Small RNA Profile in Moso Bamboo Root and Leaf Obtained by High Definition Adapters

Moso bamboo (Phyllostachy heterocycla cv. pubescens L.) is an economically important fast-growing tree. In order to gain better understanding of gene expression regulation in this important species we used next generation sequencing to profile small RNAs in leaf and roots of young seedlings. Since standard kits to produce cDNA of small RNAs are biased for certain small RNAs, we used High Definition adapters that reduce ligation bias. We identified and experimentally validated five new microRNAs and a few other small non-coding RNAs that were not microRNAs. The biological implication of microRNA expression levels and targets of microRNAs are discussed

Metal A and Metal B Sites of Nuclear RNA Polymerases Pol IV and Pol V Are Required for siRNA-Dependent DNA Methylation and Gene Silencing

Plants are unique among eukaryotes in having five multi-subunit nuclear RNA polymerases: the ubiquitous RNA polymerases I, II and III plus two plant-specific activities, nuclear RNA polymerases IV and V (previously known as Polymerases IVa and IVb). Pol IV and Pol V are not required for viability but play non-redundant roles in small interfering RNA (siRNA)-mediated pathways, including a pathway that silences retrotransposons and endogenous repeats via siRNA-directed DNA methylation. RNA polymerase activity has not been demonstrated for Polymerases IV or V in vitro, making it unclear whether they are catalytically active enzymes. Their largest and second-largest subunit sequences have diverged considerably from Pol I, II and III in the vicinity of the catalytic center, yet retain the invariant Metal A and Metal B amino acid motifs that bind magnesium ions essential for RNA polymerization. By using site-directed mutagenesis in conjunction with in vivo functional assays, we show that the Metal A and Metal B motifs of Polymerases IV and V are essential for siRNA production, siRNA-directed DNA methylation, retrotransposon silencing, and the punctate nuclear localization patterns typical of both polymerases. Collectively, these data show that the minimal core sequences of polymerase active sites, the Metal A and B sites, are essential for Pol IV and Pol V biological functions, implying that both are catalytically active

Small RNAs and the regulation of cis-natural antisense transcripts in Arabidopsis

<p>Abstract</p> <p>Background</p> <p>In spite of large intergenic spaces in plant and animal genomes, 7% to 30% of genes in the genomes encode overlapping cis-natural antisense transcripts (cis-NATs). The widespread occurrence of cis-NATs suggests an evolutionary advantage for this type of genomic arrangement. Experimental evidence for the regulation of two cis-NAT gene pairs by natural antisense transcripts-generated small interfering RNAs (nat-siRNAs) via the RNA interference (RNAi) pathway has been reported in Arabidopsis. However, the extent of siRNA-mediated regulation of cis-NAT genes is still unclear in any genome.</p> <p>Results</p> <p>The hallmarks of RNAi regulation of NATs are 1) inverse regulation of two genes in a cis-NAT pair by environmental and developmental cues and 2) generation of siRNAs by cis-NAT genes. We examined Arabidopsis transcript profiling data from public microarray databases to identify cis-NAT pairs whose sense and antisense transcripts show opposite expression changes. A subset of the cis-NAT genes displayed negatively correlated expression profiles as well as inverse differential expression changes under at least one of the examined developmental stages or treatment conditions. By searching the <it>Arabidopsis </it>Small RNA Project (ASRP) and Massively Parallel Signature Sequencing (MPSS) small RNA databases as well as our stress-treated small RNA dataset, we found small RNAs that matched at least one gene in 646 pairs out of 1008 (64%) protein-coding cis-NAT pairs, which suggests that siRNAs may regulate the expression of many cis-NAT genes. 209 putative siRNAs have the potential to target more than one gene and half of these small RNAs could target multiple members of a gene family. Furthermore, the majority of the putative siRNAs within the overlapping regions tend to target only one transcript of a given NAT pair, which is consistent with our previous finding on salt- and bacteria-induced nat-siRNAs. In addition, we found that genes encoding plastid- or mitochondrion-targeted proteins are over-represented in the Arabidopsis cis-NATs and that 19% of sense and antisense partner genes of cis-NATs share at least one common Gene Ontology term, which suggests that they encode proteins with possible functional connection.</p> <p>Conclusion</p> <p>The negatively correlated expression patterns of sense and antisense genes as well as the presence of siRNAs in many of the cis-NATs suggest that siRNA regulation of cis-NATs via the RNAi pathway is an important gene regulatory mechanism for at least a subgroup of cis-NATs in Arabidopsis.</p

