Identification of chilling stress-responsive tomato microRNAs and their target genes by high-throughput sequencing and degradome analysis

Background

MicroRNAs (miRNAs) are a class of noncoding small RNAs (sRNAs) that are 20–24 nucleotides (nt) in length. Extensive studies have indicated that miRNAs play versatile roles in plants, functioning in processes such as growth, development and stress responses. Chilling is a common abiotic stress that seriously affects plants growth and development. Recently, chilling-responsive miRNAs have been detected in several plant species. However, little is known about the miRNAs in the model plant tomato. ‘LA1777’ (Solanum habrochaites) has been shown to survive chilling stress due to its various characteristics.

Results

Here, two small RNA libraries and two degradome libraries were produced from chilling-treated (CT) and non-chilling-treated (NT) leaves of S. habrochaites seedlings. Following high-throughput sequencing and filtering, 161 conserved and 236 novel miRNAs were identified in the two libraries. Of these miRNAs, 192 increased in the response to chilling stress while 205 decreased. Furthermore, the target genes of the miRNAs were predicted using a degradome sequencing approach. It was found that 62 target genes were cleaved by 42 conserved miRNAs, while nine target genes were cleaved by nine novel miRNAs. Additionally, nine miRNAs and six target genes were validated by quantitative real-time PCR (qRT-PCR). Target gene functional analysis showed that most target genes played positive roles in the chilling response, primarily by regulating the expression of anti-stress proteins, antioxidant enzyme and genes involved in cell wall formation.

Conclusions

Tomato is an important model plant for basic biological research. In this study, numerous conserved and novel miRNAs involved in the chilling response were identified using high-throughput sequencing, and the target genes were analyzed by degradome sequencing. The work helps identify chilling-responsive miRNAs in tomato and increases the number of identified miRNAs involved in chilling stress. Furthermore, the work provides a foundation for further study of the regulation of miRNAs in the plant response to chilling stress.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-1130) contains supplementary material, which is available to authorized users.     

Identification and Characterization of Argonaute Protein, Ago2 and Its Associated Small RNAs in Schistosoma japonicum

Background

The complex life cycle of the genus Schistosoma drives the parasites to employ subtle developmentally dependent gene regulatory machineries. Small non-coding RNAs (sncRNAs) are essential gene regulatory factors that, through their impact on mRNA and genome stability, control stage-specific gene expression. Abundant sncRNAs have been identified in this genus. However, their functionally associated partners, Argonaute family proteins, which are the key components of the RNA-induced silencing complex (RISC), have not yet been fully explored.

Methodology/Principal Findings

Two monoclonal antibodies (mAbs) specific to Schistosoma japonicum Argonaute protein Ago2 (SjAgo2), but not SjAgo1 and SjAgo3, were generated. Soluble adult worm antigen preparation (SWAP) was subjected to immunoprecipitation with the mAbs and the captured SjAgo2 protein was subsequently confirmed by Western blot and mass spectrometry (MS) analysis. The small RNA population associated with native SjAgo2 in adult parasites was extracted from the immunoprecipitated complex and subjected to library construction. High-through-put sequencing of these libraries yielded a total of ≈50 million high-quality reads. Classification of these small RNAs showed that endogenous siRNAs (endo-siRNAs) generated from transposable elements (TEs), especially from the subclasses of LINE and LTR, were prominent. Further bioinformatics analysis revealed that siRNAs derived from ten types of well-defined retrotransposons were dramatically enriched in the SjAgo2-specific libraries compared to small RNA libraries constructed with total small RNAs from separated adult worms. These results suggest that a key function of SjAgo2 is to maintain genome stability through suppressing the activities of retrotransposons.

Conclusions/Significance

In this study, we identified and characterized one of the three S. japonicum Argonautes, SjAgo2, and its associated small RNAs were found to be predominantly derived from particular classes of retrotransposons. Thus, a major function of SjAgo2 appears to associate with the maintenance of genome stability via suppression of retroelements. The data advance our understanding of the gene regulatory mechanisms in the blood fluke.     

Identification and Comparative Analysis of the Tegillarca granosa Haemocytes MicroRNA Transcriptome in Response to Cd Using a Deep Sequencing Approach

Background

MicroRNAs (miRNAs) are endogenous non-coding small RNAs (sRNAs) that can base pair with their target mRNAs, which represses their translation or induces their degradation in various biological processes. To identify miRNAs regulated by heavy metal stress, we constructed two sRNA libraries for the blood clam Tegillarca granosa: one for organisms exposed to toxic levels of cadmium (Cd) and one for a control group.

