The role of Mediator in small and long noncoding RNA production in Arabidopsis thaliana

Abstract

Mediator is a conserved multi-subunit complex known to promote the transcription of protein-coding genes by RNA polymerase II (Pol II) in eukaryotes. It has been increasingly realized that Pol II transcribes a large number of intergenic loci to generate noncoding RNAs, but the role of Mediator in Pol II-mediated noncoding RNA production has been largely unexplored. The role of Mediator in noncoding RNA production in plants is particularly intriguing given that plants have evolved from Pol II two additional polymerases, Pol IV and Pol V, to specialize in noncoding RNA production and transcriptional gene silencing at heterochromatic loci. Here, we show that Mediator is required for microRNA (miRNA) biogenesis by recruiting Pol II to promoters of miRNA genes. We also show that several well-characterized heterochromatic loci are de-repressed in Mediator mutants and that Mediator promotes Pol II-mediated production of long noncoding scaffold RNAs, which serve to recruit Pol V to these loci. This study expands the function of Mediator to include Pol II-mediated intergenic transcription and implicates a role of Mediator in genome stability.

Figure 1

Isolation and characterization of mutants in Mediator genes. (A) Four-week-old plants of wild type (Col), med20a, med17, med18, and nrpb2-3. (B) Siliques from Col, med20a, med17, and med18 plants. (C) Schematic diagrams of MED20 paralogs MED20a, MED20b, and MED20c. The asterisk indicates the mutation causing a premature stop codon in the med20a mutant. Black rectangles represent exons and lines represent introns. MED20a and MED20b are composed of three exons showing 85% identity at the nucleotide level. MED20c only encodes the conserved second exon compared with MED20a and MED20b. (D) Schematic diagrams of T-DNA insertion mutants of MED17 and MED18. Large triangles represent T-DNA insertions. Black rectangles represent exons. (E) RT–PCR analysis of MED20a, MED17, and MED18 expression in med20a, med17, and med18 mutants, respectively. The images in the top row represent the indicated Mediator genes. UBIQUITIN5 (UBQ5) was used as an internal loading control. The ‘−RT' reactions were performed with the UBQ5 primers. The PCR primers used are indicated by small arrowheads in (C, D).

Figure 2

The accumulation of miRNAs and a ta-siRNA in nrpb2-3 and Mediator mutants as determined by northern blotting. (A) The accumulation of five miRNAs and a ta-siRNA (siR1511) in wild type (Col) and nrpb2-3. (B) The accumulation of 11 miRNAs and one ta-siRNA (siR1511) in Col and med20a. (C) The accumulation of four miRNAs in Col, med17, and med18. Total RNAs were extracted from inflorescences. The levels of each small RNA were normalized to those of U6 and compared with Col. The numbers below the gel images indicate the relative abundance of the small RNAs. Two to three biological replicates were performed for all small RNAs except for miR390 and miR159 and yielded similar results.

Figure 3

med20a affects the transcription of MIR genes. (A) The accumulation of six pri-miRNAs was determined by real-time RT–PCR in Col and the med20a mutant. Total RNAs were extracted from inflorescences. The pri-miRNA levels were normalized to those of UBQ5 and compared with Col. Standard deviations were calculated from three technical replicates. Three biological replicates yielded similar results. (B) GUS expression driven by the MIR167a promoter was monitored in isogenic Col and med20a transgenic lines through GUS staining. GUS expression in old flowers was reduced in med20a compared with Col (arrows). (C) The accumulation of GUS mRNA from the lines in (B) was determined by real-time RT–PCR. The GUS mRNA levels were normalized to those of UBQ5 and compared with Col. Standard deviations were calculated from three technical replicates. Two biological replicates yielded nearly identical results.

Figure 4

Pol II occupancy at miRNA and siRNA loci. Pol II occupancy at several miRNA and siRNA loci was determined by ChIP using anti-RPB2 antibodies in Col and med20a. DNA present in the immunoprecipitates was quantified by real-time PCR relative to total input DNA. (A) ChIP performed with no antibodies as negative controls. (B) ChIP with anti-RPB2 antibodies. The results were reproduced in two biological replicates. Standard deviations were calculated from three technical repeats. AtSN1_A and soloLTR_A, regions A of these two loci as depicted in Figure 5B. AtSN1_B and soloLTR_B, regions B of these two loci as in Figure 5B.

Figure 5

Mediator in TGS of repeats and transposons. (A) The accumulation of endogenous siRNAs from heterochromatic loci. Small RNA northern blotting was performed with total RNAs extracted from inflorescences from Col and med20a. Small RNA accumulation was not affected in the med20a mutant. The U6 blots served as loading controls for the overlying small RNA blots. (B) Diagrams of AtSN1, soloLTR, and siR02 genomic regions. These regions are based on analysis of transcription units by Wierzbicki et al (2008) and Zheng et al (2009). The ‘A' regions are where the siRNAs are derived, while the ‘B' regions are where scaffold transcripts are produced. (C) RT–PCR analysis of noncoding transcripts from regions A and B at siRNA loci in Col and Mediator mutants. UBQ5 was used as an internal control. The RT (−) control PCR was performed with UBQ5 primers. nrpe1-11 is a loss-of-function allele in the largest subunit of Pol V. The results shown were reproduced in three biological replicates. (D) Pol V occupancy at regions B of AtSN1 and soloLTR was determined by ChIP using anti-FLAG antibodies in pNRPE∷NRPE1-FLAG and pNRPE∷NRPE1-FLAG med20a. DNA that co-purified with Pol V was measured by real-time PCR against total input DNA. eIF4A1, a gene not bound by Pol V, served as a negative control. Standard deviations were calculated from three technical repeats. The results were reproduced in two other biological replicates shown in Supplementary Figure S8.