Deep sequencing technologies such as Illumina, SOLiD, and 454 platforms have become very powerful tools in discovering and quantifying small RNAs in diverse organisms. Sequencing small RNA fractions always identifies RNAs derived from abundant RNA species such as rRNAs, tRNAs, snRNA, and snoRNA, and they are widely considered to be random degradation products. We carried out bioinformatic analysis of deep sequenced HeLa RNA and after quality filtering, identified highly abundant small RNA fragments, derived from mature tRNAs that are likely produced by specific processing rather than from random degradation. Moreover, we showed that the processing of small RNAs derived from tRNA Gln is dependent on Dicer in vivo and that Dicer cleaves the tRNA in vitro.

Small RNAs (21-24 nt) are involved in gene regulation through translation inhibition, mRNA cleavage, or directing chromatin modifications. In rice, currently Ϸ240 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 cisantisense 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.high-throughput sequencing ͉ siRNA ͉ small RNA ͉ massively parallel signature sequencing (MPSS) M ost eukaryotes possess small RNA-based gene silencing systems that can down-regulate genes at transcriptional and posttranscriptional levels (1, 2). At least five classes of these small regulatory RNAs (21-24 nt) have been characterized, including microRNAs (miRNAs), heterochromatic siRNAs, transacting siRNAs (ta-siRNAs), natural antisense siRNAs (nat-siRNAs), and, in metazoans, the Piwi-interacting RNAs (3-7). miRNAs are processed from self-complementary transcripts by the activity of Dicer ribonucleases. siRNAs originate from longer, doublestranded (ds)RNA molecules and usually represent both strands of the RNA, although they are similar in biochemical structure to miRNAs and have some functional similarities. In plants, siRNAs typically derive from transposons, repetitive sequences, and transgenes. These siRNAs could be involved in DNA methylation and histone modifications that silence target transcription (8). Although nat-siRNAs also have been identified (9, 10), natural antisense miRNAs (nat-miRNAs) have not been reported in any system.Plant miRNAs have near-perfect pairing to their targets and therefore generally cause mRNA cleavage. Numerous studies have demonstrated the critical role of miRNAs in controlling developmental processes and organ identity. As of April 2007, the miRNA Sanger database contained 916 plant miRNAs. The list is rapidly growing as a result of new deep-sequencing technologies for small RNA discovery. In Arabidopsis, small RNAs from various mutants, tissues, and developmental stages have been analyzed by highthroughput pyrosequencing (11-15). These efforts identified at least 184 miRNAs (Ϸ70 families) in Arabidopsis. Cloning of miRNAs from lower plants such as moss indicates that some miRNAs are conserved over a long evolutionary distance. In fact, most miRNAs identified in the early studies (21/28) are conserved in more than one plant species, although some miRNAs...

Alternative splicing enhances transcriptome diversity in all eukaryotes and plays a role in plant tissue identity and stress adaptation. To catalog new maize (Zea mays) transcripts and identify genomic loci that regulate alternative splicing, we analyzed over 90 RNA-seq libraries from maize inbred lines B73 and Mo17, as well as Syn10 doubled haploid lines (progenies from B73 3 Mo17). Transcript discovery was augmented with publicly available data from 14 maize tissues, expanding the maize transcriptome by more than 30,000 and increasing the percentage of intron-containing genes that undergo alternative splicing to 40%. These newly identified transcripts greatly increase the diversity of the maize proteome, sometimes coding for entirely different proteins compared with their most similar annotated isoform. In addition to increasing proteome diversity, many genes encoding novel transcripts gained an additional layer of regulation by microRNAs, often in a tissue-specific manner. We also demonstrate that the majority of genotype-specific alternative splicing can be genetically mapped, with cis-acting quantitative trait loci (QTLs) predominating. A large number of trans-acting QTLs were also apparent, with nearly half located in regions not shown to contain genes associated with splicing. Taken together, these results highlight the currently underappreciated role that alternative splicing plays in tissue identity and genotypic variation in maize.

Alternative splicing plays a crucial role in plant development as well as stress responses. Although alternative splicing has been studied during development and in response to stress, the interplay between these two factors remains an open question. To assess the effects of drought stress on developmentally regulated splicing in maize (Zea mays), 94 RNA-seq libraries from ear, tassel, and leaf of the B73 public inbred line were constructed at four developmental stages under both well-watered and drought conditions. This analysis was supplemented with a publicly available series of 53 libraries from developing seed, embryo, and endosperm. More than 48,000 novel isoforms, often with stage-or condition-specific expression, were uncovered, suggesting that developmentally regulated alternative splicing occurs in thousands of genes. Drought induced large developmental splicing changes in leaf and ear but relatively few in tassel. Most developmental stage-specific splicing changes affected by drought were tissue dependent, whereas stage-independent changes frequently overlapped between leaf and ear. A linear relationship was found between gene expression changes in splicing factors and alternative spicing of other genes during development. Collectively, these results demonstrate that alternative splicing is strongly associated with tissue type, developmental stage, and stress condition.After transcription, the majority of eukaryotic premRNA is subjected to a series of posttranscriptional modifications, including the removal of introns to form a mature mRNA (Stamm et al., 2005). Although some introns are removed constitutively, many can be processed in a variety of alternative ways, including exon skipping, intron retention, alternative acceptor, alternative donor, and alternative position (change in both acceptor and donor positions; Lorkovic et al., 2000). These alternative splicing events form a crucial regulatory level and have the ability to alter an mRNA's stability, localization, and protein products. Alternative splicing events are controlled by a variety of cis-elements, including the presence of consensus splice sequences at the intron-exon border and intronic and exonic splicing enhancer sequences (Pertea et al., 2007). Trans-acting factors also exert a strong effect on splicing and are mostly composed of Ser/Arg-rich proteins, which typically promote intron removal, and heterogenous nuclear ribonucleoproteins, which typically inhibit it (Erkelenz et al., 2013). A host of other indirect factors can affect splicing, including transcription rate, methylation status, and any cellular conditions that alter RNA secondary structure (Kornblihtt et al

