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Noncoding and coding RNAs are key regulators of plant growth, development, and stress responses. To investigate the types of transcripts accumulated during the vegetative to reproductive transition and floral development in the Coffea arabica L., we sequenced small RNA libraries from eight developmental stages, up to anthesis. We combined these data with messenger RNA and PARE sequencing of two important development stages that marks the transition of an apparent latent to a rapid growth stage. In addition, we took advantage of multiple in silico tools to characterize genomic loci producing small RNAs such as phasiRNAs, miRNAs, and tRFs. Our differential and co-expression analysis showed that some types of small RNAs such as tRNAs, snoRNAs, snRNAs, and phasiRNAs preferentially accumulate in a stage-specific manner. Members of the miR482/miR2118 superfamily and their 21-nucleotide phasiRNAs originating from resistance genes show a robust co-expression pattern that is maintained across all the evaluated developmental stages. Finally, the majority of miRNAs accumulate in a family stage-specific manner, related to modulated hormonal responses and transcription factor expression., (© 2024 Society for Experimental Biology and John Wiley & Sons Ltd.)

Reproductive phasiRNAs (phased, small interfering RNAs) are broadly present in angiosperms and play crucial roles in sustaining male fertility. While the premeiotic 21-nt (nucleotides) phasiRNAs and meiotic 24-nt phasiRNA pathways have been extensively studied in maize ( Zea mays ) and rice ( Oryza sativa ), a third putative category of reproductive phasiRNAs-named premeiotic 24-nt phasiRNAs-have recently been reported in barley ( Hordeum vulgare ) and wheat ( Triticum aestivum ). To determine whether premeiotic 24-nt phasiRNAs are also present in maize and related species and begin to characterize their biogenesis and function, we performed a comparative transcriptome and degradome analysis of premeiotic and meiotic anthers from five maize inbred lines and three teosinte species/subspecies. Our data indicate that a substantial subset of the 24-nt phasiRNA loci in maize and teosinte are already highly expressed at the premeiotic phase. The premeiotic 24-nt phasiRNAs are similar to meiotic 24-nt phasiRNAs in genomic origin and dependence on DCL5 (Dicer-like 5) for biogenesis, however, premeiotic 24-nt phasiRNAs are unique in that they are likely i) not triggered by microRNAs, ii) not loaded by AGO18 proteins, and iii) not capable of mediating PHAS precursor cleavage. In addition, we also observed a group of premeiotic 24-nt phasiRNAs in rice using previously published data. Together, our results indicate that the premeiotic 24-nt phasiRNAs constitute a unique class of reproductive phasiRNAs and are present more broadly in the grass family (Poaceae) than previously known., Competing Interests: Competing interests statement:Reviewer X. Chen and corresponding author B.C.M. have co-authored editorial and commentary papers in 2022 and 2023, but have not had research collaborations in more than 48 mo.

Small RNAs play important roles in plant growth and development by modulating expression of genes and transposons. In many flowering plant species, male reproductive organs, the anthers, produce abundant phased small interfering RNAs (phasiRNAs). Two classes of reproductive phasiRNAs are generally known, mostly from monocots: (i) pre-meiotic 21-nucleotide (nt) phasiRNAs triggered by miR2118 and (ii) meiotic 24-nt phasiRNAs triggered by miR2275. Here, we describe conserved and non-conserved triggers of 24-nt phasiRNAs in several eudicots. We found that the abundant 24-nt phasiRNAs in the basal eudicot columbine (Aquilegia coerulea) are produced by the canonical trigger miR2275, as well as by other non-canonical triggers, miR482/2118 and miR14051. These triggering microRNAs (miRNAs) are localized in microspore mother cells and tapetal cells of meiotic and post-meiotic stage anthers. Furthermore, we identified a lineage-specific trigger (miR11308) of 24-nt phasiRNAs and an expanded number of 24-PHAS loci in wild strawberry (Fragaria vesca). We validated the presence of the miR2275-derived 24-nt phasiRNA pathway in rose (Rosa chinensis). Finally, we evaluated all eudicots that have been validated for the presence of 24-nt phasiRNAs as possible model systems in which to study the biogenesis and function of 24-nt phasiRNAs. We conclude that columbine (Aquilegia coerulea) would be a strong model because of its extensive number of 24-PHAS loci and its diversity of trigger miRNAs., (© 2021 Society for Experimental Biology and John Wiley & Sons Ltd.)

Plant small RNAs are important regulatory elements that fine-tune gene expression and maintain genome integrity by silencing transposons. Reproductive organs of monocots produce abundant phased, small interfering RNAs (phasiRNAs). The 21-nt reproductive phasiRNAs triggered by miR2118 are highly enriched in pre-meiotic anthers, and have been found in multiple eudicot species, in contrast with prior reports of monocot specificity. The 24-nt reproductive phasiRNAs are triggered by miR2275, and are highly enriched during meiosis in many angiosperms. Here, we report the widespread presence of the 21-nt reproductive phasiRNA pathway in eudicots including canonical and non-canonical microRNA (miRNA) triggers of this pathway. In eudicots, these 21-nt phasiRNAs are enriched in pre-meiotic stages, a spatiotemporal distribution consistent with that of monocots and suggesting a role in anther development. Although this pathway is apparently absent in well-studied eudicot families including the Brassicaceae, Solanaceae and Fabaceae, our work in eudicots supports an earlier singular finding in spruce, a gymnosperm, indicating that the pathway of 21-nt reproductive phasiRNAs emerged in seed plants and was lost in some lineages., (© 2021. The Author(s).)

