Arabidopsis RRP6L1 and RRP6L2 Function in FLOWERING LOCUS C Silencing via Regulation of Antisense RNA Synthesis

Abstract

The exosome complex functions in RNA metabolism and transcriptional gene silencing. Here, we report that mutations of two Arabidopsis genes encoding nuclear exosome components AtRRP6L1 and AtRRP6L2, cause de-repression of the main flowering repressor FLOWERING LOCUS C ( FLC) and thus delay flowering in early-flowering Arabidopsis ecotypes. AtRRP6L mutations affect the expression of known FLC regulatory antisense (AS) RNAs AS I and II, and cause an increase in Histone3 K4 trimethylation (H3K4me3) at FLC. AtRRP6L1 and AtRRP6L2 function redundantly in regulation of FLC and also act independently of the exosome core complex. Moreover, we discovered a novel, long non-coding, non-polyadenylated antisense transcript (ASL, for Antisense Long) originating from the FLC locus in wild type plants. The AtRRP6L proteins function as the main regulators of ASL synthesis, as these mutants show little or no ASL transcript. Unlike ASI/II, ASL associates with H3K27me3 regions of FLC, suggesting that it could function in the maintenance of H3K27 trimethylation during vegetative growth. AtRRP6L mutations also affect H3K27me3 levels and nucleosome density at the FLC locus. Furthermore, AtRRP6L1 physically associates with the ASL transcript and directly interacts with the FLC locus. We propose that AtRRP6L proteins participate in the maintenance of H3K27me3 at FLC via regulating ASL. Furthermore, AtRRP6Ls might participate in multiple FLC silencing pathways by regulating diverse antisense RNAs derived from the FLC locus.

Author Summary

Arabidopsis FLOWERING LOCUS C (FLC) delays flowering; therefore, repressing expression of FLC provides a critical mechanism to regulate flowering. This mechanism involves multiple levels of regulation, including genetic regulation by transcription factors, and epigenetic regulation by modifications of genomic DNA and histones at the FLC locus. This work examines the role of non-coding RNAs in the epigenetic regulation of FLC, finding that the different RNAs produced from the FLC locus may have different functions in altering the epigenetic landscape at the FLC locus, and revealing that AtRRP6L1 and AtRRP6L2, two components of the exosome, an RNA-processing complex, play roles in regulating these non-coding RNAs. Therefore, this work explores the complex ties between RNA processing, non-coding RNAs, and epigenetic regulation of FLC, a key repressor of flowering.