Comparative genomics of small RNA-based gene silencing mechanisms in land plants Small RNAs, 18-25 nucleotides (nt) in size, act in a homology-dependent manner to guide transcriptional and posttranscriptional RNA silencing mechanisms. The small RNAs found in a typical plant cell include a small number of highly abundant, mainly 21-nt microRNAs (miRNAs) and a large number of small interfering RNAs (siRNAs; mainly 24-nt). Both, miRNAs and siRNAs, play essential roles during development and in response to biotic and abiotic stresses. The plant RNA-silencing phenomena share four consensus biochemical steps: (1) induction by double-stranded RNA (dsRNA), (2) dsRNA processing into 18–25 nt-long sRNAs, (3) Methylation of sRNA, and (4) sRNA incorporation into effector complexes that associate with partially or fully complementary target RNA or DNA reviewed in (Chapman and Carrington, 2007; Chen, 2008; Chen et al., 2002; Voinnet, 2009). The biogenesis, targets and often the modes of action of miRNAs and siRNAs are distinct (Chen, 2009; Voinnet, 2009). Most of our current knowledge regarding the molecular basis of small RNA mediated RNA silencing phenomena comes from studies in the model plant Arabidopsis thaliana (Arabidopsis). MicroRNAs (miRNAs)
MiRNAs are cleaved from the hairpin as a duplex by a DICER-LIKE protein, but only the miRNA strand becomes associated with an ARGONAUTE protein in an RNA-induced Silencing Complex (RISC). By contrast, siRNAs are not per se encoded by the genome but are derived from long RNA molecules that have been made double-stranded by RNA dependent RNA polymerases (RDRs) (Bartel, 2004; Vazquez et al., 2010; Voinnet, 2009). Most MIRNA genes are either intronic or intergenic and are transcribed by RNA polymerase II (Pol II). MiRNA expression frequently exhibits tissue specificity and/or sensitivity to external stimuli. MiRNA transcription yields a primary miRNA transcript (pri-miRNA) that forms an imperfect fold-back structure. The pri-miRNA is processed into a stem-loop precursor (pre-miRNA) and then diced as a duplex containing the mature miRNA and an ephimerous passenger strand called miRNA*. The dsRNA ribonuclease DICER-LIKE 1 (DCL1) in combination with HYPONASTYC LEAVES 1 (HYL1; a double-stranded RNA binding protein) and SERRATE (SE; a zinc finger protein) participates in the processing steps from pri-miRNA to pre-miRNA and from pre-miRNA to miRNA. The miRNA/miRNA* duplex is methylated at the 3’ terminal nucleotides by the RNA methyltransferase HUA ENHANCER 1 (HEN1). The methylated mature miRNA selectively incorporated into a protein complex known as microRNA-RNA Induced Silencing Complex (miRNA-RISC) where ARGONAUTE 1 (AGO1) protein is absolutely required. MiRNA function is believed to be cytoplasmic following HASTY (HST) mediated transport of miRNA-RISC or methylated miRNA. By a mechanism not yet elucidated, miRNA-RISC is able to find their mRNA targets and induce translational repression and/or mRNA instability (reviewed in Chen, 2009; Jones-Rhoades et al., 2010, 2006; Ruiz-Ferrer and Voinnet, 2009). Based on the sheer abundance and diversity of plant miRNAs, it is likely that most, if not all, biological processes in plants involve at some point the action of one or more miRNAs. Studies of miRNAs in plant species that undergo major environmental stresses certainly hold great promise for the identification of new miRNAs and possibly new modes of regulation by these molecules (Sunkar et al., 2007; Voinnet, 2009). Short Interfering RNAs (siRNAs)
SiRNAs are mainly involved in epigenetic regulation. Throughout plant development, siRNAs target homologous genomic DNA sequences for cytosine methylation and histone modifications characteristic of heterochromatin through a phenomenon known as RNA‑directed DNA methylation (RdDM) (Law and Jacobsen, 2010; Matzke et al., 2009). RdDM is involved in silencing invasive nucleic acids including repetitive regions of the genome and transposable elements (Matzke et al., 2009). The biogenesis of the 24-nt siRNAs depends on the activity of RNA polymerases IV and V (Pol IV and Pol V) that are two Pol II-related enzymes complexes, limited to the plant kingdom. Single-stranded RNA (ssRNA) transcripts from transposons and repetitive regions of the genome are thought to be generated by Pol IV activity (Pikaard et al., 2008; Wierzbicki et al., 2008). RNA-DEPENDENT RNA POLYMERASE 2 (RDR2) is proposed to generate dsRNA from ssRNA transcripts. The dsRNA is processed by DICER-LIKE3 (DCL3) into 24-nt siRNAs which are bound by ARGONAUTE 4 (AGO4). AGO4 interacts with Pol V to target DOMAINS REARRANGED METHYLTRANSFERASE 2 (DRM2) and additional components to guide de novo DNA methylation and histone modifications to those genomic loci showing homology to the siRNAs (reviewed in Law and Jacobsen, 2009).