In monocots other than maize (Zea mays) and rice (Oryza sativa), the repertoire and diversity of microRNAs (miRNAs) and the populations of phased, secondary, small interfering RNAs (phasiRNAs) are poorly characterized. To remedy this, we sequenced small RNAs (sRNA) from vegetative and dissected inflorescence tissue in 28 phylogenetically diverse monocots and from several early-diverging angiosperm lineages, as well as publicly available data from 10 additional monocot species. We annotated miRNAs, small interfering RNAs (siRNAs) and phasiRNAs across the monocot phylogeny, identifying miRNAs apparently lost or gained in the grasses relative to other monocot families, as well as a number of transfer RNA fragments misannotated as miRNAs. Using our miRNA database cleaned of these misannotations, we identified conservation at the 8th, 9th, 19th, and 3'-end positions that we hypothesize are signatures of selection for processing, targeting, or Argonaute sorting. We show that 21-nucleotide (nt) reproductive phasiRNAs are far more numerous in grass genomes than other monocots. Based on sequenced monocot genomes and transcriptomes, DICER-LIKE5, important to 24-nt phasiRNA biogenesis, likely originated via gene duplication before the diversification of the grasses. This curated database of phylogenetically diverse monocot miRNAs, siRNAs, and phasiRNAs represents a large collection of data that should facilitate continued exploration of sRNA diversification in flowering plants., (© American Society of Plant Biologists 2021. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Two classes of premeiotic (21-nucleotides [nt]) and meiotic (24-nt) phased small interfering RNAs (phasiRNAs) and their patterns of accumulation have been described in maize ( Zea mays ) and rice ( Oryza sativa ) anthers. Their precise function remains unclear, but studies have shown that they support male fertility. The important role of phasiRNAs in anthers underpins our present study to characterize these small RNAs in wheat ( Triticum aestivum ) and barley ( Hordeum vulgare ) anthers. We staged anthers at every 0.2 mm of development for one wheat and two barley varieties. We isolated premeiotic (0.2, 0.4, and 0.6 mm), meiotic (0.8, 1.0, and 1.4 mm), and postmeiotic (1.8 mm) anthers, for which we then investigated accumulation patterns of RNAs, including reproductive phasiRNAs. We annotated a total of 12,821 and 2,897 PHAS loci in the wheat and barley genomes, respectively. By comparing the total number of PHAS loci in genomes of maize, rice, barley, and wheat, we identified an expansion of reproductive PHAS loci in the genomes of Poaceae subfamilies from Panicoideae to Oryzoideae and to Poideae. In addition to the two classes of premeiotic (21-nt) and meiotic (24-nt) phasiRNAs, previously described in maize and rice anthers, we characterized a group of 24-nt phasiRNAs that accumulate in premeiotic anthers. The absence of premeiotic 24-nt phasiRNAs in maize and rice suggests a divergence in grass species of the Poideae subfamily. Additionally, we performed a gene coexpression analysis describing the regulation of phasiRNA biogenesis in wheat and barley anthers. We highlight Argonaute 9 ( AGO9 ) and Argonaute 6 ( AGO6 ) as candidate binding partners of premeiotic and meiotic 24-nt phasiRNAs, respectively., (© 2020 American Society of Plant Biologists. All Rights Reserved.)

Phased secondary small interfering RNAs (phasiRNAs) constitute a major category of small RNAs in plants, but most of their functions are still poorly defined. Some phasiRNAs, known as trans -acting siRNAs, are known to target complementary mRNAs for degradation and to function in development. However, the targets or biological roles of other phasiRNAs remain speculative. New insights into phasiRNA biogenesis, their conservation, and their variation across the flowering plants continue to emerge due to the increased availability of plant genomic sequences, deeper and more sophisticated sequencing approaches, and improvements in computational biology and biochemical/molecular/genetic analyses. In this review, we survey recent progress in phasiRNA biology, with a particular focus on two classes associated with male reproduction: 21-nucleotide (accumulate early in anther ontogeny) and 24-nucloetide (produced in somatic cells during meiosis) phasiRNAs. We describe phasiRNA biogenesis, function, and evolution and define the unanswered questions that represent topics for future research., (© 2020 American Society of Plant Biologists. All rights reserved.)

Small RNAs are non-coding RNAs that play important roles in the lives of both animals and plants. They are 21- to 24-nt in length and ∼10 nm in size. Their small size and high diversity have made it challenging to develop detection methods that have sufficient resolution and specificity to multiplex and quantify. We created a method, sRNA-PAINT, for the detection of small RNAs with 20 nm resolution by combining the super-resolution method, DNA-based points accumulation in nanoscale topography (DNA-PAINT), and the specificity of locked nucleic acid (LNA) probes for the in situ detection of multiple small RNAs. The method relies on designing probes to target small RNAs that combine DNA oligonucleotides (oligos) for PAINT with LNA-containing oligos for hybridization; therefore, we developed an online tool called 'Vetting & Analysis of RNA for in situ Hybridization probes' (VARNISH) for probe design. Our method utilizes advances in DNA-PAINT methodologies, including qPAINT for quantification, and Exchange-PAINT for multiplexing. We demonstrated these capabilities of sRNA-PAINT by detecting and quantifying small RNAs in different cell layers of early developmental stage maize anthers that are important for male sexual reproduction., (© The Author(s) 2020. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Small RNAs are abundant in plant reproductive tissues, especially 24-nucleotide (nt) small interfering RNAs (siRNAs). Most 24-nt siRNAs are dependent on RNA Pol IV and RNA-DEPENDENT RNA POLYMERASE 2 (RDR2) and establish DNA methylation at thousands of genomic loci in a process called RNA-directed DNA methylation (RdDM). In Brassica rapa , RdDM is required in the maternal sporophyte for successful seed development. Here, we demonstrate that a small number of siRNA loci account for over 90% of siRNA expression during B. rapa seed development. These loci exhibit unique characteristics with regard to their copy number and association with genomic features, but they resemble canonical 24-nt siRNA loci in their dependence on RNA Pol IV/RDR2 and role in RdDM. These loci are expressed in ovules before fertilization and in the seed coat, embryo, and endosperm following fertilization. We observed a similar pattern of 24-nt siRNA expression in diverse angiosperms despite rapid sequence evolution at siren loci. In the endosperm, siren siRNAs show a marked maternal bias, and siren expression in maternal sporophytic tissues is required for siren siRNA accumulation. Together, these results demonstrate that seed development occurs under the influence of abundant maternal siRNAs that might be transported to, and function in, filial tissues., Competing Interests: The authors declare no competing interest.

We developed public web sites and resources for data access, display, and analysis of plant small RNAs. These web sites are interconnected with related data types. The current generation of these informatics tools was developed for Illumina data, evolving over more than 15 years of improvements. Our online databases have customized web interfaces to uniquely handle and display RNA-derived data from diverse plant species, ranging from Arabidopsis ( Arabidopsis thaliana ) to wheat ( Triticum spp.), including many crop and model species. The web interface displays the abundance and genomic context of data for small RNAs, parallel analysis of RNA ends/degradome reads, RNA sequencing, and even chromatin immunoprecipitation sequencing data; it also provides information about potentially novel transcripts (antisense transcripts, alternative splice isoforms, and regulatory intergenic transcripts). Numerous options are included for downloading data as tables or via web services. Interpretation of these data is facilitated by the inclusion of extensive repeat or transposon data in our genome viewer. We have developed graphical and analytical tools, including a new viewer and a query page for the analysis of phased small RNAs; these are particularly useful for understanding the complex small RNA pathways of plants. These public databases are accessible at https://mpss.danforthcenter.org., (© 2020 American Society of Plant Biologists. All Rights Reserved.)

