Analysis of transitive RNA silencing after grafting in transgenic plants with the coat protein gene of <Emphasis Type="Italic">Sweet potato feathery mottle virus</Emphasis>

We have previously reported the graft transmission of target specificity for RNA silencing using transgenic Nicotiana benthamiana plants expressing the coat protein gene (CP, including the 3′ non-translated region) of Sweet potato feathery mottle virus. Transgenic plants carrying the 5′ 200 and 400 bp regions of CP were newly produced. From these plants, two silenced and two non-silenced lines were selected to investigate the manifestation of transitive RNA silencing by graft experiments. Non-silenced scions carrying the entire transgene were grafted onto either 5′ or 3′ silencing inducer rootstocks. When non-silenced scions were grafted onto 5′ silencing inducer rootstocks, RNA silencing was induced in the non-silenced scions and spread toward the 3′ region of the transgene mRNA. Similarly, when non-silenced scions were grafted onto 3′ silencing inducer rootstocks, RNA silencing was induced in the non-silenced scions, but was restricted to the 3′ region of the transgene and did not spread to the 5′ region. In addition, results from crossing experiments, involving non-silenced and 3′ silencing inducer plants, confirmed the above finding. This indicates that RNA silencing spreads in the 5′–3′ direction, not in the 3′–5′ direction, along the transgene mRNA.     

RNA interference is ineffective as a routine method for gene silencing in chick embryos as monitored by fgf8 silencing

The in vivo accessibility of the chick embryo makes it a favoured model system for experimental developmental biology. Although the range of available techniques now extends to miss-expression of genes through in ovo electroporation, it remains difficult to knock out individual gene expression. Recently, the possibility of silencing gene expression by RNAi in chick embryos has been reported. However, published studies show only discrete quantitative differences in the expression of the endogenous targeted genes and unclear morphological alterations. To elucidate whether the tools currently available are adequate to silence gene expression sufficiently to produce a clear and specific null-like mutant phenotype, we have performed several experiments with different molecules that trigger RNAi: dsRNA, siRNA, and shRNA produced from a plasmid coexpressing green fluorescent protein as an internal marker. Focussing on fgf8 expression in the developing isthmus, we show that no morphological defects are observed, and that fgf8 expression is neither silenced in embryos microinjected with dsRNA nor in embryos microinjected and electroporated with a pool of siRNAs. Moreover, fgf8 expression was not significantly silenced in most isthmic cells transformed with a plasmid producing engineered shRNAs to fgf8. We also show that siRNA molecules do not spread significantly from cell to cell as reported for invertebrates, suggesting the existence of molecular differences between different model systems that may explain the different responses to RNAi. Although our results are basically in agreement with previously reported studies, we suggest, in contrast to them, that with currently available tools and techniques the number of cells in which fgf8 gene expression is decreased, if any, is not sufficient to generate a detectable mutant phenotype, thus making RNAi useless as a routine method for functional gene analysis in chick embryos.     

Transgenerational maintenance of transgene body CG but not CHG and CHH methylation

In plants, RNA-directed DNA methylation (RdDM) can target both transgene promoters and coding regions/gene bodies. RdDM leads to methylation of cytosines in all sequence contexts: CG, CHG and CHH. Upon segregation of the RdDM trigger, at least CG methylation can be maintained at promoter regions in the progeny. So far, it is not clear whether coding region methylation can be also maintained. We showed that the body of Potato spindle tuber viroid (PSTVd) transgene constructs became densely de novo methylated at CG, CHG and CHH sites upon PSTVd infection. In this study, we demonstrate that in viroid-free progeny plants, asymmetric CHH and CHG methylation was completely lost. However, symmetric CG methylation was stably maintained for at least two generations. Importantly, the presence of transgene body methylation did not lead to an increase of dimethylation of histone H3 lysine 9 or a decrease of acetylation of H3. Our data supports the view that CG methylation can be maintained not only in promoters but also in the body of transgenes. They further suggest that maintenance of methylation may occur independently of tested chromatin modifications.     

