Cryptic RNA-binding by PRC2 components EZH2 and SUZ12

<div><p>Polycomb repressive complex 2 (PRC2) is a histone-modifying complex that di/tri-methylates histone H3 lysine 27 (H3K27), a mark of transcriptionally repressed chromatin. However, how PRC2 is specifically recruited to its target loci remains controversial, although it has been postulated that long non-coding RNAs (lncRNAs) can function as guides. Here we purified individual components of PRC2 from human cultured cells and found that EZH2 and SUZ12 can directly bind to RNAs. In agreement with recent evidence, our results support the notion that these two PRC2 subunits have RNA-binding activity, with general preference for longer RNAs. However, the length alone does not explain their cryptic substrate preference. Our data highlight the difficulty of characterizing the RNA-binding activity of PRC2.</p></div

List of primers.

PIWI-interacting RNAs (piRNAs) guide PIWI proteins to target transposons in germline cells, thereby suppressing transposon activity to preserve genome integrity in metazoans’ gonadal tissues. Piwi, one of three Drosophila PIWI proteins, is expressed in the nucleus and suppresses transposon activity by forming heterochromatin in an RNA cleavage-independent manner. Recently, Piwi was reported to control cell metabolism in Drosophila fat body, providing an example of piRNAs acting in non-gonadal somatic tissues. However, mutant flies of the other two PIWI proteins, Aubergine (Aub) and Argonaute3 (Ago3), show no apparent phenotype except for infertility, blurring the importance of the piRNA pathway in non-gonadal somatic tissues. The silkworm, Bombyx mori, possesses two PIWI proteins, Siwi (Aub homolog) and BmAgo3 (Ago3 homolog), whereas B. mori does not have a Piwi homolog. Siwi and BmAgo3 are mainly expressed in gonadal tissues and play a role in repressing transposon activity by cleaving transposon RNA in the cytoplasm. Here, we generated Siwi and BmAgo3 loss-of-function mutants of B. mori and found that they both showed delayed larval growth and failed to become adult moths. They also exhibited defects in wing development and sexual differentiation. Transcriptome analysis revealed that loss of somatic piRNA biogenesis pathways results in abnormal expression of not only transposons but also host genes, presumably causing severe growth defects. Our results highlight the roles of non-gonadal somatic piRNAs in B. mori development.</div

GOs enriched in tissue-specific genes and corresponding expression changes.

GOs enriched in tissue-specific genes and corresponding expression changes.</p

Phenotypic characterization of <i>BmAgo3</i> and <i>Siwi</i> KO mutants.

(A) Hatching rate in crosses between BmAgo3 or Siwi heterozygous KO mutants. To obtain homozygous KO mutants, heterozygous male and female KO individuals were crossed. The average percentages of hatched, unhatched, and unfertilized eggs from 3 (WT and BmAgo3) or 4 (Siwi) batches are indicated by white, black, and gray bars, respectively. The crossing patterns are indicated in the figure. (B) Developmental delay in BmAgo3 and Siwi KO mutant larvae. Third instar larvae obtained from the parents carrying heterozygous mutations were photographed. Developmentally delayed larvae are indicated by arrowheads. Photographs of all larvae are shown in S2 Fig. (C) Detection of PIWI proteins. Whole cell extracts from the whole body of a day 0 third instar (III0) larva were separated by SDS-PAGE and immunoblotted with anti-BmAgo3, Siwi, and Tubulin antibodies. Developmentally delayed individuals were used as BmAgo3 and Siwi KO. (D) Survival curves of larvae obtained by crosses between BmAgo3 or Siwi heterozygous KO mutants. One hundred newly hatched larvae were reared using an artificial diet and the number of dead larvae was counted every day. (E) Timing of larval wandering in BmAgo3 and Siwi KO mutants. The larval wandering behavior that precedes pupation was delayed in BmAgo3 homozygous mutants by a few days. Pupae of Siwi homozygous mutants were not obtained in this experiment. The number indicates the sample size. (F) Phenotypes of BmAgo3 and Siwi KO mutant pupae. Impaired head and leg morphologies in both homozygous mutants. Female pupae of Siwi KO mutants were not obtained. Scale bars, 2 mm. (G) Abnormalities in internal tissues in BmAgo3 and Siwi KO mutant larvae. Ovaries (OV), testes (TES), and wing discs (WD) were dissected from day 4 fifth instar larvae. The positions connected to the duct are indicated by arrowheads. The margin of the wing disc is indicated by a dotted curve. Scale bars, 1 mm. (H) Developmental defect of gonads in BmAgo3 KO mutant pupae. Day 4 female pupae were dissected and the inside of the abdomen was observed under a microscope. No ovarian eggs were found in BmAgo3 KO mutant pupae. Testes were removed from day 4 male pupae and observed in phosphate-buffered saline under a microscope. Scale bars, 1 mm.</p

