| dc.description.abstract | Posttranscriptional gene silencing (PTGS) involves two kinds of small non-coding regulatory RNAs, miRNA and small interfering RNA (siRNA) that regulate gene expression in diverse biological processes in eukaryotic organisms. miRNAs originate from primary transcripts (pri-miRNAs) through sequential cleavage by Microprocessor that comprises DCL1 and DRB1/HYL1. The mode of action of siRNAs is similar to that of miRNAs. However, the prerequisite step for initiating siRNA-mediated RNA silencing in plants is the conversion of single-stranded (ss) RNA substrates to double-stranded (ds) RNAs, which is fulfilled by RNA-dependent RNA polymerase 6 (RDR6) and Suppressor of Gene Silencing 3 (SGS3). In turn, dsRNAs are processed by DCL2 or, DCL4 together with its partner DRB4, to 21–22 nt siRNAs, which are eventually loaded into AGO1 to destroy target RNAs. Notwithstanding, the biochemical partners and functional bridges of RDR6/SGS3-DCL4/DRB4 are far less understood compared with microprocessor.
FLOWER LOCUS VE (FVE), a plant homolog of mammalian retinoblastoma-associated protein (RbAp48), has been well known as an epigenetic component in nucleus. However, whether and/ how FVE is involved in PTGS is unknown.
We generated a dual LUC reporter system for miRNA and siRNA pathways. Through an Ethyl methanesulfonate (EMS) mutagenesis of the reporter line, we screened an allele of FVE (fve-8, a mutant encodes a truncated FVE protein FVE-8) displaying enhanced LUC expression level at transcription and protein levels. We observed that FVE protein is localized in both nucleus and cytoplasm. Cytoplasmic FVE (FVENES) could fully rescue the LUC signal, but not the epigenetic-related later flowering phenotype, in fve-8. Through RNA-seq and small RNA-seq, we found that FVE promoted the accumulation of transgene-derived siRNAs in several reporter lines. Through candidate search method, we found that FVE interacts with SGS3, the master regulator of PTGS and promotes SGS3 homodimerization, which is prerequisite for its function in vivo. On the other hand, the truncated FVE-8 protein does not interact with SGS3 as FVE-8 itself forms a homodimer or oligomer.
We then found that FVE is an RNA binding protein. FVE and FVE-8 show similar binding affinity to single-stranded (ss) RNA while SGS3 does not bind to ssRNA. Thus, FVE/SGS3 can form a ribonucleoprotein complex to present ssRNA to RDR6 for generation of dsRNA in vivo. By contrast, FVE-8 gains a new function by binding dsRNA with a significantly increased binding affinity, leading to its hijacking of dsRNA from the SGS3 complex.
Finally, we found that FVE/SGS3 is recruited to DCL4 complexes through interaction with the DCL4 partner, DRB4 protein. In vitro DCL4/DRB4 reconstitution assays showed that FVE directly promotes, while FVE-8 impedes, DCL2/4 activity of siRNA production in vitro.
Based on these results, we concluded that FVE has a novel role in cytoplasmic PTGS pathway. We proposed that FVE can synchronize RDR6/SGS3 and DRB4/DCL4 activity through interaction with the proteins and RNA substrates to promote the synthesis of transgene-derived siRNAs and therefore RNA silencing. | en |
