PRP8 regulates chromatin organization to enforce transcriptional gene silencing independent of DNA methylation

Transcriptional gene silencing (TGS) is critical in maintaining genome integrity in plants, during which DNA methylation plays a fundamental role. The establishment and maintenance of DNA methylation are well characterized, while the downstream events mediating transcriptional repression remain poorly understood. In this study, we performed a forward genetic screen for suppressors of hdp1-induced report gene silencing to identify components involved in TGS downstream of DNA methylation and identified Pre-mRNA processing factor 8 (PRP8), a well-known component of the spliceosome complex. Through comprehensive genetic analysis coupled with RNA sequencing and whole-genome bisulfite sequencing, we demonstrated that PRP8-mediated TGS of the transgenes along with a subset of endogenous genes and transposable elements in a DNA methylation-independent manner. Through high-throughput chromosome conformation capture (Hi-C) analysis, we demonstrated that PRP8 is required for maintaining pericentromeric heterochromatin interactions and higher-order chromatin organization. Further genetic analysis revealed that PRP8 acts synergistically with Arabidopsis MICRORCHIDIA (MORC) protein MORC6, HISTONE DEACETYLASE 6 (HDA6), MORPHEUS MOLECULE 1 (MOM1), and NUCLEAR RNA POLYMERASE D1B (NRPE1), suggesting its involvement in a previously uncharacterized TGS pathway. In summary, our findings establish PRP8 as a dual-function factor involved in both splicing and DNA methylation-independent TGS.