Analysis of Argonaute 4-Associated Long Non-Coding RNA in Arabidopsis thaliana Sheds Novel Insights into Gene Regulation through RNA-Directed DNA Methylation.

RNA-directed DNA methylation (RdDM) is a plant-specific de novo DNA methylation mechanism that requires long noncoding RNA (lncRNA) as scaffold to define target genomic loci. While the role of RdDM in maintaining genome stability is well established, how it regulates protein-coding genes remains poorly understood and few RdDM target genes have been identified. In this study, we obtained sequences of RdDM-associated lncRNAs using nuclear RNA immunoprecipitation against ARGONAUTE 4 (AGO4), a key component of RdDM that binds specifically with the lncRNA. Comparison of these lncRNAs with gene expression data of RdDM mutants identified novel RdDM target genes. Surprisingly, a large proportion of these target genes were repressed in RdDM mutants suggesting that they are normally activated by RdDM. These RdDM-activated genes are more enriched for gene body lncRNA than the RdDM-repressed genes. Histone modification and RNA analyses of several RdDM-activated stress response genes detected increased levels of active histone mark and short RNA transcript in the lncRNA-overlapping gene body regions in the ago4 mutant despite the repressed expression of these genes. These results suggest that RdDM, or AGO4, may play a role in maintaining or activating stress response gene expression by directing gene body chromatin modification preventing cryptic transcription.

Expression analysis of Cdx2 and Pou5f1 in a marsupial, the stripe-faced dunnart, during early development.

The first lineage allocation during mouse development forms the trophectoderm and inner cell mass, in which Cdx2 and Pou5f1 display reciprocal expression. Yet Cdx2 is not required for trophectoderm specification in other mammals, such as the human, cow, pig, or in two marsupials, the tammar and opossum. The role of Cdx2 and Pou5f1 in the first lineage allocation of Sminthopsis macroura, the stripe-faced dunnart, is unknown. In this study, expression of Cdx2 and Pou5f1 during oogenesis, development from cleavage to blastocyst stages, and in the allocation of the first three lineages was analyzed for this dunnart. Cdx2 mRNA was present in late antral-stage oocytes, but not present again until Day 5.5. Pou5f1 mRNA was present from primary follicles to zygotes, and then expression resumed starting at the early unilaminar blastocyst stage. All cleavage stages and the pluriblast and trophoblast cells co-expressed CDX2 and POU5F1 proteins, which persisted until early stages of hypoblast formation. Hypoblast cells also show co-localisation of POU5F1 and CDX2 once they were allocated, and this persisted during their division and migration. Our studies suggest that CDX2, and possibly POU5F1, are maternal proteins, and that the first lineage to differentiate is the trophoblast, which differentiates to trophectoderm after shell loss one day before implantation. In the stripe-faced dunnart, cleavage cells, as well as trophoblast and pluriblast cells, are polarized, suggesting the continued presence of CDX2 in both lineages until late blastocyst stages may play a role in the formation and maintenance of polarity.

Satellite RNAs interfere with the function of viral RNA silencing suppressors.

Viral satellite RNAs (satRNAs) are small subviral RNAs and depend on the helper virus for replication and spread. satRNAs can attenuate helper virus-induced symptoms, the mechanism of which remains unclear. Here, we show that two virus-encoded suppressors of RNA silencing (VSRs), Cucumber mosaic virus (CMV) 2b and Tombusvirus P19, suppress hairpin RNA (hpRNA)-induced silencing of a ß-glucuronidase (GUS) gene in Nicotiana benthamiana. This suppression can be overcome by CMV Y-satellite RNA (Y-Sat) via the Y-Sat-derived small interfering RNAs (siRNAs), which bind to the VSRs and displace the bound hpGUS-derived siRNAs. We also show that microRNA target gene expression in N. tabacum was elevated by CMV infection, presumably due to function of the 2b VSR, but this upregulation of microRNA target genes was reversed in the presence of Y-Sat. These results suggest that satRNA infection minimizes the effect of VSRs on host siRNA and microRNA-directed silencing. Our results suggest that the high abundance of satRNA-derived siRNAs contributes to symptom attenuation by binding helper virus-encoded VSRs, minimizing the capacity of the VSRs to bind host siRNA and miRNA and interfere with their function.

DNA demethylases target promoter transposable elements to positively regulate stress responsive genes in Arabidopsis.

