Molecular Insights into MpAGO1 and Its Regulatory miRNA, miR11707, in the High-Temperature Acclimation of Marchantia polymorpha.

As a model plant for bryophytes, Marchantia polymorpha offers insights into the role of RNA silencing in aiding early land plants navigate the challenges posed by high-temperature environments. Genomic analysis revealed unique ARGONAUTE1 ortholog gene (MpAGO1) in M. polymorpha, which is regulated by two species-specific microRNAs (miRNAs), miR11707.1 and miR11707.2. Comparative studies of small RNA profiles from M. polymorpha cellular and MpAGO1 immunoprecipitation (MpAGO1-IP) profiles at various temperatures, along with analyses of Arabidopsis AGO1 (AtAGO1), revealed that MpAGO1 has a low selectivity for a diverse range of small RNA species than AtAGO1. Protein structural comparisons revealed no discernible differences in the guide strand small RNA recognition middle domain, MID domain, of MpAGO1 and AtAGO1, suggesting the complexity of miRNA species specificity and necessitating further exploration. Small RNA profiling and size exclusion chromatography have pinpointed a subset of M. polymorpha miRNAs, notably miR11707, that remain in free form within the cell at 22°C but are loaded into MpAGO1 at 28°C to engage in RNA silencing. Investigations into the mir11707 gene editing (mir11707ge) mutants provided evidence of the regulation of miR11707 in MpAGO1. Notably, while MpAGO1 mRNA expression decreases at 28°C, the stability of the MpAGO1 protein and its associated miRNAs is essential for enhancing the RNA-inducing silencing complex (RISC) activity, revealing the importance of RNA silencing in enabling M. polymorpha to survive thermal stress. This study advances our understanding of RNA silencing in bryophytes and provides groundbreaking insights into the evolutionary resilience of land plants to climatic adversities.

Arabidopsis mRNA decay landscape shaped by XRN 5'-3' exoribonucleases.

5'-3' exoribonucleases (XRNs) play crucial roles in the control of RNA processing, quality, and quantity in eukaryotes. Although genome-wide profiling of RNA decay fragments is now feasible, how XRNs shape the plant mRNA degradome remains elusive. Here, we profiled and analyzed the RNA degradomes of Arabidopsis wild-type and mutant plants with defects in XRN activity. Deficiency of nuclear XRN3 or cytoplasmic XRN4 activity but not nuclear XRN2 activity greatly altered Arabidopsis mRNA decay profiles. Short excised linear introns and cleaved pre-mRNA fragments downstream of polyadenylation sites were polyadenylated and stabilized in the xrn3 mutant, demonstrating the unique function of XRN3 in the removal of cleavage remnants from pre-mRNA processing. Further analysis of stabilized XRN3 substrates confirmed that pre-mRNA 3' end cleavage frequently occurs after adenosine. The most abundant decay intermediates in wild-type plants include not only the primary substrates of XRN4 but also the products of XRN4-mediated cytoplasmic decay. An increase in decay intermediates with 5' ends upstream of a consensus motif in the xrn4 mutant suggests that there is an endonucleolytic cleavage mechanism targeting the 3' untranslated regions of many Arabidopsis mRNAs. However, analysis of decay fragments in the xrn4 mutant indicated that, except for microRNA-directed slicing, endonucleolytic cleavage events in the coding sequence rarely result in major decay intermediates. Together, these findings reveal the major substrates and products of nuclear and cytoplasmic XRNs along Arabidopsis transcripts and provide a basis for precise interpretation of RNA degradome data.

Association of Low Back Pain with Shift Work: A Meta-Analysis.

