Viral infection induces expression of novel phased microRNAs from conserved cellular microRNA precursors

RNA silencing, mediated by small RNAs including microRNAs (miRNAs) and small interfering RNAs (siRNAs), is a potent antiviral or antibacterial mechanism, besides regulating normal cellular gene expression critical for development and physiology. To gain insights into host small RNA metabolism under infections by different viruses, we used Solexa/Illumina deep sequencing to characterize the small RNA profiles of rice plants infected by two distinct viruses, Rice dwarf virus (RDV, dsRNA virus) and Rice stripe virus (RSV, a negative sense and ambisense RNA virus), respectively, as compared with those from non-infected plants. Our analyses showed that RSV infection enhanced the accumulation of some rice miRNA*s, but not their corresponding miRNAs, as well as accumulation of phased siRNAs from a particular precursor. Furthermore, RSV infection also induced the expression of novel miRNAs in a phased pattern from several conserved miRNA precursors. In comparison, no such changes in host small RNA expression was observed in RDV-infected rice plants. Significantly RSV infection elevated the expression levels of selective OsDCLs and OsAGOs, whereas RDV infection only affected the expression of certain OsRDRs. Our results provide a comparative analysis, via deep sequencing, of changes in the small RNA profiles and in the genes of RNA silencing machinery induced by different viruses in a natural and economically important crop host plant. They uncover new mechanisms and complexity of virus-host interactions that may have important implications for further studies on the evolution of cellular small RNA biogenesis that impact pathogen infection, pathogenesis, as well as organismal development.     

Alterations in SiRNA and MiRNA Expression Profiles Detected by Deep Sequencing of Transgenic Rice with SiRNA-Mediated Viral Resistance

RNA-mediated gene silencing has been demonstrated to serve as a defensive mechanism against viral pathogens by plants. It is known that specifically expressed endogenous siRNAs and miRNAs are involved in the self-defense process during viral infection. However, research has been rarely devoted to the endogenous siRNA and miRNA expression changes under viral infection if the resistance has already been genetically engineered in plants. Aiming to gain a deeper understanding of the RNA-mediated gene silencing defense process in plants, the expression profiles of siRNAs and miRNAs before and after viral infection in both wild type and transgenic anti-Rice stripe virus (RSV) rice plants were examined by small RNA high-throughput sequencing. Our research confirms that the newly generated siRNAs, which are derived from the engineered inverted repeat construct, is the major contributor of the viral resistance in rice. Further analysis suggests the accuracy of siRNA biogenesis might be affected when siRNAs machinery is excessively used in the transgenic plants. In addition, the expression levels of many known miRNAs are dramatically changed due to RSV infection on both wild type and transgenic rice plants, indicating potential function of those miRNAs involved in plant-virus interacting process.     

Identification of MiRNA from Eggplant (Solanum melongena L.) by Small RNA Deep Sequencing and Their Response to Verticillium dahliae Infection

MiRNAs are a class of non-coding small RNAs that play important roles in the regulation of gene expression. Although plant miRNAs have been extensively studied in model systems, less is known in other plants with limited genome sequence data, including eggplant (Solanum melongena L.). To identify miRNAs in eggplant and their response to Verticillium dahliae infection, a fungal pathogen for which clear understanding of infection mechanisms and effective cure methods are currently lacking, we deep-sequenced two small RNA (sRNA) libraries prepared from mock-infected and infected seedlings of eggplants. Specifically, 30,830,792 reads produced 7,716,328 unique miRNAs representing 99 known miRNA families that have been identified in other plant species. Two novel putative miRNAs were predicted with eggplant ESTs. The potential targets of the identified known and novel miRNAs were also predicted based on sequence homology search. It was observed that the length distribution of obtained sRNAs and the expression of 6 miRNA families were obviously different between the two libraries. These results provide a framework for further analysis of miRNAs and their role in regulating plant response to fungal infection and Verticillium wilt in particular.     

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.     

