Regulation of RNA silencing in Arabidopsis thaliana

In plants, RNA silencing is employed as a defence mechanism against viruses and transposons and as a process for quantitatively regulating gene expression. The universal trigger for all RNA silencing pathways is double-stranded RNA, which may be formed either by direct transcription of self-complementary RNA molecules, or by the activity of an RNA-dependent RNA polymerase (RDR) on a single-stranded template. Double-stranded RNA is diced into small interfering RNAs that can direct RNA silencing either transcriptionally, by chromatin modifications, or post-transcriptionally, by mRNA cleavage and/or translational repression. Transgene silencing represents a major problem for plant biotechnologists aiming to achieve high and stable transgene expression. It is triggered by a poorly defined class of aberrant RNA, which, upon exceeding a threshold level in the cell, sets off a silencing cascade that is both initiated and maintained by an RDR. In contrast to RNA silencing, RNA decay pathways are typically employed for qualitative regulation of gene expression. Nonsense-mediated decay (NMD) is a quality control pathway whereby prematurely terminated transcripts are recognized and subject to exonucleolytic decay. To investigate the potential interconnections that exist between RNA decay and RNA silencing pathways, we constructed a series of transgenes, each with a different premature, and out-of-frame, start codon. These transgenes were susceptible to both NMD and RNA silencing, indicating that both pathways have shared substrates. Moreover, RNA degradation by NMD reduced RNA levels such that the incidence of transgene silencing was reduced. To further explore the interconnectedness between these two pathways, we analysed the role of UPF1, a conserved and essential component of NMD, in RNA silencing pathways in Arabidopsis. We found that UPF1 was required for efficient transgene silencing, and for the proper accumulation of at least one micro-RNA. Thus, we present a previously unexplored overlap in substrate and genetic requirements between RNA silencing and RNA decay pathways in Arabidopsis. An interesting feature of endogenous genes is their inherent resistance to RDR activity. We hypothesized that introns, a feature common in endogenous genes, but often lacking in transgenes, may suppress the RDR activity. By including an intron in a GREEN FLUOESCENCE PROTEIN (GFP) transgene, the incidence of transgene silencing was reduced by more than four-fold in primary transformants. Intron-mediated suppression of transgene silencing is dependent on efficient intron splicing, and requires the ortholog of human cap-binding protein 80. Genome-wide small RNA analyses in Arabidopsis found that intronless genes are associated with substantially more small RNAs than intron-containing genes, suggesting that intron splicing may have broad roles in protecting endogenous genes from RNA silencing. That only efficiently spliced introns could suppress RNA silencing led us to investigate the fate of unspliced RNAs in Arabidopsis. We constructed a non-spliceable intron and found that it triggered transgene silencing in all primary transformants. Silencing was both transcriptional and post-transcriptional, and involved intergenic Pol II transcription. This uniquely silenced transgene resembles that observed for endogenous loci, and may represent a broader endogenous role for inefficient intron splicing

KH domain protein RCF3 is a tissue-biased regulator of the plant miRNA biogenesis cofactor HYL1

The biogenesis of microRNAs (miRNAs), which regulate mRNA abundance through posttranscriptional silencing, comprises multiple well-orchestrated processing steps. We have identified the Arabidopsis thaliana K homology (KH) domain protein REGULATOR OF CBF GENE EXPRESSION 3 (RCF3) as a cofactor affecting miRNA biogenesis in specific plant tissues. MiRNA and miRNA-target levels were reduced in apex-enriched samples of rcf3 mutants, but not in other tissues. Mechanistically, RCF3 affects miRNA biogenesis through nuclear interactions with the phosphatases C-TERMINAL DOMAIN PHOSPHATASE-LIKE1 and 2 (CPL1 and CPL2). These interactions are essential to regulate the phosphorylation status, and thus the activity, of the double-stranded RNA binding protein and DICER-LIKE1 (DCL1) cofactor HYPONASTIC LEAVES1 (HYL1)

A Role for the F-Box Protein HAWAIIAN SKIRT in Plant microRNA Function

As regulators of gene expression in multicellular organisms, microRNAs (miRNAs) are crucial for growth and development. Although a plethora of factors involved in their biogenesis and action in Arabidopsis (Arabidopsis thaliana) has been described, these processes and their fine-tuning are not fully understood. Here, we used plants expressing an artificial miRNA target mimic (MIM) to screen for negative regulators of miR156. We identified a new mutant allele of the F-box gene HAWAIIAN SKIRT (HWS; At3G61590), hws-5, as a suppressor of the MIM156-induced developmental and molecular phenotypes. In hws plants, levels of some endogenous miRNAs are increased and their mRNA targets decreased. Plants constitutively expressing full-length HWS-but not a truncated version lacking the F-box domain-display morphological and molecular phenotypes resembling those of mutants defective in miRNA biogenesis and activity. In combination with such mutants, hws loses its delayed floral organ abscission ("skirt") phenotype, suggesting epistasis. Also, the hws transcriptome profile partially resembles those of well-known miRNA mutants hyl1-2, se-3, and ago1-27, pointing to a role in a common pathway. We thus propose HWS as a novel, F-box dependent factor involved in miRNA function