Fig. 4. MHZ9-N-terminal domain binds to the 3’UTR of OsEBF1/2 mRNA.

a The RNA bases identified from purified MHZ9-N peptides are indicated in dark blue. The RNA-bound peptides are highlighted in red. b MHZ9-binding genes and binding sites detected through RNA-IP (RIP)-seq and crosslinking and immunoprecipitation followed by sequencing (CLIP-seq). Then the 1186 MHZ9-binding sites identified from the overlapped 626 genes (left) were analyzed to investigate their distribution patterns (right). c Schematic representation of the MHZ9-binding sites on OsEBF2 mRNA from RIP-seq and CLIP-seq. d Binding test of OsEBF2 mRNA UTRs by various MHZ9 truncations through RIP-qPCR assay. The protein expression levels were also examined. e Binding tests of MHZ9 to OsEBF2 mRNA 3’UTR through MS2-based BiFC assay. 12 × MS2 binding sites (12 × MBS) fused with or without OsEBF2 mRNA 3’UTR was transiently expressed with MS2-cYFP and MHZ9-nYFP in tobacco leaves and treated with 10 μL/L of ethylene or air for 16 h at 2 days post infiltration. Scale bars, 20 µm. Values are means ± SD (n = 12 biologically independent samples). f OsEIN2 effect on the binding of MHZ9 to OsEBF2 mRNA 3’UTR through RIP-qPCR. g MHZ9 effect on the association between OsEIN2 and OsEBF2 mRNA 3’UTR through RIP-qPCR. d, f, g Values are means ± SD (n = 3 biologically independent samples). The asterisks indicate significant differences compared with the corresponding control (**P < 0.01; two-tailed Student’s t-test). h MHZ9-N binds to the 3’UTR of OsEBF1/2 mRNA directly in an RNA-EMSA assay. Arrows and braces indicate shifted protein/RNA complex bands. i Roles of MHZ9 in co-localization of OsEBF2 mRNA 3’UTR and OsEIN5-RFP in P-body. 12 × MS2 binding sites (12 × MBS) fused with or without OsEBF2 mRNA 3′UTR was transiently expressed with MS2-GFP and OsEIN5-RFP in rice protoplasts of WT or mhz9 mutant. Scale bars, 2 µm. Each experiment was repeated at least three times with similar results. Source data are provided as a Source Data file.