Abstract
Chromatin remodelling factors (CHRs) typically function to alter chromatin structure. CHRs also reside in ribonucleoprotein complexes, but little is known about their RNA-related functions. Here we show that CHR2 (also known as BRM), the ATPase subunit of the large switch/sucrose non-fermentable (SWI/SNF) complex, is a partner of the Microprocessor component Serrate (SE). CHR2 promotes the transcription of primary microRNA precursors (pri-miRNAs) while repressing miRNA accumulation in vivo. Direct interaction with SE is required for post-transcriptional inhibition of miRNA accumulation by CHR2 but not for its transcriptional activity. CHR2 can directly bind to and unwind pri-miRNAs and inhibit their processing, and this inhibition requires the remodelling and helicase activity of CHR2 in vitro and in vivo. Furthermore, the secondary structures of pri-miRNAs differed between wild-type Arabidopsis thaliana and chr2 mutants. We conclude that CHR2 accesses pri-miRNAs through SE and remodels their secondary structures, preventing downstream processing by DCL1 and HYL1. Our study uncovers pri-miRNAs as a substrate of CHR2, and an additional regulatory layer upstream of Microprocessor activity to control miRNA accumulation.
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Acknowledgements
We thank A. Jerzmanowski and Y. Cui for sharing CHR2 plasmids and chr2-1 alleles; A. Millar for PMIR159a-GUS and PMIR159b-GUS lines; P. He and P. Manavella for Y3H constructs; C. Kaplan and T. Devarenne for reading the manuscript; R. Hays for technical advice; and L. Zeng and F. Raushel for equipment and facility support. This work was supported by the NSF grant MCB-1716243 and CPRIT grant RP160822 to X.Z. D.S. and Y.L. were supported by the China Scholar Council Fellowship.
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Nature thanks K. P. Hopfner, Z. Szweykowska-Kulińska and P. Waterhouse for their contribution to the peer review of this work.
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Contributions
X.Z. conceptualized the study. X.Z., Z.W. and C.C.-G. designed experiments. X.Z. performed gel filtration chromatography and immunoprecipitation with mass spectrometry. Z.W. performed all biochemistry assays, genetic and molecular studies, RIP, in vivo pri-miRNA structure probing, and part of the analysis of high-throughput sequencing data. Z.M. performed subcellular localization assays and analysed high-throughput sequencing data. C.C.-G. performed protein alignment and provided intellectual input. D.S. performed LCI assay. Y.L. performed part of the Y2H and Y3H and genotyping assays. B.Y. provided antibodies and additional reagents. B.Z. and P.L. provided the baculovirus/insect expression system and intellectual input. X.Z. and Z.W. wrote the manuscript.
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Extended data figures and tables
Extended Data Fig. 1 CHR2 is a partner of the Microprocessor component SE and represses miRNA accumulation in Arabidopsis.
a, Size-exclusion chromatography of total protein extracts from Col-0; P35S- FM-SE transgenic plants shows the FM-SE enrichment in macromolecular complexes (>680 kDa). Western blot analysis was conducted with an anti-Flag antibody. b, CHR2 peptides recovered from proteomic analysis of the SE complex but not a control immunoprecipitation from Col-0 plants. SE complexes were isolated from Col-0; P35S-FM-SE transgenic plants by two-step affinity purification, and analysed by mass spectrometry. c, Confocal microscopic images show co-localization of SE–CFP and CHR2–YFP when co-expressed in Arabidopsis protoplasts. Top, SE–CFP and CHR2–YFP, individually expressed, serve as control. Scale bar, 5 μm. d, Luciferase complementary imaging (LCI) assay shows the specific CHR2–SE interaction in Nicotiana benthamiana. The infiltration scheme in the leaf shows different combinations of constructs fused to either N-terminal (nLuc) or C-terminal (cLuc) regions of luciferase. LCI complementation (LUC), bright field, and merged photograph (Merge) are shown. The red arrows and colour bar indicate the infiltration positions and the signal intensity, respectively. e, Schematic illustration of T-DNA insertions (reverse triangles) or deletion (triangle) in CHR2 and SE genes. f, Opposite leaf polarity phenotypes in chr2-1 and se loss-of-function mutants. Pictures of 28-day-old plants are shown. Scale bar, 1 cm. g, Additional replicate of sRNA blot analysis shows enhanced accumulation of miRNAs in the chr2-1 null mutant. h, Flower developmental defects in chr2-1. Scale bar, 0.5 mm. i, sRNA blot analysis shows that miRNAs accumulated in chr2-1 flowers. j, The chr2-1 morphological defect was fully rescued by the PCHR2-gCHR2-FM transgene. Scale bar, 1 cm. k, sRNA blot analysis shows that miRNA accumulation was restored to wild-type miRNA levels in the chr2-1; PCHR2-gCHR2-FM complementation lines (top). Western blot analysis of CHR2-FM using an anti-Myc antibody is shown (bottom). l, sRNA-seq revealed that the accumulation of 259 of 365 annotated miRNAs (http://www.mirbase.org) in Arabidopsis is SE-dependent. m, Overlap of SE-regulated and CHR2-repressed miRNAs in Arabidopsis.