<i>β-glucosidase</i> genes commonly down-regulated in <i>BmAgo3</i> and <i>Siwi</i> KO.

(A) Clustering of B. mori β-glucosidase gene expression patterns. Numerous β-glucosidase genes were commonly down-regulated in BmAgo3 and Siwi KO. TPMs of B. mori β-glucosidase genes were normalized by Z-score and clustered using Heatplus R package. F: female, M: Male. (B) Alignment of amino acid sequences of B. mori β-glucosidases. (TIF)</p

Detailed phenotypes of <i>BmAgo3</i> and <i>Siwi</i> KO mutant pupae.

(A) Pupal weight of BmAgo3 and Siwi KO mutants. No female pupae were obtained from the Siwi KO mutant. Bars indicate means ± SE. The number indicates the sample size. Asterisks indicate statistical significance in Mann-Whitney test (p BmAgo3 and Siwi KO mutant pupae. Scale bars, 2 mm. (TIF)</p

All delayed larvae possessed homozygous KO alleles as shown by genotyping.

All delayed larvae possessed homozygous KO alleles as shown by genotyping.</p

CRISPR/Cas9-mediated <i>BmAgo3</i> and <i>Siwi</i> knockout.

(A) BmAgo3 sequences surrounding the sgRNA target site and predicted protein structures. The target sgRNA site is underlined, and the proto-spacer adjacent motif (PAM) highlighted by a gray box. The cleavage site is shown by an arrowhead. The deleted (−) and inserted (small letters) sequences are shown near the cleavage site. The rounded rectangles indicate the location of protein domains corresponding to Siwi proteins [91]. (B) Siwi sequences surrounding the sgRNA target site and predicted protein structures. The target sgRNA site is underlined, and the PAM highlighted by a gray box. The cleavage site is shown by an arrowhead. The deleted (−) sequences are shown near the cleavage site. The rounded rectangles indicate the location of protein domains. The PAZ domain in the Siwi KO mutant is slightly truncated (gray rounded rectangles). (TIF)</p

<i>CYP</i> genes commonly down-regulated in <i>BmAgo3</i> and <i>Siwi</i> KO.

Clustering of expression variation patterns of B. mori CYP genes in mRNA libraries in the whole body of third instar larvae. TPMs of B. mori CYP genes were normalized by Z-score and clustered using Heatplus R package. F: female, M: Male. (TIF)</p

Expression changes of tissue-specific genes in <i>BmAgo3</i> and <i>Siwi</i> KO larvae.

(A) Genes specifically expressed in the ovaries (OV), fat body (FB), and wing discs (WD) in fifth instar larvae were investigated for increased or decreased (1 2(Fold Change)) expression in BmAgo3 KO. Each tissue-specific gene was defined as that whose TPM in the WT library of one tissue was more than twice as large as that in the libraries of the other two tissues. (B) Violin plots of tissue-specific gene expression patterns in the whole body of third instar larvae. (TIF)</p