Exoribonuclease R in Mycoplasma genitalium can carry out both RNA processing and degradative functions and is sensitive to RNA ribose methylation

Mycoplasma genitalium, a small bacterium having minimal genome size, has only one identified exoribonuclease, RNase R (MgR). We have purified MgR to homogeneity, and compared its RNA degradative properties to those of its Escherichia coli homologs RNase R (EcR) and RNase II (EcII). MgR is active on a number of substrates including oligoribonucleotides, poly(A), rRNA, and precursors to tRNA. Unlike EcR, which degrades rRNA and pre-tRNA without formation of intermediate products, MgR appears sensitive to certain RNA structural features and forms specific products from these stable RNA substrates. The 3'-ends of two MgR degradation products of 23S rRNA were mapped by RT-PCR to positions 2499 and 2553, each being 1 nucleotide downstream of a 2'-O-methylation site. The sensitivity of MgR to ribose methylation is further demonstrated by the degradation patterns of 16S rRNA and a synthetic methylated oligoribonucleotide. Remarkably, MgR removes the 3'-trailer sequence from a pre-tRNA, generating product with the mature 3'-end more efficiently than EcII does. In contrast, EcR degrades this pre-tRNA without the formation of specific products. Our results suggest that MgR shares some properties of both EcR and EcII and can carry out a broad range of RNA processing and degradative functions.     

A protein containing an XYPPX repeat and a C2 domain is associated with virally induced hypersensitive cell death in plants

In this study, we characterized a Capsicum hypersensitive response (HR)-associated gene, SS52, which encodes a protein that contains an N-terminal C2 domain and a C-terminal XYPPX repeat. Expression analyses revealed that SS52 and its homologue in Arabidopsis were induced by infection with incompatible viruses, indicating the conserved function of this gene. SS52 was not induced by treatment with defense-related hormones, but was induced by abiotic stresses, including wounding. Overexpression of SS52 in tobacco plants suppressed the spread of HR cell death and restricted the spread of an incompatible virus from local lesions. Collectively, the results suggest that SS52 negatively regulates plant HR cell death.     

Gene promoter methylation in colorectal cancer and healthy adjacent mucosa specimens: Correlation with physiological and pathological characteristics,and with biomarkers of one-carbon metabolism

We evaluated the promoter methylation levels of the APC, MGMT, hMLH1, RASSF1A and CDKN2A genes in 107 colorectal cancer (CRC) samples and 80 healthy adjacent tissues. We searched for correlation with both physical and pathological features, polymorphisms of folate metabolism pathway genes (MTHFR, MTRR, MTR, RFC1, TYMS, and DNMT3B), and data on circulating folate, vitamin B12 and homocysteine, which were available in a subgroup of the CRC patients. An increased number of methylated samples were found in CRC respect to adjacent healthy tissues, with the exception of APC, which was also frequently methylated in healthy colonic mucosa. Statistically significant associations were found between RASSF1A promoter methylation and tumor stage, and between hMLH1 promoter methylation and tumor location. Increasing age positively correlated with both hMLH1 and MGMT methylation levels in CRC tissues, and with APC methylation levels in the adjacent healthy mucosa. Concerning gender, females showed higher hMLH1 promoter methylation levels with respect to males. In CRC samples, the MTR 2756AG genotype correlated with higher methylation levels of RASSF1A, and the TYMS 1494 6bp ins/del polymorphism correlated with the methylation levels of both APC and hMLH1. In adjacent healthy tissues, MTR 2756AG and TYMS 1494 6bp del/del genotypes correlated with APC and MGMT promoter methylation, respectively. Low folate levels were associated with hMLH1 hypermethylation. Present results support the hypothesis that DNA methylation in CRC depends from both physiological and environmental factors, with one-carbon metabolism largely involved in this process.     

The value-added genome: building and maintaining genomic cytosine methylation landscapes

Epigenetic marks, such as cytosine methylation and post-translational histone modifications, are important for interpreting and managing eukaryotic genomes. Recent genetic studies in plants have uncovered details on the different interwoven mechanisms that are responsible for specification of genomic cytosine methylation patterns. These mechanisms include targeting cytosine methylation using heterochromatic histone modifications and RNA guides. Genomic cytosine methylation patterns also reflect locus-specific demethylation initiated by specialized DNA glycosylases. While genetics continues to more fully define these mechanisms, genomic studies in Arabidopsis have yielded an unprecedented high-resolution view of how epigenetic marks are layered over a genome.     