Extended Data Fig. 2 CHR2 plays self-antagonistic roles in the regulation of miRNA accumulation at transcriptional and posttranscriptional levels.
a, Histochemical staining analysis of GUS activity shows comparable transcriptional levels of tested MIR genes in wild-type (WT, left, big plants) and chr2-1 (right, small plants) backgrounds. The promoters of individual MIR genes59,60,61 were fused to the FM-GUS coding sequence and transformed into a chr2-1+/− heterozygote line. Twenty-one-day-old T2 generation plants were used for GUS staining. The SAM and leaf primordia of PMIR164a- and PMIR164c -FM-GUS transgenic plants are magnified to comparable sizes. Scale bar, 1 cm. b, c, Western blot analysis shows that protein levels of FM-GUS from the native MIR promoters were higher in wild-type (+/+) than in chr2-1 (−/−) plants. Western blot assays were done with an anti-Flag antibody. Actin serves as a loading control. d, Schematic illustration of the conserved functional domains in CHR2 and the locations of the point mutations N1392A and R1417A in CHR2-1a (red arrows). e, Thin-layer chromatography (TLC) assay shows that the CHR2-1a variant has significantly reduced ATPase activity in vitro. ATPase activity was conducted with 500 ng dsDNA for 2 h. Bottom, SDS–PAGE of purified CHR2 and CHR2-1a proteins. f, Western blot analysis using an anti-Myc antibody shows comparable expression of CHR2 and CHR2-1a in the chr2-1 background. Actin serves as a loading control. g, chr2-1 morphological and miRNA accumulation defects, analysed by sRNA blot assay, were partially rescued in chr2-1a (chr2-1;PCHR2-gCHR2-1a-FM) plants. Scale bar, 1 cm. U6 serves as a loading control for sRNA blot analysis. In f, g, *shows the same samples as in Extended Data Fig. 10e. h, Morphological phenotypes of the hypomorphic chr2-1a mutant and its derived double mutants. Photographs of 21-days-old plants are shown. Scale bar, 1 cm. i, RT–qPCR analysis of pri-miRNAs in single and double mutants relative to Col-0. Total RNA was prepared from a pool of 21-day-old plants; EF-1α serves as a loading control. The relative signals of pri-miRNAs were normalized to those of wild-type Col-0, where the ratio was arbitrarily set to 1 with s.d. calculated from three biological repeats. Individual data points are shown. Data significantly different from the corresponding controls are indicated (chr2-1a versus Col-0, **P < 0.01; chr2-1a hyl1-2 versus hyl1-2, +P < 0.05, ++P < 0.01; unpaired, two-tailed Student’s t-test).
