Messages on small RNA duplexes in plants

Journal of Plant Research - Small RNA-mediated gene silencing encompasses diverse developmental events, stress responses, defense against pathogens, and maintenance of genome integrity. Extensive...     

Epigenetic modification of plants with systemic RNA viruses

Knowledge of gene function is critical to the development of new plant traits for improved agricultural and industrial applications. Viral expression vectors offer a rapid and proven method to provide epigenetic expression of foreign sequences throughout infected plants. Expression of these sequences from viral vectors can lead to gain- or loss-of-function phenotypes, allowing gene function to be determined by phenotypic or biochemical effects in the infected plant. Tobacco mosaic virus and barley stripe mosaic virus expression vectors have been developed to express foreign gene sequences in dicotyledonous and monocotyledonous hosts, respectively. Large-scale application of both viral vector systems for gene function discovery in Nicotiana and barley hosts resulted in high infection rates and produced distinctive visual phenotypes in approximately 5% of transfected plants. Novel genes expressing potential herbicide target proteins in addition to genes promoting stem elongation, leaf development and apical dominance were identified in the large-scale screening. This report illustrates the adaptability of viral vectors for gene function discovery in higher plants.     

Pathways through the small RNA world of plants

RNA silencing pathways in plants have diversified along with key gene families involved in small RNA biogenesis and effector steps. Evidence suggests that these pathways have distinct roles in plant biology.     

Phase separation of chromatin and small RNA pathways in plants

Gene expression can be modulated by epigenetic mechanisms, including chromatin modifications and small regulatory RNAs. These pathways are unevenly distributed within a cell and usually take place in specific intracellular regions. Unfortunately, the fundamental driving force and biological relevance of such spatial differentiation is largely unknown. Liquid–liquid phase separation (LLPS) is a natural propensity of demixing liquid phases and has been recently suggested to mediate the formation of biomolecular condensates that are relevant to diverse cellular processes. LLPS provides a mechanistic explanation for the self-assembly of subcellular structures by which the efficiency and specificity of certain cellular reactions are achieved. In plants, LLPS has been observed for several key factors in the chromatin and small RNA pathways. For example, the formation of facultative and obligate heterochromatin involves the LLPS of multiple relevant factors. In addition, phase separation is observed in a set of proteins acting in microRNA biogenesis and the small interfering RNA pathway. In this Focused Review, we highlight and discuss the recent findings regarding phase separation in the epigenetic mechanisms of plants.     

Essential requirements for robust signaling in Hfq dependent small RNA networks

Bacteria possess networks of small RNAs (sRNAs) that are important for modulating gene expression. At the center of many of these sRNA networks is the Hfq protein. Hfq's role is to quickly match cognate sRNAs and target mRNAs from among a large number of possible combinations and anneal them to form duplexes. Here we show using a kinetic model that Hfq can efficiently and robustly achieve this difficult task by minimizing the sequestration of sRNAs and target mRNAs in Hfq complexes. This sequestration can be reduced by two non-mutually exclusive kinetic mechanisms. The first mechanism involves heterotropic cooperativity (where sRNA and target mRNA binding to Hfq is influenced by other RNAs bound to Hfq); this cooperativity can selectively decrease singly-bound Hfq complexes and ternary complexes with non-cognate sRNA-target mRNA pairs while increasing cognate ternary complexes. The second mechanism relies on frequent RNA dissociation enabling the rapid cycling of sRNAs and target mRNAs among different Hfq complexes; this increases the probability the cognate ternary complex forms before the sRNAs and target mRNAs degrade. We further demonstrate that the performance of sRNAs in isolation is not predictive of their performance within a network. These findings highlight the importance of experimentally characterizing duplex formation in physiologically relevant contexts with multiple RNAs competing for Hfq. The model will provide a valuable framework for guiding and interpreting these experiments.     