Small RNAs (sRNAs) regulate gene expression, play important roles in epigenetic pathways, and are hypothesized to contribute to hybrid vigor in plants. Prior investigations have provided valuable insights into associations between sRNAs and heterosis, often using a single hybrid genotype or tissue, but our understanding of the role of sRNAs and their potential value to plant breeding are limited by an incomplete picture of sRNA variation between diverse genotypes and development stages. Here, we provide a deep exploration of sRNA variation and inheritance among a panel of 108 maize ( Zea mays ) samples spanning five tissues from eight inbred parents and 12 hybrid genotypes, covering a spectrum of heterotic groups, genetic variation, and levels of heterosis for various traits. We document substantial developmental and genotypic influences on sRNA expression, with varying patterns for 21-nucleotide (nt), 22-nt, and 24-nt sRNAs. We provide a detailed view of the distribution of sRNAs in the maize genome, revealing a complex makeup that also shows developmental plasticity, particularly for 22-nt sRNAs. sRNAs exhibited substantially more variation between inbreds as compared with observed variation for gene expression. In hybrids, we identify locus-specific examples of nonadditive inheritance, mostly characterized as partial or complete dominance, but rarely outside the parental range. However, the global abundance of 21-nt, 22-nt, and 24-nt sRNAs varies very little between inbreds and hybrids, suggesting that hybridization affects sRNA expression principally at specific loci rather than on a global scale. This study provides a valuable resource for understanding the potential role of sRNAs in hybrid vigor., (© 2020 American Society of Plant Biologists. All Rights Reserved.)

Background: Plants encounter pathogenic and non-pathogenic microorganisms on a nearly constant basis. Small RNAs such as siRNAs and miRNAs/milRNAs influence pathogen virulence and host defense responses. We exploited the biotrophic interaction between the powdery mildew fungus, Blumeria graminis f. sp. hordei (Bgh), and its diploid host plant, barley (Hordeum vulgare) to explore fungal and plant sRNAs expressed during Bgh infection of barley leaf epidermal cells., Results: RNA was isolated from four fast-neutron immune-signaling mutants and their progenitor over a time course representing key stages of Bgh infection, including appressorium formation, penetration of epidermal cells, and development of haustorial feeding structures. The Cereal Introduction (CI) 16151 progenitor carries the resistance allele Mla6, while Bgh isolate 5874 harbors the AVR a6 avirulence effector, resulting in an incompatible interaction. Parallel Analysis of RNA Ends (PARE) was used to verify sRNAs with likely transcript targets in both barley and Bgh. Bgh sRNAs are predicted to regulate effectors, metabolic genes, and translation-related genes. Barley sRNAs are predicted to influence the accumulation of transcripts that encode auxin response factors, NAC transcription factors, homeodomain transcription factors, and several splicing factors. We also identified phasing small interfering RNAs (phasiRNAs) in barley that overlap transcripts that encode receptor-like kinases (RLKs) and nucleotide-binding, leucine-rich domain proteins (NLRs)., Conclusions: These data suggest that Bgh sRNAs regulate gene expression in metabolism, translation-related, and pathogen effectors. PARE-validated targets of predicted Bgh milRNAs include both EKA (effectors homologous to AVR k1 and AVR a10 ) and CSEP (candidate secreted effector protein) families. We also identified barley phasiRNAs and miRNAs in response to Bgh infection. These include phasiRNA loci that overlap with a significant proportion of receptor-like kinases, suggesting an additional sRNA control mechanism may be active in barley leaves as opposed to predominant R-gene phasiRNA overlap in many eudicots. In addition, we identified conserved miRNAs, novel miRNA candidates, and barley genome mapped sRNAs that have PARE validated transcript targets in barley. The miRNA target transcripts are enriched in transcription factors, signaling-related proteins, and photosynthesis-related proteins. Together these results suggest both barley and Bgh control metabolism and infection-related responses via the specific accumulation and targeting of genes via sRNAs.

Small RNAs are key regulators in plant growth and development. One subclass, phased siRNAs (phasiRNAs) require a trigger microRNA for their biogenesis. In grasses, two pathways yield abundant phasiRNAs during anther development; miR2275 triggers one class, 24-nt phasiRNAs, coincident with meiosis, while a second class of 21-nt phasiRNAs are present in premeiotic anthers. Here we report that the 24-nt phasiRNA pathway is widely present in flowering plants, indicating that 24-nt reproductive phasiRNAs likely originated with the evolutionary emergence of anthers. Deep comparative genomic analyses demonstrated that this miR2275/24-nt phasiRNA pathway is widely present in eudicots plants, however, it is absent in legumes and in the model plant Arabidopsis, demonstrating a dynamic evolutionary history of this pathway. In Solanaceae species, 24-nt phasiRNAs were observed, but the miR2275 trigger is missing and some loci displaying 12-nt phasing. Both the miR2275-triggered and Solanaceae 24-nt phasiRNAs are enriched in meiotic stages, implicating these phasiRNAs in anther and/or pollen development, a spatiotemporal pattern consistent in all angiosperm lineages that deploy them.

Little is known about the characteristics and function of reproductive phased, secondary, small interfering RNAs (phasiRNAs) in the Poaceae, despite the availability of significant genomic resources, experimental data, and a growing number of computational tools. We utilized machine-learning methods to identify sequence-based and positional features that distinguish phasiRNAs in rice and maize from other small RNAs (sRNAs). We developed Random Forest classifiers that can distinguish reproductive phasiRNAs from other sRNAs in complex sets of sequencing data, utilizing sequence-based (k-mers) and features describing position-specific sequence biases. The classification performance attained is > 80% in accuracy, sensitivity, specificity, and positive predicted value. Feature selection identified important features in both ends of phasiRNAs. We demonstrated that phasiRNAs have strand specificity and position-specific nucleotide biases potentially influencing AGO sorting; we also predicted targets to infer functions of phasiRNAs, and computationally assessed their sequence characteristics relative to other sRNAs. Our results demonstrate that machine-learning methods effectively identify phasiRNAs despite the lack of characteristic features typically present in precursor loci of other small RNAs, such as sequence conservation or structural motifs. The 5'-end features we identified provide insights into AGO-phasiRNA interactions. We describe a hypothetical model of competition for AGO loading between phasiRNAs of different nucleotide compositions., (© 2018 The Authors. New Phytologist © 2018 New Phytologist Trust.)

Post-transcriptional gene silencing in plants results from independent activities of diverse small RNA types. In anthers of grasses, hundreds of loci yield noncoding RNAs that are processed into 21- and 24-nucleotide (nt) phased small interfering RNAs (phasiRNAs); these are triggered by miR2118 and miR2275. We characterized these 'reproductive phasiRNAs' from rice (Oryza sativa) panicles and anthers across seven developmental stages. Our computational analysis identified characteristics of the 21-nt reproductive phasiRNAs that impact their biogenesis, stability, and potential functions. We demonstrate that 21-nt reproductive phasiRNAs can function in cis to target their own precursors. We observed evidence of this cis regulatory activity in both rice and maize (Zea mays). We validated this activity with evidence of cleavage and a resulting shift in the pattern of phasiRNA production. We characterize biases in phasiRNA biogenesis, demonstrating that the Pol II-derived 'top' strand phasiRNAs are consistently higher in abundance than the bottom strand. The first phasiRNA from each precursor overlaps the miR2118 target site, and this impacts phasiRNA accumulation or stability, evident in the weak accumulation of this phasiRNA position. Additional influences on this first phasiRNA duplex include the sequence composition and length, and we show that these factors impact Argonaute loading., (© 2018 The Authors. New Phytologist © 2018 New Phytologist Trust.)