Extended Data Fig. 3 Delineation of the CHR2–SE interaction interface and identification of CHR2 E1747 as a critical residue for the specific CHR2–SE interaction by Y2H assays.
a, Rough mapping of CHR2 domains that interact with SE. b, Fine mapping of the CHR2–SE interaction interface and final identification of CHR2 E1747 as a critical residue for the CHR2–SE interaction. Note: part of the image related to CHR2 E1747A is also shown in Fig. 2a. AD, GAL4 activation domain; BD, GAL4 DNA binding domain. −LT, lacking Leu and Trp; −LTHA, lacking Leu, Trp, His and Ade. 1:5 serial dilutions are shown. c, The DESRM domain and E1747 residue of CHR2 that are involved in its interaction with SE are conserved through plants. Sequence alignment of DESRM domains of CHR2 proteins across different species (the region is rich in Asp-Glu-Ser residues, and therefore named the DES-rich motif (DESRM)). Red star indicates E1747, which is critical for the CHR2−SE interaction. Conserved neutral, acidic and basic amino acids are marked in black, blue, and red, respectively. d, Western blot analysis shows that expression levels of CHR2 variants were comparable in transformed yeast colonies. Western blot assays were conducted with anti-HA and anti-Myc antibodies to detect CHR2 and SE, respectively. Asterisks indicate non-specific bands. e, Summary of Y2H assays. Left, schematic illustration of CHR2 variants used for Y2H. The numbers indicate the positions of the amino acid residues in the constructs. Right, + and – denote positive and negative results for the CHR2−SE interaction from Y2H assays in a and b. f, Histochemical staining analysis of GUS activity from PMIR159a-FM-GUS homozygotes show that CHR2(E1747A) does not alter the transcriptional level of MIR159a in Col-0 and chr2-1;PCHR2-gCHR2(E1747A)-FM backgrounds. Scale bar, 1 cm.
Extended Data Fig. 4 CHR2 does not affect the expression of canonical components involved in miRNA biogenesis and metabolism, nor the SE- dependent pre-mRNA splicing pathway.
a, Gene Ontology analysis of differentially expressed genes in chr2-1 plants. The dataset is mined from a previously published RNA-seq database55 for the chr2 mutant. b, Re-analysis of previously published microarray data sets for chr2 mutants56,57,58 to assess the expression of numerous canonical genes involved in the miRNA pathways. c, d, RT–PCR (c) and RT−qPCR (d) show that the expression levels of the genes involved in the miRNA pathway are generally comparable between Col-0 and the chr2-1 mutant. At1g22690 and At5g27845 serve as controls for down- and upregulated genes in chr2-1. Data significantly different from Col-0 controls are indicated (* P < 0.05, ** P < 0.01; unpaired, two-tailed Student’s t-test). In b, d, the relative signals of genes were normalized to those of wild-type, where the ratio was arbitrarily set to 1 with s.d. calculated from published data (b) or three biological repeats (d). Individual data points are shown. e, Western blot analysis shows that chr2-1 does not show enhanced accumulation of Microprocessor proteins in vivo. Western blot assays were conducted using antibodies against native DCL1 and HYL1 proteins; an anti-Myc antibody was used to detect the native promoter-driven FM−SE protein. Actin serves as a control. f, g, Re-analysis of previously published RNA-seq data sets55,62,63 for chr2-1/brm-1 and se mutants shows that there is no significant overlap of abnormal intron retention events (f) and abnormal alternative splicing between se and chr2-1 mutants (g). h, RT–PCR analysis of selected marker genes shows that CHR2 is not involved in the SE-coordinated pre-mRNA splicing pathway. gDNA (genomic DNA) serves as a control.
Extended Data Fig. 5 In vitro Microprocessor assays show that CHR2 inhibits pri-miRNA processing.