Shift work (SW) is the main working schedule worldwide, and it may cause sleep disorders, breast cancer, and cardiovascular disease. Low back pain (LBP) is a common problem in the workplace; however, the association between LBP and SW remains unclear. Therefore, we conducted a meta-analysis to determine the association between SW and LBP. This study was conducted in accordance with the Preferred Reporting Items for Systematic Reviews and Meta-Analyses guidelines. The PubMed, Embase, and Web of Science databases using a set of associated keywords were queried. The inclusion criteria were as follows: (1) adult employees hired by a company or organization; (2) SW exposure; and (3) the outcome of LBP according to examination or assessment. A total of 40 studies were included that met the inclusion criteria for the meta-analysis. SW was significantly associated with LBP (odds ratio [OR]: 1.31, 95% confidence interval [CI]: 1.18−1.47, p < 0.00001). Furthermore, it was observed that LBP was significantly associated with night shift (NS) (OR: 1.49, 95% CI: 1.24−1.82, p < 0.0001) but not with rotating shift (RS) (OR: 0.96, 95% CI: 0.76−1.22, p = 0.49). Moreover, LBP was significantly associated with SW in health care workers (HCWs) (OR: 1.40, 95% CI: 1.20−1.63, p < 0.0001) but not in non-HCWs (OR: 1.19, 95% CI: 0.94−1.50, p = 0.14). SW was significantly associated with LBP. Furthermore, the subgroup analysis showed that NS, but not RS, was associated with LBP. Compared with SW in non-HCWs, SW in HCWs was significantly associated with LBP.

Cultivar-specific markers, mutations, and chimerisim of Cavendish banana somaclonal variants resistant to Fusarium oxysporum f. sp. cubense tropical race 4.

BACKGROUND: The selection of tissue culture-derived somaclonal variants of Giant Cavendish banana (Musa spp., Cavendish sub-group AAA) by the Taiwan Banana Research Institute (TBRI) has resulted in several cultivars resistant to Fusarium oxysporum f. sp. cubense tropical race 4 (Foc TR4), a destructive fungus threatening global banana production. However, the mutations in these somaclonal variants have not yet been determined. We performed an RNA-sequencing (RNA-seq) analysis of three TBRI Foc TR4-resistant cultivars: 'Tai-Chiao No. 5' (TC5), 'Tai-Chiao No. 7' (TC7), and 'Formosana' (FM), as well as their susceptible progenitor 'Pei-Chiao' (PC), to investigate the sequence variations among them and develop cultivar-specific markers. RESULTS: A group of single-nucleotide variants (SNVs) specific to one cultivar were identified from the analysis of RNA-seq data and validated using Sanger sequencing from genomic DNA. Several SNVs were further converted into cleaved amplified polymorphic sequence (CAPS) markers or derived CAPS markers that could identify the three Foc TR4-resistant cultivars among 6 local and 5 international Cavendish cultivars. Compared with PC, the three resistant cultivars showed a loss or alteration of heterozygosity in some chromosomal regions, which appears to be a consequence of single-copy chromosomal deletions. Notably, TC7 and FM shared a common deletion region on chromosome 5; however, different TC7 tissues displayed varying degrees of allele ratios in this region, suggesting the presence of chimerism in TC7. CONCLUSIONS: This work demonstrates that reliable SNV markers of tissue culture-derived and propagated banana cultivars with a triploid genome can be developed through RNA-seq data analysis. Moreover, the analysis of sequence heterozygosity can uncover chromosomal deletions and chimerism in banana somaclonal variants. The markers obtained from this study will assist with the identification of TBRI Cavendish somaclonal variants for the quality control of tissue culture propagation, and the protection of breeders' rights.

Identification of MaWRKY40 and MaDLO1 as Effective Marker Genes for Tracking the Salicylic Acid-Mediated Immune Response in Bananas.