BACKGROUND: DNA demethylases regulate DNA methylation levels in eukaryotes. Arabidopsis encodes four DNA demethylases, DEMETER (DME), REPRESSOR OF SILENCING 1 (ROS1), DEMETER-LIKE 2 (DML2), and DML3. While DME is involved in maternal specific gene expression during seed development, the biological function of the remaining DNA demethylases remains unclear. RESULTS: We show that ROS1, DML2, and DML3 play a role in fungal disease resistance in Arabidopsis. A triple DNA demethylase mutant, rdd (ros1 dml2 dml3), shows increased susceptibility to the fungal pathogen Fusarium oxysporum. We identify 348 genes differentially expressed in rdd relative to wild type, and a significant proportion of these genes are downregulated in rdd and have functions in stress response, suggesting that DNA demethylases maintain or positively regulate the expression of stress response genes required for F. oxysporum resistance. The rdd-downregulated stress response genes are enriched for short transposable element sequences in their promoters. Many of these transposable elements and their surrounding sequences show localized DNA methylation changes in rdd, and a general reduction in CHH methylation, suggesting that RNA-directed DNA methylation (RdDM), responsible for CHH methylation, may participate in DNA demethylase-mediated regulation of stress response genes. Many of the rdd-downregulated stress response genes are downregulated in the RdDM mutants nrpd1 and nrpe1, and the RdDM mutants nrpe1 and ago4 show enhanced susceptibility to F. oxysporum infection. CONCLUSIONS: Our results suggest that a primary function of DNA demethylases in plants is to regulate the expression of stress response genes by targeting promoter transposable element sequences.

Characterizing RNA-protein interaction using cross-linking and metabolite supplemented nuclear RNA-immunoprecipitation.

RNA-immunoprecipitation (RNA-IP) is a method used to isolate and identify RNA molecules specifically associated with an RNA-binding protein. Non-coding RNAs are emerging as key regulators of many biological and developmental pathways and RNA-IP has become an important tool in studying their function(s). While RNA-IP is successfully used to determine protein-RNA interaction, specific details regarding the level of this association and the metabolic requirement of this interaction which can influence the success of RNA-IP remain unclear. Here, we investigate the conditions required for efficient nuclear RNA-IP using Arabidopsis AGO4 (Argonaute 4) and siRNA binding as the study model. We showed that formaldehyde cross-linking, but not UV cross-linking, allowed for efficient pull-down of 24-nt siRNAs, suggesting that AGO4-siRNA interaction involves other protein(s). We also showed that, while formaldehyde cross-linking could also be performed on purified nuclei, ATP supplementation to the nuclei isolation buffer was needed to efficiently pull down 24-nt siRNAs. This result indicates that ATP is required for efficient siRNA loading onto AGO4. As most of the known RNA-mediated regulatory processes occur in the nucleus, our findings on cross-linking conditions and metabolite requirement for successful AGO4 nuclear RNA-IP provide a valuable insight and future consideration when studying the function of protein-RNA interactions in plants.

A novel marsupial pri-miRNA transcript has a putative role in gamete maintenance and defines a vertebrate miRNA cluster paralogous to the miR-15a/miR-16-1 cluster.

Successful maintenance, survival and maturation of gametes rely on bidirectional communication between the gamete and its supporting cells. Before puberty, factors from the gamete and its supporting cells are necessary for spermatogonial stem cell and primordial follicle oocyte maintenance. Following gametogenesis, gametes rely on factors and nutrients secreted by cells of the reproductive tracts, the epididymis and/or oviduct, to complete maturation. Despite extensive studies on female and male reproduction, many of the molecular mechanisms of germ cell maintenance remain relatively unknown, particularly in marsupial species. We present the first study and characterisation of a novel primary miRNA transcript, pri-miR-16c, in the marsupial, the stripe-faced dunnart. Bioinformatic analysis showed that its predicted processed miRNA - miR-16c - is present in a wide range of vertebrates, but not eutherians. In situ hybridisation revealed dunnart pri-miR-16c expression in day 4 (primordial germ cells) and day 7 (oogonia) pouch young, in primary oocytes and follicle cells of primordial follicles but then only in follicle cells of primary, secondary and antral follicles in adult ovaries. In the adult testis, pri-miR-16c transcripts were present in the cytoplasm of spermatogonial cells. The oviduct and the epididymis both showed expression, but not any other somatic tissues examined or conceptuses during early embryonic development. This pattern of expression suggests that pri-miR-16c function may be associated with gamete maintenance, possibly through mechanisms involving RNA transfer, until the zygote enters the uterus at the pronuclear stage.