a, SDS–PAGE of recombinant proteins purified from a Baculovirus/insect system or from E. coli. b, The in vitro-reconstituted Microprocessor processed pri-miRNA substrates as accurately and efficiently as DCL1 complex immunoprecipitated from plants in vivo. c, Pre-incubation of CHR2 with pri-miRNAs inhibited the miRNA processing in vitro. hSTING serves as a negative control. On the left, schematic illustration of in vitro reconstitution orders with recombinant CHR2, 5′ 32P-labelled pri-miR166f transcript and Microprocessor components. RT, room temperature. On the right, autoradiography of in vitro Microprocessor assays (two replicates). Black arrows indicate processed and unprocessed fragments of pri-miR166f. d, CHR2 inhibition of Microprocessor activity was positively correlated with its amount applied. Molar ratio CHR2:DCL1 is shown at the top of the autoradiograph. The reconstitution assay was conducted as in Fig. 3a. e, CHR2 inhibition of pri-miRNA processing was positively correlated with the pre-incubation time of CHR2 with pri-miRNA. The pre-incubation time of CHR2 with pri-miR166f is shown at the top of the autoradiograph. The reconstitution assay was conducted as in Fig. 3a. f, Incubation of HYL1−SE with pri-miRNAs before addition of CHR2 largely bypassed the inhibitory effect of CHR2 on pri-miRNA processing in vitro. Left, schematic representation of the in vitro reconstitution assay. Right, autoradiography of in vitro Microprocessor assays (two replicates). g, Incubation of CHR2 variant compromised in SE interaction (CHR2(E1747A)) with pri-miRNA substantially reduced pri-miRNA processing in vitro. Left, schematic representation of the in vitro reconstitution assay. Right, autoradiography of in vitro Microprocessor assays (two replicates). h, CHR2(E1747A) had only a marginal inhibitory effect on pri-miRNA processing when the substrate pri-miRNA was pre-incubated with excessive SE. Molar ratio of SE:CHR2 (or its variant) was 4:1. Left, schematic representation of in vitro reconstitution assay. Right, autoradiography of in vitro Microprocessor assays (three replicates).
Extended Data Fig. 6 Three non-exclusive hypotheses for the mechanism by which CHR2 inhibits miRNA production; and CHR2 and DCL1−HYL1 interact with different domains of SE.
a, Schematic illustration of three non-exclusive hypotheses for the mechanism by which CHR2 inhibits miRNA processing. b, Summarized Y2H results for mapping of the DCL1−SE and CHR2−SE interaction interfaces in SE protein (detailed results are shown in Extended Data Fig 7a, b). Left, schematic illustration of the truncation and deletion variants of SE and DCL1 used for Y2H. The numbers indicate the positions of the amino acid residues in the constructs. Right, + and – denote positive and negative Y2H results for the interaction with SE, respectively. c, Proposed models of the protein interactions between DCL1−HYL1 and SE and between CHR2 and SE. DCL1 occupies the zinc finger domain (ZnF) of SE through its PAZ domain; dsRNA-binding domain 2 (RBD2) in HYL1 interacts with the N-terminal and mid domains (N+Mid) of the SE core19. The DESRM motif in CHR2 contacts the GAPE domain (named for enriched Gly-Ala-Pro-Glu residues) in the unstructured C terminus of SE. Numbers show the amino acid residues comprising the indicated domains. The red, blue and cyan arrows indicate the interaction domains of the indicated proteins. d, RT–PCR shows that all genes are expressed well in the Y3H system. All transfected yeast cells were cultured in −Leu, −Trp and −Met synthetic defined broth. ScSED1 serves as a control. e, f, Y3H assays show that CHR2 does not interfere with the accessibility of DCL1−HYL1 (e) or that of U1 snRNP component (Prp40b)64,65 (f) to SE protein. 1:10 serial dilutions are shown. −LTM, lacking Leu, Trp and Met; −LTMH+5mM 3-AT, lacking Leu, Trp, Met, His and adding 5 mM 3-AT.
Extended Data Fig. 7 Mapping the regions of SE that interact with DCL1 and CHR2 by Y2H; and the GAPE domain of SE that interacts with CHR2 is conserved through plants.
a, b, Detailed Y2H results of mapping the DCL1–SE interaction interface (a) and the interaction region of SE with CHR2 (b); 1:5 serial dilutions are shown. −LT, lacking Leu and Trp; −LTHA, lacking Leu, Trp, His and Ade. c, Sequence alignment of GAPE domains of SE proteins across different species; conserved neutral, acidic and basic amino acids are marked by black, blue, and red, respectively.