Bananas are among the world's most important cash and staple crops but are threatened by various devastating pathogens. The phytohormone salicylic acid (SA) plays a key role in the regulation of plant immune response. Tracking the expression of SA-responsive marker genes under pathogen infection is important in pathogenesis elucidation. However, the common SA-responsive marker genes are not consistently induced in different banana cultivars or different organs. Here, we conducted transcriptome analysis for SA response of a banana cultivar, 'Pei-Chiao' (Cavendish, AAA genome), and identified three genes, MaWRKY40, MaWRKY70, and Downy Mildew Resistant 6 (DMR6)-Like Oxygenase 1 (MaDLO1) that are robustly induced upon SA treatment in both the leaves and roots. Consistent induction of these three genes by SA treatment was also detected in both the leaves and roots of bananas belonging to different genome types such as 'Tai-Chiao No. 7' (Cavendish, AAA genome), 'Pisang Awak' (ABB genome), and 'Lady Finger' (AA genome). Furthermore, the biotrophic pathogen cucumber mosaic virus elicited the expression of MaWRKY40 and MaDLO1 in infected leaves of susceptible cultivars. The hemibiotrophic fungal pathogen Fusarium oxysporum f. sp. cubense tropical race 4 (TR4) also consistently induced the expression of MaWRKY40 and MaDLO1 in the infected roots of the F. oxysporum f. sp. cubense TR4-resistant cultivar. These results indicate that MaWRKY40 and MaDLO1 can be used as reliable SA-responsive marker genes for the study of plant immunity in banana. Revealing SA-responsive marker genes provides a stepping stone for further studies in banana resistance to pathogens.

Impact of changing reimbursement criteria on statin treatment patterns among patients with atherosclerotic cardiovascular disease or cardiovascular risk factors.

WHAT IS KNOWN AND OBJECTIVE: Starting 1 August 2013, the eligible cholesterol level for statin reimbursement in patients with atherosclerotic cardiovascular disease (ASCVD) or cardiovascular disease (CVD)-related risk factors changed from LDL-C ≥ 130 mg/dl (or TC ≥ 200 mg/dl) to LDL-C ≥ 100 mg/dl (or TC ≥ 160 mg/dl) in Taiwan, which may modify clinician prescribing behaviours. We aimed to evaluate the impact of changing reimbursement criteria on statin treatment patterns. METHODS: A before-after cohort design was conducted using Taiwan's National Health Insurance Research Database. Differences in statin treatment patterns between the pre- and postregulation periods were compared. Two prespecified study cohorts were identified to examine the impacts of this change on those who need statins for "secondary prevention" (patients newly diagnosed with ASCVD) and those who need statins for "primary prevention" (patients newly diagnosed with CVD-related risk factors, such as diabetes mellitus [DM]). Treatment patterns measured in this study included initiation, discontinuation, switching, dose increase, dose decrease and dose maximization. RESULTS: The proportion of patients who initiated statins during the postregulation period was higher than that of patients who initiated statins during the preregulation period (eg coronary heart disease (CHD) patients, pre- vs. postregulation: 41.23% vs. 48.25%). Notably, only 30%-40% of patients initiated statin use in the postregulation period across different conditions. In addition, the proportion of patients who discontinued statins remained very high. Even in the postregulation period, more than half of CHD patients discontinued statins during the 1-year follow-up period (eg CHD patients, pre- vs. postregulation: 59.07% vs. 52.75%). WHAT IS NEW AND CONCLUSION: The new reimbursement criteria started on 1 August 2013 seemed to lower the barriers of access to the first statin prescription among patients with CHD, cerebrovascular disease (CBVD) and DM. Nevertheless, the proportion of patients who initiated statin use was suboptimal, and the proportion of patients who discontinued statins was very high in the postregulation period.

Widespread Exon Junction Complex Footprints in the RNA Degradome Mark mRNA Degradation before Steady State Translation.

Exon junction complexes (EJCs) are deposited on mRNAs during splicing and displaced by ribosomes during the pioneer round of translation. Nonsense-mediated mRNA decay (NMD) degrades EJC-bound mRNA, but the lack of suitable methodology has prevented the identification of other degradation pathways. Here, we show that the RNA degradomes of Arabidopsis (Arabidopsis thaliana), rice (Oryza sativa), worm (Caenorhabditis elegans), and human (Homo sapiens) cells exhibit an enrichment of 5' monophosphate (5'P) ends of degradation intermediates that map to the canonical EJC region. Inhibition of 5' to 3' exoribonuclease activity and overexpression of an EJC disassembly factor in Arabidopsis reduced the accumulation of these 5'P ends, supporting the notion that they are in vivo EJC footprints. Hundreds of Arabidopsis NMD targets possess evident EJC footprints, validating their degradation during the pioneer round of translation. In addition to premature termination codons, plant microRNAs can also direct the degradation of EJC-bound mRNAs. However, the production of EJC footprints from NMD but not microRNA targets requires the NMD factor SUPPRESSOR WITH MORPHOLOGICAL EFFECT ON GENITALIA PROTEIN7. Together, our results demonstrating in vivo EJC footprinting in Arabidopsis unravel the composition of the RNA degradome and provide a new avenue for studying NMD and other mechanisms targeting EJC-bound mRNAs for degradation before steady state translation.