Results

Sequencing of the two libraries and subsequent analysis revealed 215 conserved and 39 new miRNAs. Most of the new miRNAs in T. granosa were up- or down-regulated in response to Cd exposure. There were significant differences in expression between the Cd and control groups for 16 miRNAs. Of these, five miRNAs were significantly up-regulated and 11 were significantly down-regulated in the Cd stress library. Potential targets were predicted for the 16 differential miRNAs in pre-miRNAs identified according to sequence homology. Some of the predicted miRNA targets are associated with regulation of the response to stress induced by heavy metals. Five differentially expressed miRNAs (Tgr-nmiR-8, Tgr-nmiR-21, Tgr-miR-2a, Tgr-miR-10a-5p, and Tgr-miR-184b) were validated by qRT-PCR.

Conclusion

Our study is the first large-scale identification of miRNAs in T. granosa haemocytes. Our findings suggest that some miRNAs and their target genes and pathways may play critical roles in the responses of this species to environmental heavy metal stresses.     

Expression of multiple artificial microRNAs from a chicken miRNA126-based lentiviral vector

Background

The use of RNAi in both basic and translational research often requires expression of multiple siRNAs from the same vector.

Methods/Principal Findings

We have developed a novel chicken miR126-based artificial miRNA expression system that can express one, two or three miRNAs from a single cassette in a lentiviral vector. We show that each of the miRNAs expressed from the same lentiviral vector is capable of potent inhibition of reporter gene expression in transient transfection and stable integration assays in chicken fibroblast DF-1 cells. Transduction of Vero cells with lentivirus expressing two or three different anti-influenza miRNAs leads to inhibition of influenza virus production. In addition, the chicken miR126-based expression system effectively inhibits reporter gene expression in human, monkey, dog and mouse cells. These results demonstrate that the flanking regions of a single primary miRNA can support processing of three different stem-loops in a single vector.

Conclusions/Significance

This novel design expands the means to express multiple miRNAs from the same vector for potent and effective silencing of target genes and influenza virus.     

Temperature during early development has long-term effects on microRNA expression in Atlantic cod

Background

Environmental temperature has serious implications in life cycle of aquatic ectotherms. Understanding the molecular mechanisms of temperature acclimation and adaptation of marine organisms is of the uttermost importance for ecology, fisheries, and aquaculture, as it allows modeling the effects of global warming on population dynamics. Regulatory molecules are major modulators of acclimation and adaptation; among them, microRNAs (miRNAs) are versatile and substantial contributors to regulatory networks of development and adaptive plasticity. However, their role in thermal plasticity is poorly known. We have asked whether the temperature and its shift during the early ontogeny (embryonic and larval development) affect the miRNA repertoire of Atlantic cod (Gadus morhua), and if thermal experience has long-term consequences in the miRNA profile.

Results

We characterized miRNA during different developmental stages and in juvenile tissues using next generation sequencing. We identified 389 putative miRNA precursor loci, 120 novel precursor miRNAs, and 281 mature miRNAs. Some miRNAs showed stage- or tissue-enriched expression and miRNAs, such as the miR-17 ~ 92 cluster, myomiRs (miR-206), neuromiRs (miR-9, miR-124), miR-130b, and miR-430 showed differential expression in different temperature regimes. Long-term effect of embryonic incubation temperature was revealed on expression of some miRNAs in juvenile pituitary (miR-449), gonad (miR-27c, miR-30c, and miR-200a), and liver (let-7 h, miR-7a, miR-22, miR-34c, miR-132a, miR-192, miR-221, miR-451, miR-2188, and miR-7550), but not in brain. Some of differentially expressed miRNAs in the liver were confirmed using LNA-based rt-qPCR. The effect of temperature on methylation status of selected miRNA promoter regions was mostly inconclusive.

Conclusions

Temperature elevation by several degrees during embryonic and larval developmental stages significantly alters the miRNA profile, both short-term and long-term. Our results suggest that a further rise in seas temperature might affect life history of Atlantic cod.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1503-7) contains supplementary material, which is available to authorized users.     

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.