Global alteration of microRNAs and transposon-derived small RNAs in cotton (Gossypium hirsutum) during Cotton leafroll dwarf polerovirus (CLRDV) infection

Small RNAs (sRNAs) are a class of non-coding RNAs ranging from 20- to 40-nucleotides (nts) that are present in most eukaryotic organisms. In plants, sRNAs are involved in the regulation of development, the maintenance of genome stability and the antiviral response. Viruses, however, can interfere with and exploit the silencing-based regulatory networks, causing the deregulation of sRNAs, including small interfering RNAs (siRNAs) and microRNAs (miRNAs). To understand the impact of viral infection on the plant sRNA pathway, we deep sequenced the sRNAs in cotton leaves infected with Cotton leafroll dwarf virus (CLRDV), which is a member of the economically important virus family Luteoviridae. A total of 60 putative conserved cotton miRNAs were identified, including 19 new miRNA families that had not been previously described in cotton. Some of these miRNAs were clearly misregulated during viral infection, and their possible role in symptom development and disease progression is discussed. Furthermore, we found that the 24-nt heterochromatin-associated siRNAs were quantitatively and qualitatively altered in the infected plant, leading to the reactivation of at least one cotton transposable element. This is the first study to explore the global alterations of sRNAs in virus-infected cotton plants. Our results indicate that some CLRDV-induced symptoms may be correlated with the deregulation of miRNA and/or epigenetic networks.     

Endogenous Small RNA Clusters in Plants

In plants, small RNAs（sRNAs） usually refer to non-coding RNAs（ncRNAs） with lengths of 20–24 nucleotides. sRNAs are involved in the regulation of many essential processes related to plant development and environmental responses. sRNAs in plants are mainly grouped into microRNAs（miRNAs） and small interfering RNAs（siRNAs）, and the latter can be further classified into trans-acting siRNAs（ta-siRNAs）, repeat-associated siRNAs（ra-siRNAs）, natural anti-sense siRNAs（nat-siRNAs）, etc. Many sRNAs exhibit a clustered distribution pattern in the genome. Here, we summarize the features and functions of cluster-distributed sRNAs, aimed to not only provide a thorough picture of sRNA clusters（SRCs） in plants, but also shed light on the identification of new classes of functional sRNAs.     

Analyses of a Glycine max Degradome Library Identify microRNA Targets and MicroRNAs that Trigger Secondary SiRNA Biogenesis

Plant microRNAs (miRNAs) regulate gene expression mainly by guiding cleavage of target mRNAs. In this study, a degradome library constructed from different soybean (Glycine max (L.) Merr.) tissues was deep-sequenced. 428 potential targets of small interfering RNAs and 25 novel miRNA families were identified. A total of 211 potential miRNA targets, including 174 conserved miRNA targets and 37 soybean-specific miRNA targets, were identified. Among them, 121 targets were first discovered in soybean. The signature distribution of soybean primary miRNAs (pri-miRNAs) showed that most pri-miRNAs had the characteristic pattern of Dicer processing. The biogenesis of TAS3 small interfering RNAs (siRNAs) was conserved in soybean, and nine Auxin Response Factors were identified as TAS3 siRNA targets. Twenty-three miRNA targets produced secondary small interfering RNAs (siRNAs) in soybean. These targets were guided by five miRNAs: gma-miR393, gma-miR1508, gma-miR1510, gma-miR1514, and novel-11. Multiple targets of these secondary siRNAs were detected. These 23 miRNA targets may be the putative novel TAS genes in soybean. Global identification of miRNA targets and potential novel TAS genes will contribute to research on the functions of miRNAs in soybean.     

Identification of novel small RNAs in tomato (<Emphasis Type="Italic">Solanum lycopersicum</Emphasis>)

To date, the majority of plant small RNAs (sRNA) have been identified in rice, poplar and Arabidopsis. To identify novel tomato sRNAs potentially involved in tomato specific processes such as fruit development and/or ripening, we cloned 4,018 sRNAs from tomato fruit tissue at the mature green stage. From this pool of sRNAs, we detected tomato homologues of nine known miRNAs, including miR482; a poplar miRNA not conserved in Arabidopsis or rice. We identified three novel putative miRNAs with flanking sequence that could be folded into a stem-loop precursor structure and which accumulated as 19-24nt RNA. One of these putative miRNAs (Put-miRNA3) exhibited significantly higher expression in fruit compared with leaf tissues, indicating a specific role in fruit development processes. We also identified nine sRNAs that accumulated as 19–24nt RNA species in tomato but genome sequence was not available for these loci. None of the nine sRNAs or three putative miRNAs possessed a homologue in Arabidopsis that had a precursor with a predicted stem-loop structure or that accumulated as a sRNA species, suggesting that the 12 sRNAs we have identified in tomato may have a species specific role in this model fruit species. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Transposable element-associated microRNA hairpins produce 21-nt sRNAs integrated into typical microRNA pathways in rice

