Evidence for Antisense Transcription Associated with MicroRNA Target mRNAs in Arabidopsis

Antisense transcription is a pervasive phenomenon, but its source and functional significance is largely unknown. We took an expression-based approach to explore microRNA (miRNA)-related antisense transcription by computational analyses of published whole-genome tiling microarray transcriptome and deep sequencing small RNA (smRNA) data. Statistical support for greater abundance of antisense transcription signatures and smRNAs was observed for miRNA targets than for paralogous genes with no miRNA cleavage site. Antisense smRNAs were also found associated with MIRNA genes. This suggests that miRNA-associated “transitivity” (production of small interfering RNAs through antisense transcription) is more common than previously reported. High-resolution (3 nt) custom tiling microarray transcriptome analysis was performed with probes 400 bp 5′ upstream and 3′ downstream of the miRNA cleavage sites (direction relative to the mRNA) for 22 select miRNA target genes. We hybridized RNAs labeled from the smRNA pathway mutants, including hen1-1, dcl1-7, hyl1-2, rdr6-15, and sgs3-14. Results showed that antisense transcripts associated with miRNA targets were mainly elevated in hen1-1 and sgs3-14 to a lesser extent, and somewhat reduced in dcl11-7, hyl11-2, or rdr6-15 mutants. This was corroborated by semi-quantitative reverse transcription PCR; however, a direct correlation of antisense transcript abundance in MIR164 gene knockouts was not observed. Our overall analysis reveals a more widespread role for miRNA-associated transitivity with implications for functions of antisense transcription in gene regulation. HEN1 and SGS3 may be links for miRNA target entry into different RNA processing pathways

Antiviral Silencing and Suppression of Gene Silencing in Plants

RNA silencing is an evolutionary conserved sequence-specific gene inactivation mechanism that contributes to the control of development, maintains heterochromatin, acts in stress responses, DNA repair and defends against invading nucleic acids like transposons and viruses. In plants RNA silencing functions as one of the main immune systems. RNA silencing process involves the small RNAs and trans factor components like Dicers, Argonautes and RNA-dependent RNA poly- merases. To deal with host antiviral silencing responses viruses evolved mecha- nisms to avoid or counteract this, most notably through expression of viral suppressors of RNA silencing. Due to the overlap between endogenous and antiviral silencing pathways while blocking antiviral pathways viruses also impact endogenous silencing processes. Here we provide an overview of antiviral silencing pathway, host factors implicated in it and the crosstalk between antiviral and endogenous branches of silencing. We summarize the current status of knowledge about the viral counter-defense strategies acting at various steps during virus infection in plants with the focus on representative, well studied silencing suppres- sor proteins. Finally we discuss future challenges of the antiviral silencing and counter-defense research field

A DELLA in Disguise: SPATULA Restrains the Growth of the Developing Arabidopsis Seedling[C][W]

This study examines the role of the PHYTOCHROME INTERACTING FACTOR3 homolog SPATULA (SPT) in the control of the developing seedling and shows that SPT is a potent regulator of cotyledon size, acting in parallel to DELLAs. As DELLAs negatively regulate SPT abundance, the light regulation of DELLAs drives the DELLA-SPT counterbalance, enforcing growth restraint across a range of ambient light conditions that are prevalent in nature

Mobile silencing in plants: what is the signal and what defines the target

RNA-mediated silencing in plants can spread from cell to cell and over a long distance, and such mobile silencing has been extensively studied in the past decade. However, major questions remain as to what is the exact nature of the mobile silencing signals, how the components of the RNA-directed DNA methylation pathway are involved, and why systemic spread of silencing has only been observed for transgenes but not endogenous genes. In this review, we provide an overview of the current knowledge on mobile gene silencing in plants and present a model where systemic silencing involves long nuclear RNA transcripts that serve as a template to amplify primary siRNA signals

The UGT73C5 of Arabidopsis thaliana glucosylates brassinosteroids

Steroid hormones are essential for development, and the precise control of their homeostasis is a prerequisite for normal growth. UDP-glycosyltransferases (UGTs) are considered to play an important regulatory role in the activity of steroids in mammals and insects. This study provides an indication that a UGT accepting plant steroids as substrates functions in brassinosteroid (BR) homeostasis. The UGT73C5 of Arabidopsis thaliana catalyses 23-O-glucosylation of the BRs brassinolide (BL) and castasterone. Transgenic plants overexpressing UGT73C5 displayed BR-deficient phenotypes and contained reduced amounts of BRs. The phenotype, which was already apparent in seedlings, could be rescued by application of BR. In feeding experiments with BL, wild-type seedlings converted BL to the 23-O-glucoside; in the transgenic lines silenced in UGT73C5 expression, no 23-O-glucoside was detected, implying that this UGT is the only enzyme that catalyzes BL-23-O-glucosylation in seedlings. Plant lines in which UGT73C5 expression was altered also displayed hypocotyl phenotypes previously described for seedlings in which BR inactivation by hydroxylation was changed. These data support the hypothesis that 23-O-glucosylation of BL is a function of UGT73C5 in planta, and that glucosylation regulates BR activity

RNA-Mediated Silencing in Algae: Biological Roles and Tools for Analysis of Gene Function ▿

Algae are a large group of aquatic, typically photosynthetic, eukaryotes that include species from very diverse phylogenetic lineages, from those similar to land plants to those related to protist parasites. The recent sequencing of several algal genomes has provided insights into the great complexity of these organisms. Genomic information has also emphasized our lack of knowledge of the functions of many predicted genes, as well as the gene regulatory mechanisms in algae. Core components of the machinery for RNA-mediated silencing show widespread distribution among algal lineages, but they also seem to have been lost entirely from several species with relatively small nuclear genomes. Complex sets of endogenous small RNAs, including candidate microRNAs and small interfering RNAs, have now been identified by high-throughput sequencing in green, red, and brown algae. However, the natural roles of RNA-mediated silencing in algal biology remain poorly understood. Limited evidence suggests that small RNAs may function, in different algae, in defense mechanisms against transposon mobilization, in responses to nutrient deprivation and, possibly, in the regulation of recently evolved developmental processes. From a practical perspective, RNA interference (RNAi) is becoming a promising tool for assessing gene function by sequence-specific knockdown. Transient gene silencing, triggered with exogenously synthesized nucleic acids, and/or stable gene repression, involving genome-integrated transgenes, have been achieved in green algae, diatoms, yellow-green algae, and euglenoids. The development of RNAi technology in conjunction with system level “omics” approaches may provide the tools needed to advance our understanding of algal physiological and metabolic processes