A plasmid-based system for expressing small interfering RNA libraries in mammalian cells

Background  

RNA interference (RNAi) is an evolutionarily conserved process that functions to inhibit gene expression. The use of RNAi in mammals as a tool to study gene function has rapidly developed in the last couple of years since the discovery that the function-inhibiting units of RNAi are short 21–25 nt double-stranded RNAs (siRNAs) derived from their longer template. The use of siRNAs allows for gene-specific knock-down without induction of the non-specific interferon response in mammalian cells. Multiple systems have been developed to introduce siRNAs into mammals. One of the most appealing of these techniques is the use of vectors containing polymerase III promoters to drive expression of hairpin siRNAs. However, there are multiple limitations to using hairpin siRNA vectors including the observation that some are unstable in bacteria and are difficult to sequence.     

Double-stranded RNA in rice: A novel RNA replicon in plants

The entire sequence of 13952 nucleotides of a plasmid-like, double-stranded RNA (dsRNA) from rice was assembled from more than 50 independent cDNA clones. The 5 non-coding region of the coding (sense) strand spans over 166 nucleotides, followed by one long open reading frame (ORF) of 13716 nucleotides that encodes a large putative polyprotein of 4572 amino acid residues, and by a 70-nucleotide 3 noncoding region. This ORF is apparently the longest reported to date in the plant kingdom. Amino acid sequence comparisons revealed that the large putative polyprotein includes an RNA helicase-like domain and an RNA-dependent RNA polymerase (replicase)-like domain. Comparisons of the amino acid sequences of these two domains and of the entire genetic organization of the rice dsRNA with those found in potyviruses and the CHV1-713 dsRNA of chestnut blight fungus suggest that the rice dsRNA is located evolutionarily between potyviruses and the CHV1-713 dsRNA. This plasmid-like dsRNA in rice seems to constitute a novel RNA replicon in plants.     

A MAPK pathway mediates ethylene signaling in plants

Ethylene signal transduction involves ETR1, a two-component histidine protein kinase receptor. ETR1 functions upstream of the negative regulator CTR1. The similarity of CTR1 to members of the Raf family of mitogen-activated protein kinase kinase kinases (MAPKKKs) suggested that ethylene signaling in plants involves a MAPK pathway, but no direct evidence for this has been provided. Here we show that distinct MAPKs are activated by the ethylene precursor aminocyclopropane-1-carboxylic acid (ACC) in Medicago and ARABIDOPSIS: In Medicago, the ACC-activated MAPKs were SIMK and MMK3, while in Arabidopsis MPK6 and another MAPK were identified. Medicago SIMKK specifically mediated ACC-induced activation of SIMK and MMK3. Transgenic Arabidopsis plants overexpressing SIMKK have constitutive MPK6 activation and ethylene-induced target gene expression. SIMKK overexpressor lines resemble ctr1 mutants in showing a triple response phenotype in the absence of ACC. Whereas MPK6 was not activated by ACC in etr1 mutants, ein2 and ein3 mutants showed normal activation profiles. In contrast, ctr1 mutants showed constitutive activation of MPK6. These data indicate that a MAPK cascade is part of the ethylene signal transduction pathway in plants.     

Light signaling in plants

Light signals have profound morphogenic effects on plant development. Signals perceived by the red/far‐red absorbing phytochrome family of photoreceptors and the blue/green/ UV‐A absorbing cryptochrome photoreceptor converge on a group of pleiotropic gene products defined by the COP/DET loci to control the pattern of development. The signaling pathway, although still undefined, includes several classic signaling molecules, such as G‐proteins, calcium, calmodulin, and cGMP. A separate signaling pathway is involved in the modulation of the phototropic response. Additional mutants have been identified that affect subsets of light signaling responses. This review provides an overview of our current understanding of the light signaling process, in particular recent genetic and biochemical advances.     

Poly(A)-associated RNA in plants

The RNA associated with poly(A) sequences from Euglena gracilis and Vicia faba has been isolated by binding to millipore filters and characterized by sedimentation velocity centrifugation and electrophoretic mobility. Poly(A)-associated RNA as isolated in solution was highly aggregated. When denatured, it sedimented as a broad peak with a mean value of 16-18 S. This RNA was shown to be covalently linked to poly(A) sequences which are 150-250 nucleotides long. Our size estimates for plant poly(A) and poly(A)-associated RNA are similar to those obtained for animal cells.