MicroRNAs (miRNAs) are key regulators of gene expression. A handful of miRNAs are broadly conserved in land plants, while the majority are lineage specific; this review describes the processes by which new miRNAs are hypothesized to have emerged. Two major models describe miRNA origins, firstly, de novo emergence via inverted duplication of target gene fragments, and secondly, the expansion and neofunctionalization of existing miRNA families. The occasional acquisition of target sites by previously un-targeted genes adds further dynamism to the process by which miRNAs may shift roles during evolution. Additional factors guiding miRNA evolution include functional constraints on their length and the importance of precursor conservation that is observed in regions above or below the mature miRNA duplex; these regions represent recognition sites for components of biogenesis machinery and direct precursor processing. Insights into the mechanisms of miRNA emergence and divergence are important for understanding plant genome evolution and the impact of miRNA regulatory networks., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

The molecular basis of transgene susceptibility to silencing is poorly characterized in plants; thus, we evaluated several transgene design parameters as means to reduce heritable transgene silencing. Analyses of Arabidopsis plants with transgenes encoding a microalgal polyunsaturated fatty acid (PUFA) synthase revealed that small RNA (sRNA)-mediated silencing, combined with the use of repetitive regulatory elements, led to aggressive transposon-like silencing of canola-biased PUFA synthase transgenes. Diversifying regulatory sequences and using native microalgal coding sequences (CDSs) with higher GC content improved transgene expression and resulted in a remarkable trans-generational stability via reduced accumulation of sRNAs and DNA methylation. Further experiments in maize with transgenes individually expressing three crystal (Cry) proteins from Bacillus thuringiensis (Bt) tested the impact of CDS recoding using different codon bias tables. Transgenes with higher GC content exhibited increased transcript and protein accumulation. These results demonstrate that the sequence composition of transgene CDSs can directly impact silencing, providing design strategies for increasing transgene expression levels and reducing risks of heritable loss of transgene expression.

Small RNAs, including microRNAs (miRNAs) and small interfering RNAs (siRNAs), play a variety of important regulatory roles in many eukaryotes. Their small size has made it challenging to study them directly in live cells. Here we describe an RNA-based fluorescent sensor for small RNA detection both in vitro and in vivo, adaptable for any small RNA. It utilizes an sxRNA switch for detection of miRNA-mRNA interactions combined with a fluorophore-binding sequence 'Spinach', a GFP-like RNA aptamer for which the RNA-fluorophore complex exhibits strong and consistent fluorescence under an excitation wavelength. Two example sensors, FASTmiR171 and FASTmiR122, can rapidly detect and quantify the levels of miR171 and miR122 in vitro. The sensors can determine relative levels of miRNAs in total RNA extracts with sensitivity similar to small RNA sequencing and northern blots. FASTmiR sensors were also used to estimate the copy number range of miRNAs in total RNA extracts. To localize and analyze the spatial distribution of small RNAs in live, single cells, tandem copies of FASTmiR122 were expressed in different cell lines. FASTmiR122 was able to quantitatively detect the differences in miR122 levels in Huh7 and HEK293T cells demonstrating its potential for tracking miRNA expression and localization in vivo., (© The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.)

In plants, miR390 directs the production of tasiRNAs from TRANS-ACTING SIRNA3 ( TAS3 ) transcripts to regulate AUXIN RESPONSIVE FACTOR ( ARF ) genes, critical for auxin signaling; these tasiRNAs are known as tasiARFs. To understand the evolution of this miR390- TAS3 - ARF pathway, we characterized homologs of these three genes from thousands of plant species, from bryophytes to angiosperms. We found the lower-stem region of MIR390 genes, critical for accurate DICER-LIKE1 processing, is conserved in sequence in seed plants. We propose a model for the transition of functional tasiRNA sequences in TAS3 genes occurred at the emergence of vascular plants, in which the two miR390 target sites of TAS3 genes showed distinct pairing patterns. Based on the cleavability of miR390 target sites and the distance between target site and tasiARF, we inferred a potential bidirectional processing mechanism exists for some TAS3 genes. We also demonstrated a tight mutual selection between tasiARF and its target genes and that ARGONAUTE7, the partner of miR390, was specified later than other factors in the pathway. All these data illuminate the evolutionary path of the miR390- TAS3 - ARF pathway in land plants and demonstrate the significant variation that occurs in this functionally important and archetypal regulatory circuit., (© 2017 American Society of Plant Biologists. All rights reserved.)

Dissection of the genetic pathways and mechanisms by which anther development occurs in grasses is crucial for both a basic understanding of plant development and for examining traits of agronomic importance such as male sterility. In rice, MULTIPLE SPOROCYTES1 (MSP1), a leucine-rich-repeat receptor kinase, plays an important role in anther development by limiting the number of sporocytes. OsTDL1a (a TPD1-like gene in rice) encodes a small protein that acts as a cofactor of MSP1 in the same regulatory pathway. In this study, we analyzed small RNA and mRNA changes in different stages of spikelets from wild-type rice, and from msp1 and ostdl1a mutants. Analysis of the small RNA data identified miRNAs demonstrating differential abundances. miR2275 was depleted in the two rice mutants; this miRNA is specifically enriched in anthers and functions to trigger the production of 24-nt phased secondary siRNAs (phasiRNAs) from PHAS loci. We observed that the 24-nt phasiRNAs as well as their precursor PHAS mRNAs were also depleted in the two mutants. An analysis of co-expression identified three Argonaute-encoding genes (OsAGO1d, OsAGO2b, and OsAGO18) that accumulate transcripts coordinately with phasiRNAs, suggesting a functional relationship. By mRNA in situ analysis, we demonstrated a strong correlation between the spatiotemporal pattern of these OsAGO transcripts and phasiRNA accumulations., (© The Author 2016. Published by Oxford University Press on behalf of the Society for Experimental Biology.)

Plant small RNAs play important roles in transcriptional and posttranscriptional regulation, with ongoing work demonstrating their functions in diverse pathways. Their roles in defense responses are a topic of active investigation, particularly the rich set of micro (mi)RNAs that target disease resistance genes such as nucleotide binding/leucine-rich repeat (NB-LRR) genes. The miRNA-NB-LRR interactions result in the production of phased, secondary small interfering (phasi)RNAs, and phasiRNAs function in both cis and trans to propagate negative regulatory effects across additional members of the target gene family. Yet, while phasiRNAs have the capacity to trigger targeted decay of specific targets, both in cis and trans, their functional relevance in NB-LRR regulation remains largely a matter of speculation.