Extended Data Fig. 8 CHR2 binds to nucleic acids in vitro and in vivo.
a, EMSA shows CHR2 bound strongly to 5′ end 32P-labelled pre-miRNA but weakly to ssRNA. Arrows indicate the mobility of protein−RNA complexes or labelled free RNA. b, c, EMSA shows the mobility pattern of the CHR2−pri-miRNA (b) and CHR2−pre-miRNA complexes (c). HYL1 and hSTING are positive and negative controls, respectively. Cold probes, used for the binding competition, are unlabelled pri-miRNA and pre-miRNAs, accordingly. d, e, EMSA shows that CHR2 bound to dsDNA but not ssDNA. Arrows indicate the mobility of protein–DNA complexes or free DNA. f, The binding affinity (apparent Kd) of CHR2-dsDNA was calculated from EMSA image quantification with s.d. from three replicates. Individual data points are shown. g, EMSA shows the mobility pattern of SE–pri-miRNA complex. h, EMSA shows the mobility pattern of HYL1-pri-miRNA complex. i, EMSA shows that HYL1 readily sequestered pri-miRNA from SE, CHR2 or CHR2−SE complexes. j, Western blot analysis of CHR2−FM protein immunoprecipitated from hyl1-2 chr2-1;PCHR2-gCHR2-FM plants using an anti-Myc antibody. Histone 3 serves as a control for input. k, RT−qPCR shows that none or faint signals were detected in the RNase-treated RIP samples. The result indicated that the nucleic acids recovered from the CHR2 immunoprecipitate shown in Fig. 4d were indeed RNAs. The relative signals of pri-miRNAs were normalized to the ones of the input initially and then to the control immunoprecipitate without RNase treatment, where the ratio was arbitrarily set to 1 with s.d. from three biological repeats. Individual data points are shown. ND, not detected.
Extended Data Fig. 9 Effect of CHR2 mutations on remodelling/helicase and ATPase activities.
a, SDS–PAGE of purified CHR2 variants. b, Schematic diagram of preparation of the 32P-labelled and nicked pri-miRNA. c, Remodelling/helicase activity of CHR2 and CHR2(D1355A/R1385A) on nicked pri-miRNAs. Note: D1355A R1385A substitutions in the nucleotide binding domains compromised the remodelling activity of CHR2 on pri-miRNA. HYL1 served as a negative control. d, Time-course of remodelling/helicase activity of CHR2 and its variants on 32P-labelled pri-miRNA. e, Remodelling/helicase activity of CHR2 on 32P-labelled dsDNA. f, Time-course of remodelling/helicase activity of CHR2 and its variants on 32P-labelled dsDNA. g, TLC assays showed that dsDNA and pri-miRNA stimulated ATPase activity of CHR2 and its ATPase mutant (G1009A/K1012A) in a dosage-dependent manner. h, TLC assays show the time-course of ATPase activity of CHR2 and its ATPase mutant (G1009A/K1012A) in the absence (none) or presence of dsDNA (167 bp in length) and pri-miRNA (150 nt in length with ~50 base-pairing). i, Quantification of ATP hydrolysis rates of CHR2 and its ATPase mutant (G1009A/K1012A) in the absence (none) or presence of dsDNA and pri-miRNA with s.d. from three replicates. Individual data points are shown.
Extended Data Fig. 10 Effect of CHR2 mutations on binding affinity to nucleic acids, Microprocessor cleavage efficiency of pri-miRNA, and rescue of morphological and miRNA accumulation defects in chr2-1 plants.
a, Binding curves of CHR2 variants with dsDNA and pri-miRNA. Apparent Kd values (appKd) were calculated from EMSA image quantification with s.d. from three replicates. b, Microprocessor assays shows that the remodelling-compromised CHR2 mutant (G1009A/K1012A) failed to efficiently inhibit pri-miRNA processing. Left, schematic illustration of in vitro reconstitution orders with recombinant CHR2 or its variant, 5′ 32P-labelled pri-miR166f transcript and Microprocessor components. RT, room temperature. Middle and right panels, autoradiography of the in vitro Microprocessor assays (two replicates). Black arrows indicate processed and unprocessed fragments of pri-miR166f. c, Diagram of point mutations in the CHR2 ATP and nucleotide binding domains. d, Remodelling-compromised CHR2 mutants partially rescued the morphological defect of chr2-1 plants. Scale bar, 1 cm. e, Remodelling-compromised CHR2 mutants partially rescued chr2-1 miRNA accumulation defects. sRNA blot analysis (top) and western blot assays of CHR2 and variants (bottom). f, Western blot analysis of GUS protein in PMIR159b-FM-GUS homozygotes (n > 30) in different backgrounds shows that CHR2(G1009A/K1012A) decreased MIR159b transcription.