microRNAs (miRNAs) are a class of small RNAs (sRNAs) of ～21 nucleotides (nt) in length processed from foldback hairpins by DICER-LIKE1 (DCL1) or DCL4. They regulate the expression of target mRNAs by base pairing through RNA-induced silencing complex (RISC). In the RISC, ARGONAUTE1 (AGO1) is the key protein that cleaves miRNA targets at position ten of a miRNA:target duplex. The authenticity of many annotated rice miRNA hairpins is under debate because of their homology to repeat sequences. Some of them, like miR1884b, have been removed from the current release of miRBase based on incomplete information. In this study, we investigated the association of transposable element (TE)-derived miRNAs with typical miRNA pathways (DCL1/4- and AGO1-dependent) using publicly available deep sequencing datasets. Seven miRNA hairpins with 13 unique sRNAs were specifically enriched in AGO1 immunoprecipitation samples and relatively reduced in DCL1/4 knockdown genotypes. Interestingly, these species are ～21-nt long, instead of 24-nt as annotated in miRBase and the literature. Their expression profiles meet current criteria for functional annotation of miRNAs. In addition, diagnostic cleavage tags were found in degradome datasets for predicted target mRNAs. Most of these miRNA hairpins share significant homology with miniature inverted-repeat transposable elements, one type of abundant DNA transposons in rice. Finally, the root-specific production of a 24-nt miRNA-like sRNA was confirmed by RNA blot for a novel EST that maps to the 3′-UTR of a candidate pseudogene showing extensive sequence homology to miR1884b hairpin. Our data are consistent with the hypothesis that TEs can serve as a driving force for the evolution of some MIRNAs, where co-opting of DICER-LIKE1/4 processing and integration into AGO1 could exapt transcribed TE-associated hairpins into typical miRNA pathways.     

萱草microRNAs生物信息学及与冷冻相关microRNAs的分析

microRNA是一类长度为16-29nt的非蛋白质编码的内源小分子RNA（sRNA）,在植物生长发育以及逆境胁迫响应等过程中发挥着重要作用。本文利用基于HiSeq原理的sRNA深度测序技术,结合生物信息学方法对萱草根系中已知miRNA的类型、丰度以及部分与冷冻胁迫相关的已知miRNA的功能进行了分析。结果表明,在10℃常温和2-5℃低温条件下萱草根系中分别有14843184和16072575条序列信息,代表14064385和15309725种sRNA片段,且sRNA均呈现正态分布特征;在非编码RNA中转运RNA（tRNA）、核糖体RNA（rRNA）所占比例较大。低温sRNA组中得到注释的sRNA有67411种,共计799994条sRNAff／段;常温NsRNA组中,得到注释的sRNA有66524种,共计1055466条sRNA片段。冷冻胁迫下,萱草通过提高miR393、miR397、miR396的表达量和降4NmiR319的表达量来增强其抗冻性。本研究为后续揭示萱草低温应答蛋白合成的调控机理,筛选抗冻关键调控基因提供了丰富的数据。     

Analysis of plant-derived miRNAs in animal small RNA datasets

ABSTRACT: BACKGROUND: Plants contain significant quantities of small RNAs (sRNAs) derived from various sRNA biogenesis pathways. Many of these sRNAs play regulatory roles in plants. Previous analysis revealed that numerous sRNAs in corn, rice and soybean seeds have high sequence similarity to animal genes. However, exogenous RNA is considered to be unstable within the gastrointestinal tract of many animals, thus limiting potential for any adverse effects from consumption of dietary RNA. A recent paper reported that putative plant miRNAs were detected in animal plasma and serum, presumably acquired through ingestion, and may have a functional impact in the consuming organisms. RESULTS: To address the question of how common this phenomenon could be, we searched for plant miRNAs sequences in public sRNA datasets from various tissues of mammals, chicken and insects. Our analyses revealed that plant miRNAs were present in the animal sRNA datasets, and significantly miR168 was extremely over-represented. Furthermore, all or nearly all (>96%) miR168 sequences were monocot derived for most datasets, including datasets for two insects reared on dicot plants in their respective experiments. To investigate if plant-derived miRNAs, including miR168, could accumulate and move systemically in insects, we conducted insect feeding studies for three insects including corn rootworm, which has been shown to be responsive to plant-produced long double-stranded RNAs. CONCLUSIONS: Our analyses suggest that the observed plant miRNAs in animal sRNA datasets can originate in the process of sequencing, and that accumulation of plant miRNAs via dietary exposure is not universal in animals.     

Identification and Characterization of ABA-Responsive MicroRNAs in Rice

MicroRNAs(miRNAs) are endogenous non-coding small RNAs that silence genes through mRNA degradation or translational inhibition.The phytohormone abscisic acid(ABA) is essential for plant development and adaptation to abiotic and biotic stresses.In Arabidopsis,miRNAs are implicated in ABA functions.However,ABA-responsive miRNAs have not been systematically studied in rice.Here high throughput sequencing of small RNAs revealed that 107 miRNAs were differentially expressed in the rice ABA deficient mutant,Osabal.Of these,13 were confirmed by stem-loop RT-PCR.Among them,miR1425-5P,miR169 a,miR169n,miR390-5P,miR397 a and miR397 b were up-regulated,but miR162 b reduced in expression in Osabal.The targets of these 13 miRNAs were predicted and validated by gene expression profiling.Interestingly,the expression levels of these miRNAs and their targets were regulated by ABA.Cleavage sites were detected on 7 of the miRNA targets by 5'-Rapid Amplification of cDNA Ends(5'-RACE).Finally,miR162 b and its target OsTREl were shown to affect rice resistance to drought stress,suggesting that miR162 b increases resistance to drought by targeting OsTREl.Our work provides important information for further characterization and functional analysis of ABA-responsive miRNAs in rice.