MicroRNAs (miRNAs) are important regulatory molecules in most eukaryotes and identification of their target mRNAs is essential for their functional analysis. Whereas conventional methods rely on ...computational prediction and subsequent experimental validation of target RNAs, we directly sequenced >28,000,000 signatures from the 5′ ends of polyadenylated products of miRNA-mediated mRNA decay, isolated from inflorescence tissue of Arabidopsis thaliana, to discover novel miRNA-target RNA pairs. Within the set of ∼27,000 transcripts included in the 8,000,000 nonredundant signatures, several previously predicted but nonvalidated targets of miRNAs were found. Like validated targets, most showed a single abundant signature at the miRNA cleavage site, particularly in libraries from a mutant deficient in the 5′-to-3′ exonuclease AtXRN4. Although miRNAs in Arabidopsis have been extensively investigated, working in reverse from the cleaved targets resulted in the identification and validation of novel miRNAs. This versatile approach will affect the study of other aspects of RNA processing beyond miRNA-target RNA pairs.
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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 from plants are known to be highly complex and abundant, with this complexity proportional to genome size. Most endogenous siRNAs in Arabidopsis are dependent on RNA-DEPENDENT RNA ...POLYMERASE 2 (RDR2) for their biogenesis. Recent work has demonstrated that the maize MEDIATOR OF PARAMUTATION1 (mop1) gene is a predicted ortholog of RDR2. The mop1 gene is required for establishment of paramutation and maintenance of transcriptional silencing of transposons and transgenes, suggesting the potential involvement of small RNAs. We analyzed small RNAs in wild-type maize and in the isogenic mop¹⁻¹ loss-of-function mutant by using Illumina's sequencing-by-synthesis (SBS) technology, which allowed us to characterize the complement of maize small RNAs to considerable depth. Similar to rdr2 in Arabidopsis, in mop¹⁻¹, the 24-nucleotide (nt) endogenous heterochromatic short-interfering siRNAs were dramatically reduced, resulting in an enrichment of miRNAs and transacting siRNAs. In contrast to the Arabidopsis rdr2 mutant, the mop¹⁻¹ plants retained a highly abundant heterochromatic ≈22-nt class of small RNAs, suggesting a second mechanism for heterochromatic siRNA production. The enrichment of miRNAs and loss of 24-nt heterochromatic siRNAs in mop¹⁻¹ should be advantageous for miRNA discovery as the maize genome becomes more fully sequenced.
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Small RNAs (21-24 nt) are involved in gene regulation through translation inhibition, mRNA cleavage, or directing chromatin modifications. In rice, currently almost equal to240 microRNAs (miRNAs) ...have been annotated. We sequenced more than four million small RNAs from rice and identified another 24 miRNA genes. Among these, we found a unique class of miRNAs that derive from natural cis-antisense transcript pairs. This configuration generates miRNAs that can perfectly match their targets. We provide evidence that the miRNAs function by inducing mRNA cleavage in the middle of their complementary site. Their production requires Dicer-like 1 (DCL1) activity, which is essential for canonical miRNA biogenesis. All of the natural antisense miRNAs (nat-miRNAs) identified in this study have large introns in their precursors that appear critical for nat-miRNA evolution and for the formation of functional miRNA loci. These findings suggest that other natural cis-antisense loci with similar exon-intron arrangements could be another source of miRNA genes.
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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 ∼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.
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Polyadenylation sites mark the ends of mRNA transcripts. Alternative polyadenylation (APA) may alter sequence elements and/or the coding capacity of transcripts, a mechanism that has been ...demonstrated to regulate gene expression and transcriptome diversity. To study the role of APA in transcriptome dynamics, we analyzed a large-scale data set of RNA "tags" that signify poly(A) sites and expression levels of mRNA. These tags were derived from a wide range of tissues and developmental stages that were mutated or exposed to environmental treatments, and generated using digital gene expression (DGE)-based protocols of the massively parallel signature sequencing (MPSS-DGE) and the Illumina sequencing-by-synthesis (SBS-DGE) sequencing platforms. The data offer a global view of APA and how it contributes to transcriptome dynamics. Upon analysis of these data, we found that ∼60% of Arabidopsis genes have multiple poly(A) sites. Likewise, ∼47% and 82% of rice genes use APA, supported by MPSS-DGE and SBS-DGE tags, respectively. In both species, ∼49%-66% of APA events were mapped upstream of annotated stop codons. Interestingly, 10% of the transcriptomes are made up of APA transcripts that are differentially distributed among developmental stages and in tissues responding to environmental stresses, providing an additional level of transcriptome dynamics. Examples of pollen-specific APA switching and salicylic acid treatment-specific APA clearly demonstrated such dynamics. The significance of these APAs is more evident in the 3034 genes that have conserved APA events between rice and Arabidopsis.