Engineering Plant Resistance to Tomato Yellow Leaf Curl Thailand Virus Using a Phloem-Specific Promoter Expressing Hairpin RNA.

Transgenic approaches employing RNA interference (RNAi) strategies have been successfully applied to generate desired traits in plants; however, variations between RNAi transgenic siblings and the ability to quickly apply RNAi resistance to diverse cultivars remain challenging. In this study, we assessed the promoter activity of a cauliflower mosaic virus 35S promoter (35S) and a phloem-specific promoter derived from rice tungro bacilliform virus (RTBV) and their efficacy to drive RNAi against the endogenous glutamate-1-semialdehyde aminotransferase gene (GSA) that acts as a RNAi marker, through chlorophyll synthesis inhibition, and against tomato yellow leaf curl Thailand virus (TYLCTHV), a begomovirus (family Geminiviridae) reported to be the prevalent cause of tomato yellow leaf curl disease (TYLCD) in Taiwan. Transgenic Nicotiana benthamiana expressing hairpin RNA of GSA driven by either the 35S or RTBV promoter revealed that RTBV::hpGSA induced stronger silencing along the vein and more uniformed silencing phenotype among its siblings than 35S::hpGSA. Analysis of transgenic N. benthamiana, 35S::hpTYLCTHV, and RTBV::hpTYLCTHV revealed that, although 35S::hpTYLCTHV generated a higher abundance of small RNA than RTBV::hpTYLCTHV, RTBV::hpTYLCTHV transgenic plants conferred better TYLCTHV resistance than 35S::hpTYLCTHV. Grafting of wild-type (WT) scions to TYLCTHV RNAi rootstocks allowed transferable TYLCTHV resistance to the scion. A TYLCTHV-inoculation assay showed that noninfected WT scions were only observed when grafted to RTBV::hpTYLCTHV rootstocks but not 35S::hpTYLCTHV nor WT rootstocks. Together, our findings demonstrate an approach that may be widely applied to efficiently confer TYLCD resistance.

Cucumber mosaic virus-induced gene silencing in banana.

Banana (Musa spp.) is one of the world's most important staple and cash crops. Despite accumulating genetic and transcriptomic data, low transformation efficiency in agronomically important Musa spp. render translational researches in banana difficult by using conventional knockout approaches. To develop tools for translational research in bananas, we developed a virus induced-gene silencing (VIGS) system based on a banana-infecting cucumber mosaic virus (CMV) isolate, CMV 20. CMV 20 genomic RNA 1, 2, and 3, were separately cloned in Agrobacterium pJL89 binary vectors, and a cloning site was introduced on RNA 2 immediately after the 2a open reading frame to insert the gene targeted for silencing. An efficient Agrobacterium inoculation method was developed for banana, which enabled the CMV 20 VIGS vector infection rate to reach 95% in our experiments. CMV 20-based silencing of Musa acuminata cv. Cavendish (AAA group) glutamate 1-semialdehyde aminotransferase (MaGSA) produced a typical chlorotic phenotype and silencing of M. acuminata phytoene desaturase (MaPDS) produced a photobleachnig phenotype. We show this approach efficiently reduced GSA and PDS transcripts to 10% and 18% of the control, respectively. The high infection rate and extended silencing of this VIGS system will provide an invaluable tool to accelerate functional genomic studies in banana.