Long non-coding RNA-mediated mechanisms independent of the RNAi pathway in animals and plants.

Recent advances in the field of RNA research have provided compelling evidence implicating long non-coding RNA molecules in many diverse and substantial biological processes that include transcriptional and post-transcriptional regulation of gene expression, genomic imprinting, modulation of protein activity and subcellular localization and cellular structural maintenance. While long non-coding RNAs have been most extensively studied in animal species, studies of long non-coding RNA in plants begin to emerge showing some conservation of mechanisms. This review aims to provide an overview of significant and recently identified long non-coding RNA-mediated mechanisms in both animal and plant species.

Cloning and characterization of a new gene from the PAT protein family, in a marsupial, the stripe-faced dunnart (Sminthopsis macroura).

Recent studies of PAT proteins in Drosophila and Xenopus have revealed significant roles for this family of proteins in the polarized transport of lipid droplets and maternal determinants during early embryogenesis. In mammals, PAT proteins are known to function mainly in lipid metabolism, yet research has yet to establish a role for PAT proteins in mammalian embryogenesis. Oocytes and early cleavage stages in Sminthopsis macroura show obvious polarized cytoplasmic distribution of organelles, somewhat similar to Drosophila and Xenopus, suggesting that a PAT protein may also be involved in S. macroura embryonic development. In the present study, we identified a new marsupial gene for PAT family proteins, DPAT, from S. macroura. Expression analyses by RT-PCR and whole mount fluorescent in situ hybridization revealed that DPAT expression was specific to oocytes and cleavage stage conceptuses. Analysis of the localization of lipid droplets during S. macroura early embryonic development found a polarized distribution of lipid droplets at the two- and four-cell stage, and an asymmetric enrichment in blastomeres on one side of conceptuses from two- to eight-cell stage. Lipid droplets largely segregate to pluriblast cells at the 16-cell stage, suggesting a role in pluriblast lineage allocation.

Induced ovulation mimics the time-table of natural development in the stripe-faced dunnart, Sminthopsis macroura and results in the birth of fertile young.

Induced ovulation maximizes captive breeding success, increasing productivity and facilitating the contribution of otherwise infertile animals to the genetic pool. In marsupials, induced ovulation to produce fertile young is unknown. Here we present an induction protocol efficient in inducing non-cycling and non-reproductive females to cycle, mate, ovulate, and conceive. Ovulation was induced in Sminthopsis macroura using an initial injection of 0.06 IU equine serum gonadotropin (eSG)/g (time 0), followed on day 4 by 0.04 IU eSG/g. Using this induction regime, the timing of follicular and embryonic development mimics natural cycles and results in the birth of viable, fertile young. Response to induction is not significantly affected by animal age, making this protocol an effective conservation tool. We have established a time-table of development following induction, providing a source of precisely timed research material. This is the first induced ovulation protocol in any marsupial to result in demonstrated fertile offspring and to allow the reliable collection of known-age samples during both the follicular phase and the gestation period.

Identification of novel and known ovary-specific genes including ZP2, in a marsupial, the stripe-faced dunnart.

During the early stages of oogenesis, oocyte-specific factors, synthesized by and stored within the oocyte, play critical roles during oogenesis, folliculogenesis, fertilization and early embryonic development in the mouse. The identification of marsupial maternal factors, expressed specifically in the ovary or oocyte, may provide an insight into the conserved evolutionary mechanisms that drive mammalian oocyte development to cleavage stages. In this study, 10 clones including dunnart ZP2 and c-mos, isolated by cDNA representational difference analysis, were validated by RT-PCR for ovary-specific expression. This novel combination of techniques to isolate ovary-specific genes has identified three novel genes with ovary-specific expression. Both dunnart ZP2 and c-mos exhibited ovary-specific expression, making this study the first isolation of c-mos in a marsupial species. Dunnart ZP2 expression was examined in detail by in situ hybridization and results indicate oocyte-specific expression of dunnart ZP2 in the cytoplasm of oocytes of primordial, primary and secondary follicles with expression being highest in oocytes of primary follicles. ZP2 was not expressed in granulosa cells of any follicles.