Small, non-coding microRNAs (miRNAs) have been implicated in many biological processes, including the development of the nervous system. However, the roles of miRNAs in natural behavioral and neuronal plasticity are not well understood. To help address this we characterized the microRNA transcriptome in the adult worker honey bee head and investigated whether changes in microRNA expression levels in the brain are associated with division of labor among honey bees, a well-established model for socially regulated behavior. We determined that several miRNAs were downregulated in bees that specialize on brood care (nurses) relative to foragers. Additional experiments showed that this downregulation is dependent upon social context; it only occurred when nurse bees were in colonies that also contained foragers. Analyses of conservation patterns of brain-expressed miRNAs across Hymenoptera suggest a role for certain miRNAs in the evolution of the Aculeata, which includes all the eusocial hymenopteran species. Our results support the intriguing hypothesis that miRNAs are important regulators of social behavior at both developmental and evolutionary time scales.

MicroRNAs (miRNAs) are a class of small RNAs, which typically function by guiding cleavage of target mRNAs. They are known to play roles in a variety of plant processes including development, responses to environmental stresses and senescence. To identify senescence regulation of miRNAs in Arabidopsis thaliana, eight small RNA libraries were constructed and sequenced at four different stages of development and senescence from both leaves and siliques, resulting in more than 200 million genome-matched sequences. Parallel analysis of RNA ends libraries, which enable the large-scale examination of miRNA-guided cleavage products, were constructed and sequenced, resulting in over 750 million genome-matched sequences. These large datasets led to the identification a new senescence-inducible small RNA locus, as well as new regulation of known miRNAs and their target genes during senescence, many of which have established roles in nutrient responsiveness and cell structural integrity. In keeping with remobilization of nutrients thought to occur during senescence, many miRNAs and targets had opposite expression pattern changes between leaf and silique tissues during the progression of senescence. Taken together, these findings highlight the integral role that miRNAs may play in the remobilization of resources and alteration of cellular structure that is known to occur in senescence.

(P.J.G.).MicroRNAs (miRNAs) are a class of small RNAs that typically function by guiding the cleavage of target messenger RNAs. They have been shown to play major roles in a variety of plant processes, including development, and responses to pathogens and environmental stresses. To identify new miRNAs and regulation in Arabidopsis (Arabidopsis thaliana), 27 small RNA libraries were constructed and sequenced from various tissues, stresses, and small RNA biogenesis mutants, resulting in 95 million genome-matched sequences. The use of rdr2 to enrich the miRNA population greatly enhanced this analysis and led to the discovery of new miRNAs arising from both known and new precursors, increasing the total number of Arabidopsis miRNAs by about 10%. Parallel Analysis of RNA Ends data provide evidence that the majority guide target cleavage. Many libraries represented novel stress/tissue conditions, such as submergence-stressed flowers, which enabled the identification of new stress regulation of both miRNAs and their targets, all of which were validated in wild-type plants. By combining small RNA expression analysis with ARGONAUTE immunoprecipitation data and global target cleavage data from Parallel Analysis of RNA Ends, a much more complete picture of Arabidopsis miRNAs was obtained. In particular, the discovery of ARGONAUTE loading and target cleavage biases gave important insights into tissue-specific expression patterns, pathogen responses, and the role of sequence variation among closely related miRNA family members that would not be evident without this combinatorial approach.Small RNAs are a type of noncoding RNA that regulate gene expression at the transcriptional or posttranscriptional level. The two major classes of small RNAs are small interfering RNAs (siRNAs) and microRNAs (miRNAs). While both classes are similar in size (20-24 nucleotides [nt]), siRNAs and miRNAs have different biogenesis pathways and gene regulation mechanisms. In plants, siRNAs are generated from double-stranded RNAs that are synthesized by RNA-dependent RNA polymerase (RDR) proteins (Xie et al., 2004). These long double-stranded precursors are processed into 21-to 24-nt siRNAs by a member of the DICER-LIKE (DCL) family of proteins, typically DCL2, DCL3, or DCL4. Most siRNAs direct chromatin modifications and DNA methylation to regulate transcription (Lippman and Martienssen, 2004). In contrast, miRNAs are excised from precursors that are transcribed by RNA polymerase II as single-stranded precursors. These precursors have regions of self-complementarity and adopt a stem-loop secondary structure. DCL1 protein recognizes the secondary structure and processes the precursor into a miRNA:miRNA-star (miRNA*) duplex (Park et al., 2002). From the duplex, the miRNA associates with an ARGONAUTE (AGO) protein in an RNAinduced silencing complex, where the miRNA base pairs with a complementary sequence in a target mRNA, directing posttranscriptional regulation of the mRNA through cleavage or translational repression (Bohmert et al., 1998).

MicroRNAs (miRNAs) are small regulatory noncoding RNAs varying in length between 20 and 24 nucleotides. They play a key role during plant development by negatively regulating gene expression at the posttranscriptional level. Moreover, recent studies reported several miRNAs associated with abiotic stress responses. Small RNA cloning and high-throughput deep sequencing methods provide expression profiles of not only known miRNAs, but also novel miRNAs. In this chapter, we describe the methods used to identify and characterize abiotic stress-associated miRNAs and their target genes.