DCL2- and RDR6-dependent transitive silencing of SMXL4 and SMXL5 in Arabidopsis dcl4 mutants causes defective phloem transport and carbohydrate over-accumulation.

DICER-LIKE (DCL) enzymes process double-stranded RNA into small RNAs that act as regulators of gene expression. Arabidopsis DCL4 and DCL2 each allow the post-transcriptional gene silencing (PTGS) of viruses and transgenes, but primary PTGS-prone DCL4 outcompetes transitive PTGS-prone DCL2 in wild-type plants. This hierarchy likely prevents DCL2 having any detrimental effects on endogenous genes. Indeed, dcl4 mutants exhibit developmental defects and increased sensitivity to genotoxic stress. In this study, the mechanism underlying dcl4 defects was investigated using genetic, biochemical and high-throughput sequencing approaches. We show that the purple phenotype of dcl4 leaves correlates with carbohydrate over-accumulation and defective phloem transport, and depends on the activity of SUPPRESSOR OF GENE SILENCING 3, RNA-DEPENDENT RNA POLYMERASE 6 (RDR6) and DCL2. This phenotype correlates with the downregulation of two genes expressed in the apex and the vasculature, SMAX1-LIKE 4 (SMXL4) and SMXL5, and the accumulation of DCL2- and RDR6-dependent small interfering RNAs derived from these two genes. Supporting a causal effect, smxl4 smxl5 double mutants exhibit leaf pigmentation, enhanced starch accumulation and defective phloem transport, similar to dcl4 plants. Overall, this study elucidates the detrimental action of DCL2 when DCL4 is absent, and indicates that DCL4 outcompeting DCL2 in wild-type plants is crucial to prevent the degradation of endogenous transcripts by DCL2- and RDR6-dependent transitive PTGS.

Global Analysis of Truncated RNA Ends Reveals New Insights into Ribosome Stalling in Plants.

High-throughput approaches for profiling the 5' ends of RNA degradation intermediates on a genome-wide scale are frequently applied to analyze and validate cleavage sites guided by microRNAs (miRNAs). However, the complexity of the RNA degradome other than miRNA targets is currently largely uncharacterized, and this limits the application of RNA degradome studies. We conducted a global analysis of 5'-truncated mRNA ends that mapped to coding sequences (CDSs) of Arabidopsis thaliana, rice (Oryza sativa), and soybean (Glycine max). Based on this analysis, we provide multiple lines of evidence to show that the plant RNA degradome contains in vivo ribosome-protected mRNA fragments. We observed a 3-nucleotide periodicity in the position of free 5' RNA ends and a bias toward the translational frame. By examining conserved peptide upstream open reading frames (uORFs) of Arabidopsis and rice, we found a predominance of 5' termini of RNA degradation intermediates that were separated by a length equal to a ribosome-protected mRNA fragment. Through the analysis of RNA degradome data, we discovered uORFs and CDS regions potentially associated with stacked ribosomes in Arabidopsis. Furthermore, our analysis of RNA degradome data suggested that the binding of Arabidopsis ARGONAUTE7 to a noncleavable target site of miR390 might directly hinder ribosome movement. This work demonstrates an alternative use of RNA degradome data in the study of ribosome stalling.

Asymmetric bulges and mismatches determine 20-nt microRNA formation in plants.

Plant microRNAs (miRNAs) are predominantly 21 nucleotides (nt) long but non-canonical lengths of 22 and 20 nt are commonly observed in diverse plant species. While miRNAs longer than 21 nt can be attributed to the neglect of unpaired bases within asymmetric bulges by the ruler function of dicer-like 1 (DCL1), how 20-nt miRNA is generated remains obscure. Analysis of small RNA data revealed that 20-nt miRNA can be divided into 3 main groups featured by atypical 3' overhangs or shorter duplex regions. Asymmetric bulges or mismatches at specific positions are commonly observed within each group and were shown to be crucial for 20-nt miRNA formation. Analysis of DCL1 cleavage sites on 20-nt miRNA precursors suggests that these determinants might alter precursor structure or trigger 3'-end decay of mature miRNA. The results herein advance our understanding of miRNA biogenesis and demonstrate that the effect of asymmetric bulges on miRNA length could be position-dependent.