Agrobacterium tumefaciens is a plant pathogen that has the natural ability of delivering and integrating a piece of its own DNA into plant genome. Although bacterial non-coding RNAs (ncRNAs) have ...been shown to regulate various biological processes including virulence, we have limited knowledge of how Agrobacterium ncRNAs regulate this unique inter-Kingdom gene transfer. Using whole transcriptome sequencing and an ncRNA search algorithm developed for this work, we identified 475 highly expressed candidate ncRNAs from A. tumefaciens C58, including 101 trans-encoded small RNAs (sRNAs), 354 antisense RNAs (asRNAs), 20 5' untranslated region (UTR) leaders including a RNA thermosensor and 6 riboswitches. Moreover, transcription start site (TSS) mapping analysis revealed that about 51% of the mapped mRNAs have 5' UTRs longer than 60 nt, suggesting that numerous cis-acting regulatory elements might be encoded in the A. tumefaciens genome. Eighteen asRNAs were found on the complementary strands of virA, virB, virC, virD, and virE operons. Fifteen ncRNAs were induced and 7 were suppressed by the Agrobacterium virulence (vir) gene inducer acetosyringone (AS), a phenolic compound secreted by the plants. Interestingly, fourteen of the AS-induced ncRNAs have putative vir box sequences in the upstream regions. We experimentally validated expression of 36 ncRNAs using Northern blot and Rapid Amplification of cDNA Ends analyses. We show functional relevance of two 5' UTR elements: a RNA thermonsensor (C1_109596F) that may regulate translation of the major cold shock protein cspA, and a thi-box riboswitch (C1_2541934R) that may transcriptionally regulate a thiamine biosynthesis operon, thiCOGG. Further studies on ncRNAs functions in this bacterium may provide insights and strategies that can be used to better manage pathogenic bacteria for plants and to improve Agrobacterum-mediated plant transformation.
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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 ∼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.
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Milling yield and eating quality are two important grain quality traits in rice. To identify the genes involved in these two traits, we performed a deep transcriptional analysis of developing seeds ...using both massively parallel signature sequencing (MPSS) and sequencing-by-synthesis (SBS). Five MPSS and five SBS libraries were constructed from 6-day-old developing seeds of Cypress (high milling yield), LaGrue (low milling yield), Ilpumbyeo (high eating quality), YR15965 (low eating quality), and Nipponbare (control).
The transcriptomes revealed by MPSS and SBS had a high correlation co-efficient (0.81 to 0.90), and about 70% of the transcripts were commonly identified in both types of the libraries. SBS, however, identified 30% more transcripts than MPSS. Among the highly expressed genes in Cypress and Ilpumbyeo, over 100 conserved cis regulatory elements were identified. Numerous specifically expressed transcription factor (TF) genes were identified in Cypress (282), LaGrue (312), Ilpumbyeo (363), YR15965 (260), and Nipponbare (357). Many key grain quality-related genes (i.e., genes involved in starch metabolism, aspartate amino acid metabolism, storage and allergenic protein synthesis, and seed maturation) that were expressed at high levels underwent alternative splicing and produced antisense transcripts either in Cypress or Ilpumbyeo. Further, a time course RT-PCR analysis confirmed a higher expression level of genes involved in starch metabolism such as those encoding ADP glucose pyrophosphorylase (AGPase) and granule bound starch synthase I (GBSS I) in Cypress than that in LaGrue during early seed development.
This study represents the most comprehensive analysis of the developing seed transcriptome of rice available to date. Using two high throughput sequencing methods, we identified many differentially expressed genes that may affect milling yield or eating quality in rice. Many of the identified genes are involved in the biosynthesis of starch, aspartate family amino acids, and storage proteins. Some of the differentially expressed genes could be useful for the development of molecular markers if they are located in a known QTL region for milling yield or eating quality in the rice genome. Therefore, our comprehensive and deep survey of the developing seed transcriptome in five rice cultivars has provided a rich genomic resource for further elucidating the molecular basis of grain quality in rice.
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Gene duplication is an important mechanism for evolution of new genes. In plants, a special group of transposable elements, called Pack-MULEs or transduplicates, is able to duplicate and amplify ...genes or gene fragments on a large scale. Despite the abundance of Pack-MULEs, the functionality of these duplicates is not clear. Here, we present a comprehensive analysis of expression and purifying selection on 2809 Pack-MULEs in rice (Oryza sativa), which are derived from 1501 parental genes. At least 22% of the Pack-MULEs are transcribed, and 28 Pack-MULEs have direct evidence of translation. Chimeric Pack-MULEs, which contain gene fragments from multiple genes, are much more frequently expressed than those derived only from a single gene. In addition, Pack-MULEs are frequently associated with small RNAs. The presence of these small RNAs is associated with a reduction in expression of both the Pack-MULEs and their parental genes. Furthermore, an assessment of the selection pressure on the Pack-MULEs using the ratio of nonsynonymous (Ka) and synonymous (Ks) substitution rates indicates that a considerable number of Pack-MULEs likely have been under selective constraint. The Ka/Ks values of Pack-MULE and parental gene pairs are lower among Pack-MULEs that are expressed in sense orientations. Taken together, our analysis suggests that a significant number of Pack-MULEs are expressed and subjected to purifying selection, and some are associated with small RNAs. Therefore, at least a subset of Pack-MULEs are likely functional and have great potential in regulating gene expression as well as providing novel coding capacities.
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