In eudicot plants, the miR482/miR2118 superfamily regulates and instigates the production of phased secondary small interfering RNAs (siRNAs) from NB-LRR (nucleotide binding leucine-rich repeat) genes that encode disease resistance proteins. In grasses, this miRNA family triggers siRNA production specifically in reproductive tissues from long noncoding RNAs. To understand this functional divergence, we examined the small RNA population in the ancient gymnosperm Norway spruce (Picea abies). As many as 41 miRNA families in spruce were found to trigger phasiRNA (phased, secondary siRNAs) production from diverse PHAS loci, with a remarkable 19 miRNA families capable of targeting over 750 NB-LRR genes to generate phasiRNAs. miR482/miR2118, encoded in spruce by at least 24 precursor loci, targets not only NB-LRR genes to trigger phasiRNA production (as in eudicots) but also noncoding PHAS loci, generating phasiRNAs preferentially in male or female cones, reminiscent of its role in the grasses. These data suggest a dual function of miR482/miR2118 present in gymnosperms that was selectively yet divergently retained in flowering plants. A few MIR482/MIR2118 precursors possess an extremely long stem-loop structure, one arm of which shows significant sequence similarity to spruce NB-LRR genes, suggestive of an evolutionary origin from NB-LRR genes through gene duplication. We also characterized an expanded miR390-TAS3 (TRANS-ACTING SIRNA GENE 3)-ARF (AUXIN RESPONSIVE FACTOR) pathway, comprising 18 TAS3 genes of diverse features. Finally, we annotated spruce miRNAs and their targets. Taken together, these data expand our understanding of phasiRNA network in plants and the evolution of plant miRNAs, particularly miR482/miR2118 and its functional diversification., (© The Author 2015. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.)

RNA-directed DNA methylation in Arabidopsis thaliana is driven by the plant-specific RNA Polymerase IV (Pol IV). It has been assumed that a Pol IV transcript can give rise to multiple 24-nt small interfering RNAs (siRNAs) that target DNA methylation. Here, we demonstrate that Pol IV-dependent RNAs (P4RNAs) from wild-type Arabidopsis are surprisingly short in length (30 to 40 nt) and mirror 24-nt siRNAs in distribution, abundance, strand bias, and 5'-adenine preference. P4RNAs exhibit transcription start sites similar to Pol II products and are featured with 5'-monophosphates and 3'-misincorporated nucleotides. The 3'-misincorporation preferentially occurs at methylated cytosines on the template DNA strand, suggesting a co-transcriptional feedback to siRNA biogenesis by DNA methylation to reinforce silencing locally. These results highlight an unusual mechanism of Pol IV transcription and suggest a "one precursor, one siRNA" model for the biogenesis of 24-nt siRNAs in Arabidopsis., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Maize anthers, the male reproductive floral organs, express two classes of phased small-interfering RNAs (phasiRNAs). PhasiRNA precursors are transcribed by RNA polymerase II and map to low-copy, intergenic regions similar to PIWI-interacting RNAs (piRNAs) in mammalian testis. From 10 sequential cohorts of staged maize anthers plus mature pollen we find that 21-nt phased siRNAs from 463 loci appear abruptly after germinal and initial somatic cell fate specification and then diminish, whereas 24-nt phasiRNAs from 176 loci coordinately accumulate during meiosis and persist as anther somatic cells mature and haploid gametophytes differentiate into pollen. Male-sterile ocl4 anthers defective in epidermal signaling lack 21-nt phasiRNAs. Male-sterile mutants with subepidermal defects--mac1 (excess meiocytes), ms23 (defective pretapetal cells), and msca1 (no normal soma or meiocytes)--lack 24-nt phasiRNAs. ameiotic1 mutants (normal soma, no meiosis) accumulate both 21-nt and 24-nt phasiRNAs, ruling out meiotic cells as a source or regulator of phasiRNA biogenesis. By in situ hybridization, miR2118 triggers of 21-nt phasiRNA biogenesis localize to epidermis; however, 21-PHAS precursors and 21-nt phasiRNAs are abundant subepidermally. The miR2275 trigger, 24-PHAS precursors, and 24-nt phasiRNAs all accumulate preferentially in tapetum and meiocytes. Therefore, each phasiRNA type exhibits independent spatiotemporal regulation with 21-nt premeiotic phasiRNAs dependent on epidermal and 24-nt meiotic phasiRNAs dependent on tapetal cell differentiation. Maize phasiRNAs and mammalian piRNAs illustrate putative convergent evolution of small RNAs in male reproduction.

Parallel analysis of RNA ends (PARE) is a technique utilizing high-throughput sequencing to profile uncapped, mRNA cleavage or decay products on a genome-wide basis. Tools currently available to validate miRNA targets using PARE data employ only annotated genes, whereas important targets may be found in unannotated genomic regions. To handle such cases and to scale to the growing availability of PARE data and genomes, we developed a new tool, 'sPARTA' (small RNA-PARE target analyzer) that utilizes a built-in, plant-focused target prediction module (aka 'miRferno'). sPARTA not only exhibits an unprecedented gain in speed but also it shows greater predictive power by validating more targets, compared to a popular alternative. In addition, the novel 'seed-free' mode, optimized to find targets irrespective of complementarity in the seed-region, identifies novel intergenic targets. To fully capitalize on the novelty and strengths of sPARTA, we developed a web resource, 'comPARE', for plant miRNA target analysis; this facilitates the systematic identification and analysis of miRNA-target interactions across multiple species, integrated with visualization tools. This collation of high-throughput small RNA and PARE datasets from different genomes further facilitates re-evaluation of existing miRNA annotations, resulting in a 'cleaner' set of microRNAs., (© The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Background: The wild grass Brachypodium distachyon has emerged as a model system for temperate grasses and biofuel plants. However, the global analysis of miRNAs, molecules known to be key for eukaryotic gene regulation, has been limited in B. distachyon to studies examining a few samples or that rely on computational predictions. Similarly an in-depth global analysis of miRNA-mediated target cleavage using parallel analysis of RNA ends (PARE) data is lacking in B. distachyon., Results: B. distachyon small RNAs were cloned and deeply sequenced from 17 libraries that represent different tissues and stresses. Using a computational pipeline, we identified 116 miRNAs including not only conserved miRNAs that have not been reported in B. distachyon, but also non-conserved miRNAs that were not found in other plants. To investigate miRNA-mediated cleavage function, four PARE libraries were constructed from key tissues and sequenced to a total depth of approximately 70 million sequences. The roughly 5 million distinct genome-matched sequences that resulted represent an extensive dataset for analyzing small RNA-guided cleavage events. Analysis of the PARE and miRNA data provided experimental evidence for miRNA-mediated cleavage of 264 sites in predicted miRNA targets. In addition, PARE analysis revealed that differentially expressed miRNAs in the same family guide specific target RNA cleavage in a correspondingly tissue-preferential manner., Conclusions: B. distachyon miRNAs and target RNAs were experimentally identified and analyzed. Knowledge gained from this study should provide insights into the roles of miRNAs and the regulation of their targets in B. distachyon and related plants.

