Mbf1 ensures Polycomb silencing by protecting E(z) mRNA from degradation by Pacman

Abstract

Under stress conditions, the coactivator Multiprotein bridging factor 1 (Mbf1) translocates from the cytoplasm into the nucleus to induce stress-response genes. However, its role in the cytoplasm, where it is mainly located, has remained elusive. Here, we show that Drosophila Mbf1 associates with E(z) mRNA and protects it from degradation by the exoribonuclease Pacman (Pcm), thereby ensuring Polycomb silencing. In genetic studies, loss of mbf1 function enhanced a Polycomb phenotype in Polycomb group mutants, and was accompanied by a significant reduction in E(z) mRNA expression. Furthermore, a pcm mutation suppressed the Polycomb phenotype and restored the expression level of E(z) mRNA, while pcm overexpression exhibited the Polycomb phenotype in the mbf1 mutant but not in the wild-type background. In vitro, Mbf1 protected E(z) RNA from Pcm activity. Our results suggest that Mbf1 buffers fluctuations in Pcm activity to maintain an E(z) mRNA expression level sufficient for Polycomb silencing.

Keywords: Drosophila melanogaster; E(z); Enhancer of zeste; Exoribonuclease; Mbf1; Pcm; Polycomb silencing.

Fig. 1.

Compromised E(z) expression in the mbf1 mutant in a Polycomb group mutant background. (A) Genetic interactions between mbf1 and Polycomb group mutants. The P-element vector P{mbf1⁺} expresses wild-type Mbf1 from a transgene. *P<0.01 (Fisher's exact test). (B) RT-qPCR analysis of the indicated Polycomb group mRNAs in whole extracts from third instar male larvae. Data are mean±s.d., relative to the wild-type mRNA level; *P<0.01 (Student's t-test). (C) Immunofluorescence analyses of indicated Polycomb group proteins in wing discs of third instar larvae. (D) Western blot analyses of E(z) in wing or leg discs. Numbers indicate relative E(z) levels normalized to those of Spt16. (E) RT-qPCR analysis of E(z) mRNA in the nuclear or cytoplasmic fraction of wing discs. NS, not significant; *P<0.01 (Student's t-test). (F) Mbf1 binds to E(z) mRNA. RIP samples from wild-type or mbf1² embryonic extracts were analyzed by RT-qPCR. Data are mean±s.d. of fold-change versus control IgG; *P<0.01 (Student's t-test).

Fig. 2.

Functional relationship among mbf1, E(z) and pcm. (A) pcm is downregulated by Psc and Pc. Expression of the indicated genes in third instar male larvae was analyzed by RT-qPCR in the wild-type or Polycomb group mutant background. pcm does not appear to be a direct target of Polycomb silencing (Zeng et al., 2012). Data are mean±s.d. relative to the wild-type mRNA level; *P<0.01 (Student's t-test). (B) Western blot analysis of Pcm in wing discs from the indicated lines. Numbers indicate relative Pcm levels normalized to Tubulin levels. (C) pcm mutation suppresses the extra sex comb phenotype. *P<0.01 (Fisher's exact test). (D) pcm mutation restores the E(z) mRNA level in Psc¹/+ or Psc¹/+; mbf1²/+. The E(z) mRNA levels in third instar male larvae of the indicated lines were analyzed by RT-qPCR. Data are mean±s.d. relative to the wild-type mRNA level. NS, not significant; *P<0.01 (Student's t-test). (E) (Top) Misexpression of Ubx in the wing disc of Psc¹/+; mbf1²/+ and its suppression by pcm^Δ1. Arrows indicate Ubx-positive spots. (Bottom) Immunostaining of E(z) protein in the wing discs shown above. (Right) Adult wing defect (arrowheads) in Psc¹/+; mbf1²/+ and its suppression by pcm^Δ1. The number of wings with the defect among the total number of wings examined is indicated. **P<0.05 (Fisher's exact test).

Fig. 3.

Mbf1 protein directly counteracts 5′-3′ exoribonuclease activity in vivo and in vitro. (A) mbf1²/hs-pcm double heterozygotes exhibit the Polycomb phenotype in the wild-type Polycomb group background. *P<0.01. (B) Recombinant Drosophila Mbf1 and Pcm preparations were resolved by 5-20% SDS-PAGE and the gel stained with Coomassie Brilliant Blue. (C) Recombinant Drosophila Mbf1 inhibits Pcm activity in vitro. In vitro-transcribed E(z) RNA [IVTed E(z) RNA] was used as substrate for Pcm. Reactions included the indicated components, and purified RNAs were resolved on a 1.5% agarose gel. dsDNA marker size is indicated (bp). Amounts of Mbf1 added: +, 2.5 µg; ++, 5 µg; ++++, 10 µg.

Fig. 4.

Conceivable functions of cytoplasmic Mbf1 protein via binding to mRNAs. (A) Model for Mbf1-ensured Polycomb silencing. In wild-type and mbf1 mutant lines, Pcm is not upregulated. Therefore, the steady-state level of E(z) mRNA is well balanced irrespective of Mbf1 expression. In Polycomb group mutants, Pcm expression is upregulated so that E(z) mRNA could become susceptible to Pcm attack. However, Mbf1 protects E(z) mRNA to ensure robustness of Polycomb silencing. In mbf1 and Polycomb group double mutants, loss of Mbf1 allows extensive degradation of E(z) mRNA by derepressed Pcm, thereby affecting Polycomb silencing. (B) The enrichment of four representative mRNAs (GstD5, Ide, Tep2 and Pebp1) identified in the RIP-seq results was confirmed by RIP RT-qPCR analysis. Results for E(z) and RpL30 mRNAs from Fig. 1F are included for comparison. Data are mean±s.d. of fold-change versus control IgG; *P<0.01. (C,D) RT-qPCR analysis of the indicated mRNAs in whole extracts of third instar male larvae from Psc¹/+; mbf1²/+ (C) or pcm^Δ1/Y (D). Data are mean±s.d. relative to the wild-type mRNA level; *P<0.01. All P-values obtained using Student's t-test. (E) Gene ontology analysis of the RNA-seq results is consistent with the known Mbf1 functions. The number of genes in each term is indicated.