Extended Data Fig. 11 CHR2 remodels the folding of pri-miRNAs or pri-miRNA complexes in vivo in various ways.
a, Schematic diagram of two DMS-based methods for probing the secondary structures of RNA in vivo. SSIV, reverse transcriptase SuperScript IV (Thermo Fisher). b, Schematic structure of pri-miRNA. c, A primer-extension assay of DMS−cDNA truncations of 18S rRNA validated the reliability of the approach of the DMS−primer extension in the experiment. d, An additional replicate of primer-extension assay for DMS−cDNA truncations shows that CHR2 altered the folding of the miRNA/* duplex and upper stem region of pri-miR164b. Red arrows, nucleotides that had stronger DMS activities and thus more mispairings in pri-miR164b from hyl1-2 chr2-1;PCHR2-gCHR2-FM plants than those from hyl1-2 chr2-1a. e, DMS−MaPseq analysis shows that DMS mutation profiling of UBQ4 transcripts was essentially identical in Col-0 and chr2-1 plants. The DMS mutation frequencies (mismatch/total) of A and C residues are plotted along the nucleotide sequences probed. Background mutations in the samples that were not treated with DMS are shown (top). f, Background mutations in pri-miR166a in Col-0 and chr2-1 samples that were not treated with DMS are shown. DMS−MaPseq results of other tested pri-miRNAs including pri-miR159a, pri-miR159b, pri-miR160a, pri-miR166b, pri-miR168a, pri-miR168b, and pri-miR319b in the untreated samples were largely identical to that of pri-miR166a, and are not shown here. g, h, DMS–MaPseq analysis of additional pri-miRNAs illustrates that CHR2 can alter nucleotide pairings and/or protein protection in terminal loops and upper stems of pri-miR166a (g) and pri-miR168a (h). Top, DMS mutation frequencies of A and C residues (mismatch/total) from targeted pri-miRNAs averaged from two biological replicates are plotted along pri-miRNA sequence. Position 1 corresponds to the 5′ end of the modelled regions. Bottom, overall and zoomed secondary structures of the targeted pri-miRNAs are predicted from DMS activities. Colour-coded nucleotides display different DMS activity on pri-miRNAs between Col and chr2-1 plants. The miRNA and * strands are marked in purple and cyan, respectively.
Extended Data Fig. 12 Additional evidence for the CHR2 function in remodelling the folding of pri-miRNAs or pri-miRNA complexes in vivo in various ways.
a–d, DMS–MaPseq analysis of additional pri-miRNAs illustrates that CHR2 can alter nucleotide pairings and/or protein protection in the lower stem of pri-miR168b (a), the terminal loop and upper stem of pri-miR319b (b), and variable parts of pri-miR160a (c) and pri-miR166b (d). Top, DMS mutation frequencies of A and C residues (mismatch/total) from targeted pri-miRNAs averaged from two biological replicates are plotted along pri-miRNA sequence. Position 1 corresponds to the 5′ end of the modelled regions. Bottom, the overall and zoomed secondary structures of the targeted pri-miRNAs are predicted from DMS activities. Colour-coded nucleotides display different DMS activity on pri-miRNAs between Col and chr2-1 plants. The miRNA and * strands are marked in purple and cyan, respectively.
Supplementary information
Supplementary Information
This file contains the Supplementary Results and Discussion and Supplementary Figure 1
Supplementary Table 1
List of miRNAs regulated in chr2-1 and se-2 compared to Col
Supplementary Table 2
List of MIRs bound by CHR2
Supplementary Table 3
Alternative splicing in chr2-1 mutant
Supplementary Table 4
A list of primers used in the study
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Wang, Z., Ma, Z., Castillo-González, C. et al. SWI2/SNF2 ATPase CHR2 remodels pri-miRNAs via Serrate to impede miRNA production. Nature 557, 516–521 (2018). https://doi.org/10.1038/s41586-018-0135-x
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DOI: https://doi.org/10.1038/s41586-018-0135-x
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