Genome-Wide Identification of Reverse Complementary microRNA Genes in Plants

MicroRNAs (miRNAs) are ∼21-nucleotide small RNAs (sRNAs) with essential regulatory roles in plants. They are generated from stem-loop-structured precursors through two sequential Dicer-like 1 (DCL1)-mediated cleavages. To date, hundreds of plant miRNAs have been uncovered. However, the question, whether the sequences reverse complementary (RC) to the miRNA precursors could form hairpin-like structures and produce sRNA duplexes similar to the miRNA/miRNA* pairs has not been solved yet. Here, we interrogated this possibility in 16 plant species based on sRNA high-throughput sequencing data and secondary structure prediction. A total of 59 RC sequences with great potential to form stem-loop structures and generate miRNA/miRNA*-like duplexes were identified in ten plants, which were named as RC-miRNA precursors. Unlike the canonical miRNAs, only a few cleavage targets of the RC-miRNAs were identified in Arabidopsis (Arabidopsis thaliana) and rice (Oryza sativa), and none in Soybean (Glycine max) based on degradome data. Surprisingly, the genomic regions surrounding some of the RC-miRNA target recognition sites were observed to be specifically methylated in both Arabidopsis and rice. Taken together, we reported a new class of miRNAs, called RC-miRNAs, which were generated from the antisense strands of the miRNA precursors. Based on the results, we speculated that the mature RC-miRNAs might have subtle regulatory activity through target cleavages, but might possess short interfering RNA-like activity by guiding sequence-specific DNA methylation.     

Systematic Analysis of Small RNAs Associated with Human Mitochondria by Deep Sequencing: Detailed Analysis of Mitochondrial Associated miRNA

Mitochondria are one of the central regulators of many cellular processes beyond its well established role in energy metabolism. The inter-organellar crosstalk is critical for the optimal function of mitochondria. Many nuclear encoded proteins and RNA are imported to mitochondria. The translocation of small RNA (sRNA) including miRNA to mitochondria and other sub-cellular organelle is still not clear. We characterized here sRNA including miRNA associated with human mitochondria by cellular fractionation and deep sequencing approach. Mitochondria were purified from HEK293 and HeLa cells for RNA isolation. The sRNA library was generated and sequenced using Illumina system. The analysis showed the presence of unique population of sRNA associated with mitochondria including miRNA. Putative novel miRNAs were characterized from unannotated sRNA sequences. The study showed the association of 428 known, 196 putative novel miRNAs to mitochondria of HEK293 and 327 known, 13 putative novel miRNAs to mitochondria of HeLa cells. The alignment of sRNA to mitochondrial genome was also studied. The targets were analyzed using DAVID to classify them in unique networks using GO and KEGG tools. Analysis of identified targets showed that miRNA associated with mitochondria regulates critical cellular processes like RNA turnover, apoptosis, cell cycle and nucleotide metabolism. The six miRNAs (counts >1000) associated with mitochondria of both HEK293 and HeLa were validated by RT-qPCR. To our knowledge, this is the first systematic study demonstrating the associations of sRNA including miRNA with mitochondria that may regulate site-specific turnover of target mRNA important for mitochondrial related functions.     

miRNA在植物病原调控方面的研究进展

miRNA是一类在真核生物体内普遍存在的,长度在22 nt左右的单链小RNA分子。大量研究发现,miRNA可广泛参与植物的生长发育、新陈代谢、物质运输、逆境响应及病原防御等多种生理生化过程。目前已经从植物中鉴定到大量的miRNA,但其中参与植物病原调控相关miRNA的研究较少。miRNA作为一种重要的转录调控因子,可参与调控病原相关分子模式触发的免疫反应和效应因子触发的免疫反应。拟南芥中,miRNA393通过靶向生长素受体基因对植物生长素进行负调控,从而在抵御细菌侵染方面发挥作用;水稻中,miRNA528可响应水稻条纹病毒(RSV)的侵染,人为提高miRNA528水平有助于维持水稻对RSV的抗性。阐述了miRNA的作用机制,与植物细菌、真菌和病毒侵染相关的miRNA研究进展,总结了葡萄,苹果,梨和桃等具有重要经济价值果树中病原调控相关miRNA研究情况,旨在为今后miRNA在植物,特别是在果树抗病研究方面提供全面的理论依据。     

Identification of a movement protein of the tenuivirus rice stripe virus

Rice stripe virus (RSV) is the type member of the genus Tenuivirus. RSV has four single-stranded RNAs and causes severe disease in rice fields in different parts of China. To date, no reports have described how RSV spreads within host plants or the viral and/or host factor(s) required for tenuivirus movement. We investigated functions of six RSV-encoded proteins using trans-complementation experiments and biolistic bombardment. We demonstrate that NSvc4, encoded by RSV RNA4, supports the intercellular trafficking of a movement-deficient Potato virus X in Nicotiana benthamiana leaves. We also determined that upon biolistic bombardment or agroinfiltration, NSvc4:enhanced green fluorescent protein (eGFP) fusion proteins localize predominantly near or within the walls of onion and tobacco epidermal cells. In addition, the NSvc4:eGFP fusion protein can move from initially bombarded cells to neighboring cells in Nicotiana benthamiana leaves. Immunocytochemistry using tissue sections from RSV-infected rice leaves and an RSV NSvc4-specific antibody showed that the NSvc4 protein accumulated in walls of RSV-infected leaf cells. Gel retardation assays revealed that the NSvc4 protein interacts with single-stranded RNA in vitro, a common feature of many reported plant viral movement proteins (MPs). RSV NSvc4 failed to interact with the RSV nucleocapsid protein using yeast two-hybrid assays. Taken together, our data indicate that RSV NSvc4 is likely an MP of the virus. This is the first report describing a tenuivirus MP.     