Research at the epilab
The three defining features functioning at the core of all small RNA silencing effector modules in plants include the presence of an Argonaute (AGO) family member, the guide strand of a small RNA and the methylation of the small RNA by the RNA methyltransferase HEN1 (Chen, 2008; Chen et al., 2002; Mallory and Vaucheret, 2010; Tolia and Joshua-Tor, 2007). AGOs are highly specialized small RNA-binding proteins that participate in different small RNA silencing modules. The number and diversity of AGO proteins greatly varies among organisms. A single AGO protein exists in fission yeast (Schizosaccharomyces pombe); two AGO proteins are found in the fruit fly (Drosophila melanogaster); four AGO exist in humans; five AGOs are found in the worm Caenorhabditis elegans. In plants the diversification is even greater as 10 AGOs are found in Arabidopsis thaliana, 14 in Zea mays and 18 in rice (Oryza sativa) (Mallory and Vaucheret, 2010). Plant AGOs have diversified in terms of the binding of small RNAs. Each AGO binds a specific subset of small RNAs (Mallory and Vaucheret, 2010; Tolia and Joshua-Tor, 2007; Vaucheret, 2008). AGOs were discovered in Arabidopsis more than 10 years ago and despite their importance in gene silencing, the function of only some members of the family has been characterized (Mallory and Vaucheret, 2010; Vaucheret, 2008). Marchantia polymorpha is a liverwort that is a descendent of the earliest terrestrial plants. Marchantia plants are morphologically simple, they grow as a thallus (flat-sheet structures) with rhizoids (root like cells) growing on the lower surface and repetitive units adapted for photosyntesis and gas exchange on the upper surface. Like other bryophytes, the haploid generation (gametophyte) is the dominant phase of the life cycle. Marchantia has both sexual and asexual reproductive mechanisms. Vegetative asexual propagules (gemmas) are formed inside specialized structures (gemma cups) that by abiotic mechanical factors (i.e. raindrops) are dispersed. The sex of Marchantia plants is determined by the presence of cytologically distinct sex chromosomes, males having one very small Y chromosome and no X chromosome and females having one X chromosome and no Y chromosome (Lorbeer, 1934). The male and female thalli look alike, but males and females can be distinguished easily by differences in the morphology of the sexual structure each produces. Male antheridiophores or female archegoniophores arise from the upper surface of the thallus. Antheridiophores produce sperm-forming antheridia, and archegoniophores produce egg-forming archegonia. Genetic crosses are easily performed by transferring sperms from a drop of water incubated onto the atheridiophores to the archegoniphores reviewed in (Tanurdzic and Banks, 2004). Marchantia has a relatively small genome size of ~280 Mbp distributed among eigtht autosomes plus one sex chromosome (Okada et al., 2000; Okada et al., 2001). The Marchantia genome is currently being sequenced (http://www.jgi.doe.gov/sequencing/cspseqplans2008.html). There are currently extensive collections of Expressed Sequence Tags (ESTs) (~2 million) and the sequence of the Y chromosome has been released (Okada et al., 2001). In addition to its rapid growth, relative simplicity of genetic networks, its ability to be propagated vegetatively by gemma cups and sexually through spores, combined with the growing set of genetic manipulation, culture and microscopy techniques, make Marchantia polymorpha a major model organism for developmental biology, genetics and functional genomics investigations. In collaboration with Dr. John Bowman from Monash University in Australia, Dr. Takayuki Kochi from the University of Kyoto, Dr. Ishizaki Kimitsune from Kobe University, in Japan and Dr. Xuemei Chen from UC Riverside; we were able to identify the core components of both miRNAs and siRNAs silencing pathways in Marchantia polymorpha and in a close relative Marchantia paleacea. Currently, we are characterizing both the miRNA and the RdDM pathway through functional genomics and high through put sequencing of mRNAs and small RNAs libraries, obtained from different tissues and growth conditions.
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