Helicase associated 2 domain is essential for helicase activity of RNA helicase A

RNA helicase A (RHA), a DExD/H box protein, plays critical roles in a wide variety of cellular or viral functions. RHA contains a conserved core helicase domain that is flanked by five other domains. Two double-stranded RNA binding domains (dsRBD1 and dsRBD2) are at the N-terminus, whereas HA2 (helicase associated 2), OB-fold (oligonucleotide- or oligosaccharide-binding fold), and RGG (repeats of arginine and glycine–glycine residues) domains are at the C-terminus. The role of these domains in the helicase activity of RHA is still elusive due to the difficulty of obtaining enzymatically active mutant RHA. Here, we purified a series of mutant RHAs containing deletions in either N-terminus or C-terminus. Analysis of these mutant RHAs reveals that the dsRBDs are not required for RNA unwinding, but can enhance the helicase activity by promoting the binding of RHA to substrate RNA. In contrast, deletion of C-terminal domains including RGG, OB-fold, and HA2 does not significantly affect the binding of RHA to substrate RNA. However, HA2 is essential for the RNA unwinding by RHA whereas the RGG and OB-fold are dispensable. The results indicate that the core helicase domain alone is not enough for RHA to execute the unwinding activity.     

The "resurrection method" for modification of specific proteins in higher plants

We describe a new method designated "the resurrection method" by which a modified protein is expressed in higher plants in place of the original protein. The modified gene constructed by introducing synonymous codon substitutions throughout the original gene to prevent the sequence-specific degradation of its mRNA during RNA silencing is expressed while the expression of the original gene is suppressed. Here, we report the successful alteration of the biochemical properties of green fluorescent protein expressed in transgenic Nicotiana benthamiana, suggesting that this method could be useful for gene control in living plants.     

Virus-like particles in Eimeria tenella are associated with multiple RNA segments

Total nucleic acids from sporulated oocysts of Eimeria tenella isolated from Changchun in China were found to contain three extrachromosomal double-stranded RNA segments (dsRNAs) of 1.4, 2.4 and 3.6 kb in sizes. These RNAs were resistant to RNase A digestion under high salt concentration (0.3 M NaCl). RNA-dependent RNA polymerase (RDRP) activity was detected in crude extracts of E. tenella sporulated oocysts containing these nucleic acid species. Virus-like particles (VLPs) were shown to have a diameter of approximately 38 nm under Electron Microscopy (EM) after purification by sucrose density gradient centrifugation. In keeping with the nomenclature generally adopted for protozoan viruses, we have named this isolate as E. tenella virus (ETV) which is the first virus isolated from E. tenella.     

miR-27a suppresses triglyceride accumulation and affects gene mRNA expression associated with fat metabolism in dairy goat mammary gland epithelial cells

MicroRNAs (miRNAs), a well-defined group of small RNAs containing about 22 nucleotides, participate in various biological metabolic processes. miR-27a is a miRNA that is known to regulate fat synthesis and differentiation in preadipocyte cells. However, little is known regarding the role that miR-27a plays in regulating goat milk fat synthesis. In this study, we determined the miR-27a expression profile in goat mammary gland and found that miR-27a expression was correlated with the lactation cycle. Additionally, prolactin promoted miR-27a expression in goat mammary gland epithelial cells. Further functional analysis showed that over-expression of miR-27a down-regulated triglyceride accumulation and decreased the ratio of unsaturated/saturated fatty acid in mammary gland epithelial cells. miR-27a also significantly affected mRNA expression related to milk fat metabolism. Specifically, over-expression of miR-27a reduced gene mRNA expression associated with triglyceride synthesis by suppressing PPARγ protein levels. This study provides the first experimental evidence that miR-27a regulates triglyceride synthesis in goat mammary gland epithelial cells and improves our understanding about the importance of miRNAs in milk fat synthesis.