MicroRNAs (miRNAs) are small RNAs that derive from endogenous precursors harboring foldback structures. Plant miRNA precursors are quite variable in their size and shape. Still, the miRNA processing machinery, consisting of DICER-LIKE1 (DCL1) and accessory proteins recognize structural features on the precursors to cleave them at specific places releasing the mature miRNAs. The identification of miRNA processing intermediates in plants has mostly relied on a modified 5' RACE method, designed to detect the 5' end of uncapped RNAs. However, this method is time consuming and is, therefore, only practical for the analysis of a handful miRNAs. Here, we present a modification of this approach in order to perform genome-wide analysis of miRNA processing intermediates. Briefly, a reverse transcription is performed with a mixture of specific primers designed against all known miRNA precursors. miRNA processing intermediates are then specifically amplified to generate a library and subjected to deep sequencing. This method, called SPARE (Specific Parallel Amplification of 5' RNA Ends) allows the identification of processing intermediates for most of the Arabidopsis miRNAs. The results enable the determination of the DCL1 processing direction and the cleavage sites introduced by miRNA processing machinery in the precursors. The SPARE method can be easily adapted to detect miRNA-processing intermediates in other systems., (Copyright © 2013 Elsevier Inc. All rights reserved.)

MicroRNAs (miRNAs) derive from longer precursors with fold-back structures. While animal miRNA precursors have homogenous structures, plant precursors comprise a collection of fold-backs with variable size and shape. Here, we design an approach to systematically analyze miRNA processing intermediates and characterize the biogenesis of most of the evolutionarily conserved miRNAs present in Arabidopsis thaliana. We found that plant miRNAs are processed by four mechanisms, depending on the sequential direction of the processing machinery and the number of cuts required to release the miRNA. Classification of the precursors according to their processing mechanism revealed specific structural determinants for each group. We found that the complexity of the miRNA processing pathways occurs in both ancient and evolutionarily young sequences and that members of the same family can be processed in different ways. We observed that different structural determinants compete for the processing machinery and that alternative miRNAs can be generated from a single precursor. The results provide an explanation for the structural diversity of miRNA precursors in plants and new insights toward the understanding of the biogenesis of small RNAs.

Plant small RNAs are 3' methylated by the methyltransferase HUA1 ENHANCER1 (HEN1). In plant hen1 mutants, 3' modifications of small RNAs, including oligo-uridylation (tailing), are associated with accelerated degradation of microRNAs (miRNAs). By sequencing small RNAs of the wild type and hen1 mutants from Arabidopsis thaliana, rice (Oryza sativa), and maize (Zea mays), we found 3' truncation prior to tailing is widespread in these mutants. Moreover, the patterns of miRNA truncation and tailing differ substantially among miRNA families but are conserved across species. The same patterns are also observable in wild-type libraries from a broad range of species, only at lower abundances. ARGONAUTE (AGO1), even with defective slicer activity, can bind these truncated and tailed variants of miRNAs. An ago1 mutation in hen1 suppressed such 3' modifications, indicating that they occur while miRNAs are in association with AGO1, either during or after RNA-induced silencing complex assembly. Our results showed AGO1-bound miRNAs are actively 3' truncated and tailed, possibly reflecting the activity of cofactors acting in conserved patterns in miRNA degradation.

Plant genomes are the source of large numbers of small RNAs, generated via a variety of genetically separable pathways. Several of these pathways converge in the production of phased, secondary, small interfering RNAs (phasiRNAs), originally designated as trans-acting small interfering RNAs or tasiRNAs. PhasiRNA biogenesis requires the involvement of microRNAs as well as the cellular machinery for the production of siRNAs. PhasiRNAs in Arabidopsis thaliana have been well described for their ability to function in trans to suppress target transcript levels. Plant genomic data from an expanding set of species have demonstrated that Arabidopsis is relatively sparing in its use of phasiRNAs, while other genomes contain hundreds or even thousands of phasiRNA-generating loci. In the dicots, targets of those phasiRNAs include several large or conserved families of genes, such as those encoding disease resistance proteins or transcription factors. Suppression of nucleotide-binding, leucine-rich repeat (NB-LRR) disease resistance genes by small RNAs is particularly unusual because of a high level of redundancy. In this review, we discuss plant phasiRNAs and the possible mechanistic significance of phasiRNA-based regulation of the NB-LRRs.

Non-coding RNAs, especially small RNAs, play important roles in many biological processes. Several small RNA types, including microRNAs (miRNAs) and small interfering RNAs (siRNAs), are well-described in rice (Oryza sativa), although much remains to be learned about their function. Many small RNAs along with their targets have been characterized with deep sequencing technologies. Some special classes of these small RNAs have been found to be unique to rice or within the larger group of grasses. The functional and biological roles of numerous plants small RNAs have been described in detail, including functions as varied as the regulation of tissue development, phase transition, or abiotic and biotic stress resistance. Mutant analysis has proven useful in the genetic identification of components involved in small RNA biogenesis and also in identification of regulatory functions of small RNAs. Although many small RNAs have been identified by deep sequencing in rice, their precise regulatory functions and cell-type specificity are in many cases still unknown., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Trans-acting small interfering RNAs (tasiRNAs) are a major class of small RNAs performing essential biological functions in plants. The first reported tasiRNA pathway, that of miR173-TAS1/2, produces tasiRNAs regulating a set of pentatricopeptide repeat (PPR) genes and has been characterized only in Arabidopsis thaliana to date. Here, we demonstrate that the microRNA (miRNA)-trans-acting small interfering RNA gene (TAS)-pentatricopeptide repeat-containing gene (PPR)-small interfering RNA pathway is a highly dynamic and widespread feature of eudicots. Nine eudicot plants, representing six different plant families, have evolved similar tasiRNA pathways to initiate phased small interfering RNA (phasiRNA) production from PPR genes. The PPR phasiRNA production is triggered by different 22-nucleotide miRNAs, including miR7122, miR1509, and fve-PPRtri1/2, and through distinct mechanistic strategies exploiting miRNA direct targeting or indirect targeting through TAS-like genes (TASL), one-hit or two-hit, or even two layers of tasiRNA-TASL interactions. Intriguingly, although those miRNA triggers display high sequence divergence caused by the occurrence of frequent point mutations and splicing shifts, their corresponding MIRNA genes show pronounced identity to the Arabidopsis MIR173, implying a common origin of this group of miRNAs (super-miR7122). Further analyses reveal that super-miR7122 may have evolved from a newly defined miR4376 superfamily, which probably originated from the widely conserved miR390. The elucidation of this evolutionary path expands our understanding of the course of miRNA evolution, especially for relatively conserved miRNA families.