Prokaryotic expression and polyclonal antibody preparation of novel ZLG10 protein involved in infection of RSV on SPC-A1 cells

Differentially expressed genes between normal SPC-A1 cells and SPC-A1 cells infected by RSV were investigated using differential display. The novel zlg10 gene codes for a novel protein, ZLG10, which has previously been reported to be up-regulated in RSV-infected SPC-A1 cells. Its putative open reading frame was also identified. To better understand the structure, function, and possible role of ZLG10 as a potential candidate for diagnosis and vaccine studies, the intact region encoding ZLG10 was obtained by PCR and expressed in Escherichia coli as a GST-fusion protein. After purification, GST-ZLG10 fusion protein was used to immunize the adult rabbits following standard protocols. Consequently, we found that the produced antiserum of the novel fusion protein significantly suppressed the infection by RSV on SPC-A1 cells by using neutral red uptake assay and quantitative measurement. Together, our data demonstrate that ZLG10, a novel protein expressed and purified in this report, might be a potential effective therapeutic candidate for treating RSV infections.     

水稻MicroRNA的预测及实验验证

根据已报道水稻pre-miRNA的序列与结构信息,利用支持向量机（support vector machine, SVM）方法在miRNA前体上预测成熟区,产生一个模型——mature-SVM.它预测水稻成熟区的敏感性和特异性分别为86.7% 和100%;然后,用这个模型对从水稻基因组中筛选出的46.501条pre-miRNA进行成熟链预测,此外再根据miRNA的作用原理用blast程序所进一步的筛选,得到了127条pre-miRNA及成熟miRNA;除去其中已知的21条,最后得到106条候选的新的水稻miRNA. 从中随机挑取10条进行Northern验证,结果有4条miRNA得到确认.     

Sex specific expression and distribution of small RNAs in papaya

Background

Regulatory function of small non-coding RNAs (sRNA) in response to environmental and developmental cues has been established. Additionally, sRNA, also plays an important role in maintaining the heterochromatin and centromere structures of the chromosome. Papaya, a trioecious species with recently evolved sex chromosomes, has emerged as an excellent model system to study sex determination and sex chromosome evolution in plants. However, role of small RNA in papaya sex determination is yet to be explored.

Results

We analyzed the high throughput sRNAs reads in the Illumina libraries prepared from male, female, and hermaphrodite flowers of papaya. Using the sRNA reads, we identified 29 miRNAs that were not previously reported from papaya. Including this and two previous studies, a total of 90 miRNAs has been identified in papaya. We analyzed the expression of these miRNAs in each sex types. A total of 65 miRNAs, including 31 conserved and 34 novel mirNA, were detected in at least one library. Fourteen of the 65 miRNAs were differentially expressed among different sex types. Most of the miRNA expressed higher in male flowers were related to the auxin signaling pathways, whereas the miRNAs expressed higher in female flowers were the potential regulators of the apical meristem identity genes. Aligning the sRNA reads identified the sRNA hotspots adjacent to the gaps of the X and Y chromosomes. The X and Y chromosomes sRNA hotspots has a 7.8 and 4.4 folds higher expression of sRNA, respectively, relative to the chromosome wide average. Approximately 75% of the reads aligned to the X chromosome hotspot was identical to that of the Y chromosome hotspot.

Conclusion

By analyzing the large-scale sRNA sequences from three sex types, we identified the sRNA hotspots flanking the gaps of papaya X, Y, and Y^h chromosome. The sRNAs expression patterns in these regions were reminiscent of the pericentromeric region indicating that the only remaining gap in each of these chromosomes is likely the centromere. We also identified 14 differentially expressed miRNAs in male, female and hermaphrodite flowers of papaya. Our results provide valuable information toward understanding the papaya sex determination.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-20) contains supplementary material, which is available to authorized users.