Sense and Antisense Transcripts of Convergent Gene Pairs in Arabidopsis thaliana Can Share a Common Polyadenylation Region

The Arabidopsis genome contains a large number of gene pairs that encode sense and antisense transcripts with overlapping 3′ regions, indicative for a potential role of natural antisense transcription in regulating sense gene expression or transcript processing. When we mapped poly(A) transcripts of three plant gene pairs with long overlapping antisense transcripts, we identified an unusual transcript composition for two of the three gene pairs. Both genes pairs encoded a class of long sense transcripts and a class of short sense transcripts that terminate within the same polyadenylation region as the antisense transcripts encoded by the opposite strand. We find that the presence of the short sense transcript was not dependent on the expression of an antisense transcript. This argues against the assumption that the common termination region for sense and antisense poly(A) transcripts is the result of antisense-specific regulation. We speculate that for some genes evolution may have especially favoured alternative polyadenylation events that shorten transcript length for gene pairs with overlapping sense/antisense transcription, if this reduces the likelihood for dsRNA formation and transcript degradation

IDN2 and Its Paralogs Form a Complex Required for RNA–Directed DNA Methylation

IDN2/RDM12 has been previously identified as a component of the RNA–directed DNA methylation (RdDM) machinery in Arabidopsis thaliana, but how it functions in RdDM remains unknown. By affinity purification of IDN2, we co-purified two IDN2 paralogs IDP1 and IDP2 (IDN2 PARALOG 1 and 2). The coiled-coil domain between the XS and XH domains of IDN2 is essential for IDN2 homodimerization, whereas the IDN2 C-terminal XH domain but not the coiled-coil domain is required for IDN2 interaction with IDP1 and IDP2. By introducing the wild-type IDN2 sequence and its mutated derivatives into the idn2 mutant for complementation testing, we demonstrated that the previously uncharacterized IDN2 XH domain is required for the IDN2-IDP1/IDP2 complex formation as well as for IDN2 function. IDP1 is required for de novo DNA methylation, siRNA accumulation, and transcriptional gene silencing, whereas IDP2 has partially overlapping roles with IDP1. Unlike IDN2, IDP1 and IDP2 are incapable of binding double-stranded RNA, suggesting that the roles of IDP1 and IDP2 are different from those of IDN2 in the IDN2-IDP1/IDP2 complex and that IDP1 and IDP2 are essential for the functioning of the complex in RdDM

Stress-Induced Activation of Heterochromatic Transcription

Constitutive heterochromatin comprising the centromeric and telomeric parts of chromosomes includes DNA marked by high levels of methylation associated with histones modified by repressive marks. These epigenetic modifications silence transcription and ensure stable inheritance of this inert state. Although environmental cues can alter epigenetic marks and lead to modulation of the transcription of genes located in euchromatic parts of the chromosomes, there is no evidence that external stimuli can globally destabilize silencing of constitutive heterochromatin. We have found that heterochromatin-associated silencing in Arabidopsis plants subjected to a particular temperature regime is released in a genome-wide manner. This occurs without alteration of repressive epigenetic modifications and does not involve common epigenetic mechanisms. Such induced release of silencing is mostly transient, and rapid restoration of the silent state occurs without the involvement of factors known to be required for silencing initiation. Thus, our results reveal new regulatory aspects of transcriptional repression in constitutive heterochromatin and open up possibilities to identify the molecular mechanisms involved