Beyond cleaved small RNA targets: unraveling the complexity of plant RNA degradome data.

BACKGROUND: Degradation is essential for RNA maturation, turnover, and quality control. RNA degradome sequencing that integrates a modified 5'-rapid amplification of cDNA ends protocol with next-generation sequencing technologies is a high-throughput approach for profiling the 5'-end of uncapped RNA fragments on a genome-wide scale. The primary application of degradome sequencing has been to identify the truncated transcripts that result from endonucleolytic cleavage guided by microRNAs or small interfering RNAs. As many pathways are involved in RNA degradation, degradome data should contain other RNA species besides the cleavage remnants of small RNA targets. Nevertheless, no systematic approaches have been established to explore the hidden complexity of plant degradome. RESULTS: Through analyzing Arabidopsis and rice RNA degradome data, we recovered 11 short motifs adjacent to predominant and abundant uncapped 5'-ends. Uncapped ends associated with several of these short motifs were more prevalent than those targeted by most miRNA families especially in the 3' untranslated region of transcripts. Through genome-wide analysis, five motifs showed preferential accumulation of uncapped 5'-ends at the same position in Arabidopsis and rice. Moreover, the association of uncapped 5'-ends with a CA-repeat motif and a motif recognized by Pumilio/Fem-3 mRNA binding factor (PUF) proteins was also found in non-plant species, suggesting that common mechanisms are present across species. Based on these motifs, potential sources of RNA ends that constitute degradome data were proposed and further examined. The 5'-end of small nucleolar RNAs could be precisely captured by degradome sequencing. Position-specific enrichment of uncapped 5'-ends was seen upstream of motifs recognized by several RNA binding proteins especially for the binding site of PUF proteins. False uncapped 5'-ends produced from capped transcripts through non-specific PCR amplification were common artifacts among degradome datasets. CONCLUSIONS: The complexity of plant RNA degradome data revealed in this study may contribute to the alternative applications of degradome in RNA research.

A microRNA superfamily regulates nucleotide binding site-leucine-rich repeats and other mRNAs.

Analysis of tomato (Solanum lycopersicum) small RNA data sets revealed the presence of a regulatory cascade affecting disease resistance. The initiators of the cascade are microRNA members of an unusually diverse superfamily in which miR482 and miR2118 are prominent members. Members of this superfamily are variable in sequence and abundance in different species, but all variants target the coding sequence for the P-loop motif in the mRNA sequences for disease resistance proteins with nucleotide binding site (NBS) and leucine-rich repeat (LRR) motifs. We confirm, using transient expression in Nicotiana benthamiana, that miR482 targets mRNAs for NBS-LRR disease resistance proteins with coiled-coil domains at their N terminus. The targeting causes mRNA decay and production of secondary siRNAs in a manner that depends on RNA-dependent RNA polymerase 6. At least one of these secondary siRNAs targets other mRNAs of a defense-related protein. The miR482-mediated silencing cascade is suppressed in plants infected with viruses or bacteria so that expression of mRNAs with miR482 or secondary siRNA target sequences is increased. We propose that this process allows pathogen-inducible expression of NBS-LRR proteins and that it contributes to a novel layer of defense against pathogen attack.

Widespread translational control contributes to the regulation of Arabidopsis photomorphogenesis.

Environmental 'light' has a vital role in regulating plant growth and development. Transcriptomic profiling has been widely used to examine how light regulates mRNA levels on a genome-wide scale, but the global role of translational regulation in the response to light is unknown. Through a transcriptomic comparison of steady-state and polysome-bound mRNAs, we reveal a clear impact of translational control on thousands of genes, in addition to transcriptomic changes, during photomorphogenesis. Genes encoding ribosomal protein are preferentially regulated at the translational level, which possibly contributes to the enhanced translation efficiency. We also reveal that mRNAs regulated at the translational level share characteristics of longer half-lives and shorter cDNA length, and that transcripts with a cis-element, TAGGGTTT, in their 5' untranslated region have higher translatability. We report a previously neglected aspect of gene expression regulation during Arabidopsis photomorphogenesis. The identities and molecular signatures associated with mRNAs regulated at the translational level also offer new directions for mechanistic studies of light-triggered translational enhancement in Arabidopsis.

