Despite a much higher proportion of intragenic heterochromatin-containing genes in crop genomes, the importance of intragenic heterochromatin in crop development remains unclear.Intragenic heterochromatin can be recognized by a protein complex, ASI1-AIPP1-EDM2 (AAE) complex, to regulate alternative polyadenylation.• Here, we investigated the impact of rice ASI1 on global poly(A) site usage through poly(A) sequencing and ASI1-dependent regulation on rice development.• We found that OsASI1 is essential for rice pollen development and flowering. OsASI1 dysfunction has an important impact on global poly(A) site usage, which is closely related to heterochromatin marks. Intriguingly, OsASI1 interacts with the intronic heterochromatin of OsXRNL, a nuclear XRN family exonuclease gene involved in the processing of miRNA precursor, to promote the processing of full-length OsXRNL and regulates miRNA abundance. Accepted ArticleThis article is protected by copyright. All rights reservedWe found that OsASI1-mediated regulation of pollen development partially depends on OsXRNL. Finally, we characterized rice AAE complex and its involvement in alternative polyadenylation and pollen development.• Our findings help to elucidate an epigenetic mechanism governing miRNA abundance and rice development, and provide a valuable resource for studying the epigenetic mechanisms of many important processes in crops.

Dimethylated histone H3 Lys9 (H3K9me2) is a conserved heterochromatic mark catalyzed by SUPPRESSOR OF VARIEGATION 3-9 HOMOLOG (SUVH) methyltransferases in plants. However, the mechanism underlying the locus specificity of SUVH enzymes has long been elusive. Here, we show that a conserved N-terminal motif is essential for SUVH6-mediated H3K9me2 deposition in planta. The SUVH6 N-terminal peptide can be recognized by the bromo-adjacent homology (BAH) domain of the RNA- and chromatin-binding protein ANTI-SILENCING 1 (ASI1), which has been shown to function in a complex to confer gene expression regulation. Structural data indicate that a classic aromatic cage of ASI1-BAH domain specifically recognizes an arginine residue of SUVH6 through extensive hydrogen bonding interactions. A classic aromatic cage of ASI1 specifically recognizes an arginine residue of SUVH6 through extensive cation-π interactions, playing a key role in recognition. The SUVH6-ASI1 module confers locus-specific H3K9me2 deposition at most SUVH6 target loci and gives rise to distinct regulation of gene expression depending on the target loci, either conferring transcriptional silencing or posttranscriptional processing of mRNA. More importantly, such mechanism is conserved in multiple plant species, indicating a coordinated evolutionary process between SUVH6 and ASI1. In summary, our findings uncover a conserved mechanism for the locus specificity of H3K9 methylation in planta. These findings provide mechanistic insights into the delicate regulation of H3K9 methylation homeostasis in plants.