At least three pathways control maintenance of DNA cytosine methylation in Arabidopsis thaliana. However, the RNA-directed DNA methylation (RdDM) pathway is solely responsible for establishment of this silencing mark. We previously described INVOLVED IN DE NOVO 2 (IDN2) as being an RNA-binding RdDM component that is required for DNA methylation establishment. In this study, we describe the discovery of two partially redundant proteins that are paralogous to IDN2 and that form a stable complex with IDN2 in vivo. Null mutations in both genes, termed IDN2-LIKE 1 and IDN2-LIKE 2 (IDNL1 and IDNL2), result in a phenotype that mirrors, but does not further enhance, the idn2 mutant phenotype. Genetic analysis suggests that this complex acts in a step in the downstream portion of the RdDM pathway. We also have performed structural analysis showing that the IDN2 XS domain adopts an RNA recognition motif (RRM) fold. Finally, genome-wide DNA methylation and expression analysis confirms the placement of the IDN proteins in an RdDM pathway that affects DNA methylation and transcriptional control at many sites in the genome. Results from this study identify and describe two unique components of the RdDM machinery, adding to our understanding of DNA methylation control in the Arabidopsis genome.

Higher plants have evolved multiple proteins in the RNase III family to produce and regulate different classes of small RNAs with specialized molecular functions. In rice (Oryza sativa), numerous genomic clusters are targeted by one of two microRNAs (miRNAs), miR2118 and miR2275, to produce secondary small interfering RNAs (siRNAs) of either 21 or 24 nucleotides in a phased manner. The biogenesis requirements or the functions of the phased small RNAs are completely unknown. Here we examine the rice Dicer-Like (DCL) family, including OsDCL1, -3a, -3b and -4. By deep sequencing of small RNAs from different tissues of the wild type and osdcl4-1, we revealed that the processing of 21-nucleotide siRNAs, including trans-acting siRNAs (tasiRNA) and over 1000 phased small RNA loci, was largely dependent on OsDCL4. Surprisingly, the processing of 24-nucleotide phased small RNA requires the DCL3 homolog OsDCL3b rather than OsDCL3a, suggesting functional divergence within DCL3 family. RNA ligase-mediated 5' rapid amplification of cDNA ends and parallel analysis of RNA ends (PARE)/degradome analysis confirmed that most of the 21- and 24-nucleotide phased small RNA clusters were initiated from the target sites of miR2118 and miR2275, respectively. Furthermore, the accumulation of the two triggering miRNAs requires OsDCL1 activity. Finally, we show that phased small RNAs are preferentially produced in the male reproductive organs and are likely to be conserved in monocots. Our results revealed significant roles of OsDCL4, OsDCL3b and OsDCL1 in the 21- and 24-nucleotide phased small RNA biogenesis pathway in rice., (© 2011 The Authors. The Plant Journal © 2011 Blackwell Publishing Ltd.)

Small RNAs have a variety of important roles in plant development, stress responses, and other processes. They exert their influence by guiding mRNA cleavage, translational repression, and chromatin modification. To identify previously unknown rice (Oryza sativa) microRNAs (miRNAs) and those regulated by environmental stress, 62 small RNA libraries were constructed from rice plants and used for deep sequencing with Illumina technology. The libraries represent several tissues from control plants and plants subjected to different environmental stress treatments. More than 94 million genome-matched reads were obtained, resulting in more than 16 million distinct small RNA sequences. This allowed an evaluation of ~400 annotated miRNAs with current criteria and the finding that among these, ~150 had small interfering RNA-like characteristics. Seventy-six new miRNAs were found, and miRNAs regulated in response to water stress, nutrient stress, or temperature stress were identified. Among the new examples of miRNA regulation were members of the same miRNA family that were differentially regulated in different organs and had distinct sequences Some of these distinct family members result in differential target cleavage and provide new insight about how an agriculturally important rice phenotype could be regulated in the panicle. This high-resolution analysis of rice miRNAs should be relevant to plant miRNAs in general, particularly in the Poaceae.

RNA-directed DNA methylation (RdDM) is a small interfering RNA (siRNA)-mediated epigenetic modification that contributes to transposon silencing in plants. RdDM requires a complex transcriptional machinery that includes specialized RNA polymerases, named Pol IV and Pol V, as well as chromatin remodelling proteins, transcription factors, RNA binding proteins, and other plant-specific proteins whose functions are not yet clarified. In Arabidopsis thaliana, DICER-LIKE3 and members of the ARGONAUTE4 group of ARGONAUTE (AGO) proteins are involved, respectively, in generating and using 24-nt siRNAs that trigger methylation and transcriptional gene silencing of homologous promoter sequences. AGO4 is the main AGO protein implicated in the RdDM pathway. Here we report the identification of the related AGO6 in a forward genetic screen for mutants defective in RdDM and transcriptional gene silencing in shoot and root apical meristems in Arabidopsis thaliana. The identification of AGO6, and not AGO4, in our screen is consistent with the primary expression of AGO6 in shoot and root growing points.

Paramutation is the epigenetic transfer of information between alleles that leads to the heritable change of expression of one allele. Paramutation at the b1 locus in maize requires seven noncoding tandem repeat (b1TR) sequences located approximately 100 kb upstream of the transcription start site of b1, and mutations in several genes required for paramutation implicate an RNA-mediated mechanism. The mediator of paramutation (mop1) gene, which encodes a protein closely related to RNA-dependent RNA polymerases, is absolutely required for paramutation. Herein, we investigate the potential function of mop1 and the siRNAs that are produced from the b1TR sequences. Production of siRNAs from the b1TR sequences depends on a functional mop1 gene, but transcription of the repeats is not dependent on mop1. Further nuclear transcription assays suggest that the b1TR sequences are likely transcribed predominantly by RNA polymerase II. To address whether production of b1TR-siRNAs correlated with paramutation, we examined siRNA production in alleles that cannot undergo paramutation. Alleles that cannot participate in paramutation also produce b1TR-siRNAs, suggesting that b1TR-siRNAs are not sufficient for paramutation in the tissues analyzed. However, when b1TR-siRNAs are produced from a transgene expressing a hairpin RNA, b1 paramutation can be recapitulated. We hypothesize that either the b1TR-siRNAs or the dsRNA template mediates the trans-communication between the alleles that establishes paramutation. In addition, we uncovered a role for mop1 in the biogenesis of a subset of microRNAs (miRNAs) and show that it functions at the level of production of the primary miRNA transcripts.