22-Nucleotide RNAs trigger secondary siRNA biogenesis in plants.

The effect of RNA silencing in plants can be amplified if the production of secondary small interfering RNAs (siRNAs) is triggered by the interaction of microRNAs (miRNAs) or siRNAs with a long target RNA. miRNA and siRNA interactions are not all equivalent, however; most of them do not trigger secondary siRNA production. Here we use bioinformatics to show that the secondary siRNA triggers are miRNAs and transacting siRNAs of 22 nt, rather than the more typical 21-nt length. Agrobacterium-mediated transient expression in Nicotiana benthamiana confirms that the siRNA-initiating miRNAs, miR173 and miR828, are effective as triggers only if expressed in a 22-nt form and, conversely, that increasing the length of miR319 from 21 to 22 nt converts it to an siRNA trigger. We also predicted and validated that the 22-nt miR771 is a secondary siRNA trigger. Our data demonstrate that the function of small RNAs is influenced by size, and that a length of 22 nt facilitates the triggering of secondary siRNA production.

Mining small RNA sequencing data: a new approach to identify small nucleolar RNAs in Arabidopsis.

Small nucleolar RNAs (snoRNAs) are noncoding RNAs that direct 2'-O-methylation or pseudouridylation on ribosomal RNAs or spliceosomal small nuclear RNAs. These modifications are needed to modulate the activity of ribosomes and spliceosomes. A comprehensive repertoire of snoRNAs is needed to expand the knowledge of these modifications. The sequences corresponding to snoRNAs in 18-26-nt small RNA sequencing data have been rarely explored and remain as a hidden treasure for snoRNA annotation. Here, we showed the enrichment of small RNAs at Arabidopsis snoRNA termini and developed a computational approach to identify snoRNAs on the basis of this characteristic. The approach successfully uncovered the full-length sequences of 144 known Arabidopsis snoRNA genes, including some snoRNAs with improved 5'- or 3'-end annotation. In addition, we identified 27 and 17 candidates for novel box C/D and box H/ACA snoRNAs, respectively. Northern blot analysis and sequencing data from parallel analysis of RNA ends confirmed the expression and the termini of the newly predicted snoRNAs. Our study especially expanded on the current knowledge of box H/ACA snoRNAs and snoRNA species targeting snRNAs. In this study, we demonstrated that the use of small RNA sequencing data can increase the complexity and the accuracy of snoRNA annotation.

Bioinformatic prediction and experimental validation of a microRNA-directed tandem trans-acting siRNA cascade in Arabidopsis.

Small RNAs play pivotal roles in regulating gene expression in higher eukaryotes. Among them, trans-acting siRNAs (ta-siRNAs) are a class of small RNAs that regulate plant development. The biogenesis of ta-siRNA depends on microRNA-targeted cleavage followed by the DCL4-mediated production of small RNAs phased in 21-nt increments relative to the cleavage site on both strands. To find TAS genes, we have used these characteristics to develop the first computational algorithm that allows for a comprehensive search and statistical evaluation of putative TAS genes from any given small RNA database. A search in Arabidopsis small RNA massively parallel signature sequencing (MPSS) databases with this algorithm revealed both known and previously unknown ta-siRNA-producing loci. We experimentally validated the biogenesis of ta-siRNAs from two PPR genes and the trans-acting activity of one of the ta-siRNAs. The production of ta-siRNAs from the identified PPR genes was directed by the cleavage of a TAS2-derived ta-siRNA instead of by microRNAs as was reported previously for TAS1a, -b, -c, TAS2, and TAS3 genes. Our results indicate the existence of a small RNA regulatory cascade initiated by miR173-directed cleavage and followed by the consecutive production of ta-siRNAs from two TAS genes.