Identification of miRNA from Porphyra yezoensis by High-Throughput Sequencing and Bioinformatics Analysis

BACKGROUND: miRNAs are a class of non-coding, small RNAs that are approximately 22 nucleotides long and play important roles in the translational level regulation of gene expression by either directly binding or cleaving target mRNAs. The red alga, Porphyra yezoensis is one of the most important marine economic crops worldwide. To date, only a few miRNAs have been identified in green unicellar alga and there is no report about Porphyra miRNAs. METHODOLOGY/PRINCIPAL FINDINGS: To identify miRNAs in Porphyra yezoensis, a small RNA library was constructed. Solexa technology was used to perform high throughput sequencing of the library and subsequent bioinformatics analysis to identify novel miRNAs. Specifically, 180,557,942 reads produced 13,324 unique miRNAs representing 224 conserved miRNA families that have been identified in other plants species. In addition, seven novel putative miRNAs were predicted from a limited number of ESTs. The potential targets of these putative miRNAs were also predicted based on sequence homology search. CONCLUSIONS/SIGNIFICANCE: This study provides a first large scale cloning and characterization of Porphyra miRNAs and their potential targets. These miRNAs belong to 224 conserved miRNA families and 7 miRNAs are novel in Porphyra. These miRNAs add to the growing database of new miRNA and lay the foundation for further understanding of miRNA function in the regulation of Porphyra yezoensis development

Characterization of Unique Small RNA Populations from Rice Grain

Small RNAs (∼20 to 24 nucleotides) function as naturally occurring molecules critical in developmental pathways in plants and animals [1], [2]. Here we analyze small RNA populations from mature rice grain and seedlings by pyrosequencing. Using a clustering algorithm to locate regions producing small RNAs, we classified hotspots of small RNA generation within the genome. Hotspots here are defined as 1 kb regions within which small RNAs are significantly overproduced relative to the rest of the genome. Hotspots were identified to facilitate characterization of different categories of small RNA regulatory elements. Included in the hotspots, we found known members of 23 miRNA families representing 92 genes, one trans acting siRNA (ta-siRNA) gene, novel siRNA-generating coding genes and phased siRNA generating genes. Interestingly, over 20% of the small RNA population in grain came from a single foldback structure, which generated eight phased 21-nt siRNAs. This is reminiscent of a newly arising miRNA derived from duplication of progenitor genes [3], [4]. Our results provide data identifying distinct populations of small RNAs, including phased small RNAs, in mature grain to facilitate characterization of small regulatory RNA expression in monocot species

Global Identification and Characterization of Transcriptionally Active Regions in the Rice Genome

Genome tiling microarray studies have consistently documented rich transcriptional activity beyond the annotated genes. However, systematic characterization and transcriptional profiling of the putative novel transcripts on the genome scale are still lacking. We report here the identification of 25,352 and 27,744 transcriptionally active regions (TARs) not encoded by annotated exons in the rice (Oryza. sativa) subspecies japonica and indica, respectively. The non-exonic TARs account for approximately two thirds of the total TARs detected by tiling arrays and represent transcripts likely conserved between japonica and indica. Transcription of 21,018 (83%) japonica non-exonic TARs was verified through expression profiling in 10 tissue types using a re-array in which annotated genes and TARs were each represented by five independent probes. Subsequent analyses indicate that about 80% of the japonica TARs that were not assigned to annotated exons can be assigned to various putatively functional or structural elements of the rice genome, including splice variants, uncharacterized portions of incompletely annotated genes, antisense transcripts, duplicated gene fragments, and potential non-coding RNAs. These results provide a systematic characterization of non-exonic transcripts in rice and thus expand the current view of the complexity and dynamics of the rice transcriptome