RNA-directed DNA methylation (RdDM) in plants requires two RNA polymerase (Pol) II-related RNA polymerases, namely Pol IV and Pol V. A genetic screen designed to reveal factors that are important for RdDM in a developmental context in Arabidopsis identified DEFECTIVE IN MERISTEM SILENCING 4 (DMS4). Unlike other mutants defective in RdDM, dms4 mutants have a pleiotropic developmental phenotype. The DMS4 protein is similar to yeast IWR1 (interacts with RNA polymerase II), a conserved putative transcription factor that interacts with Pol II subunits. The DMS4 complementary DNA partly complements the K1 killer toxin hypersensitivity of a yeast iwr1 mutant, suggesting some functional conservation. In the transgenic system studied, mutations in DMS4 directly or indirectly affect Pol IV-dependent secondary short interfering RNAs, Pol V-mediated RdDM, Pol V-dependent synthesis of intergenic non-coding RNA and expression of many Pol II-driven genes. These data suggest that DMS4 might be a regulatory factor for several RNA polymerases, thus explaining its diverse roles in the plant.

Small RNAs play an important role in plant development, stress responses, and epigenetic regulation, primarily through their role in transcriptional and post-transcriptional silencing of specific target genes and loci. Most if not all plants utilize these small RNA signaling networks. We have developed a deep-sequencing based dataset of plant small RNAs, based on the hypothesis that comparisons among the complex pool of small RNAs from diverse plants will identify novel types of conserved, regulated, or species-specific molecules. A database containing upward of hundreds of millions of plant small RNA sequences is being created for comparative analyses. This small RNA database will allow the experimental characterization of the majority of the biologically important small RNAs for a range of plant species. This database can be accessed from our website (http://smallrna.udel.edu/). A variety of web-based tools have been developed for analyses of these data. Here, we focus on these tools, and we describe how the users can implement these tools to analyze and interpret the small RNA data and how the users could use similar approaches for other sets of plant small RNAs from diverse species.

Background: Miscanthus x giganteus (Mxg) is a perennial grass that produces superior biomass yields in temperate environments. The essentially uncharacterized triploid genome (3n = 57, x = 19) of Mxg is likely critical for the rapid growth of this vegetatively propagated interspecific hybrid., Results: A survey of the complex Mxg genome was conducted using 454 pyrosequencing of genomic DNA and Illumina sequencing-by-synthesis of small RNA. We found that the coding fraction of the Mxg genome has a high level of sequence identity to that of other grasses. Highly repetitive sequences representing the great majority of the Mxg genome were predicted using non-cognate assembly for de novo repeat detection. Twelve abundant families of repeat were observed, with those related to either transposons or centromeric repeats likely to comprise over 95% of the genome. Comparisons of abundant repeat sequences to a small RNA survey of three Mxg organs (leaf, rhizome, inflorescence) revealed that the majority of observed 24-nucleotide small RNAs are derived from these repetitive sequences. We show that high-copy-number repeats match more of the small RNA, even when the amount of the repeat sequence in the genome is accounted for., Conclusions: We show that major repeats are present within the triploid Mxg genome and are actively producing small RNAs. We also confirm the hypothesized origins of Mxg, and suggest that while the repeat content of Mxg differs from sorghum, the sorghum genome is likely to be of utility in the assembly of a gene-space sequence of Mxg.

MicroRNAs (miRNAs) are approximately 21 nucleotide noncoding RNAs produced by Dicer-catalyzed excision from stem-loop precursors. Many plant miRNAs play critical roles in development, nutrient homeostasis, abiotic stress responses, and pathogen responses via interactions with specific target mRNAs. miRNAs are not the only Dicer-derived small RNAs produced by plants: A substantial amount of the total small RNA abundance and an overwhelming amount of small RNA sequence diversity is contributed by distinct classes of 21- to 24-nucleotide short interfering RNAs. This fact, coupled with the rapidly increasing rate of plant small RNA discovery, demands an increased rigor in miRNA annotations. Herein, we update the specific criteria required for the annotation of plant miRNAs, including experimental and computational data, as well as refinements to standard nomenclature.

Identification of all expressed transcripts in a sequenced genome is essential both for genome analysis and for realization of the goals of systems biology. We used the transcriptional profiling technology called 'massively parallel signature sequencing' to develop a comprehensive expression atlas of rice (Oryza sativa cv Nipponbare). We sequenced 46,971,553 mRNA transcripts from 22 libraries, and 2,953,855 small RNAs from 3 libraries. The data demonstrate widespread transcription throughout the genome, including sense expression of at least 25,500 annotated genes and antisense expression of nearly 9,000 annotated genes. An additional set of approximately 15,000 mRNA signatures mapped to unannotated genomic regions. The majority of the small RNA data represented lower abundance short interfering RNAs that match repetitive sequences, intergenic regions and genes. Among these, numerous clusters of highly regulated small RNAs were readily observed. We developed a genome browser (http://mpss.udel.edu/rice) for public access to the transcriptional profiling data for this important crop.

The class of small RNAs known as microRNAs (miRNAs) has a demonstrated role in the negative regulation of gene expression in both plants and animals. These small molecules have been shown to play a critical role in a wide range of developmental and physiological pathways. Although hundreds of different miRNAs have now been identified using cloning and computational approaches, characterization of their targets and biological roles has been more limited. New sequencing technologies promise to accelerate the sequencing of small RNAs and additional genetic and genomic strategies are being applied to assess their regulatory function on RNA targets. These technologies will enable the identification of large numbers of small RNAs from diverse species, and comparative genomics approaches based on these data are likely to identify additional miRNAs. Combined with bioinformatics and experimental approaches to separate miRNAs from short-interfering RNAs (siRNAs), the pace of miRNA discovery is likely to accelerate, leading to an improved understanding of miRNA function and biological significance.

MPSS (massively parallel signature sequencing) is a sequencing-based technology that uses a unique method to quantify gene expression level, generating millions of short sequence tags per library. We have created a series of databases for four species (Arabidopsis, rice, grape and Magnaporthe grisea, the rice blast fungus). Our MPSS databases measure the expression level of most genes under defined conditions and provide information about potentially novel transcripts (antisense transcripts, alternative splice isoforms and regulatory intergenic transcripts). A modified version of MPSS has been used to perform deep profiling of small RNAs from Arabidopsis, and we have recently adapted our database to display these data. Interpretation of the small RNA MPSS data is facilitated by the inclusion of extensive repeat data in our genome viewer. All the data and the tools introduced in this article are available at http://mpss.udel.edu.

Small RNAs play important regulatory roles in most eukaryotes, but only a small proportion of these molecules have been identified. We sequenced more than two million small RNAs from seedlings and the inflorescence of the model plant Arabidopsis thaliana. Known and new microRNAs (miRNAs) were among the most abundant of the nonredundant set of more than 75,000 sequences, whereas more than half represented lower abundance small interfering RNAs (siRNAs) that match repetitive sequences, intergenic regions, and genes. Individual or clusters of highly regulated small RNAs were readily observed. Targets of antisense RNA or miRNA did not appear to be preferentially associated with siRNAs. Many genomic regions previously considered featureless were found to be sites of numerous small RNAs.