope images and western blots.

Source Data Extended Data Fig. 8

Unprocessed western blots, confocal microscope images and RT–qPCR source.

Source Data Extended Data Fig. 9

Unprocessed western blots and northern blots.

Source Data Extended Data Fig. 10

Unprocessed plant images, western blots, DNA gels and northern blots.

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Zhang, B., You, C., Zhang, Y. et al. Linking key steps of microRNA biogenesis by TREX-2 and the nuclear pore complex in Arabidopsis. Nat. Plants 6, 957–969 (2020). https://doi.org/10.1038/s41477-020-0726-z

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