Identification of novel stress-regulated microRNAs from Oryza sativa L.

MicroRNAs (miRNAs) are a type of small non-coding RNA found in eukaryotes. They play a key role in gene expression by down-regulating gene expression and are involved in the environment stress response in plants. Although a large number of miRNAs have been identified from Arabidopsis, few studies have focused on Oryza sativa miRNAs, especially on stress-related miRNAs. Five cDNA libraries of small RNAs from rice seedlings treated with cold, dehydration, salinity, and abscisic acid (ABA), as well as wild-type seedlings, were constructed. Seven rice novel miRNAs were identified by Northern analysis, and their expression patterns under different stress treatments were determined. Results showed that the expression of several novel miRNAs was regulated by one or more stress treatments. Our identification of novel stress-related miRNAs in rice suggests that these miRNAs might be involved in rice stress response pathways.     

siRNAs from miRNA sites mediate DNA methylation of target genes

Arabidopsis microRNA (miRNA) genes (MIR) give rise to 20- to 22-nt miRNAs that are generated predominantly by the type III endoribonuclease Dicer-like 1 (DCL1) but do not require any RNA-dependent RNA Polymerases (RDRs) or RNA Polymerase IV (Pol IV). Here, we identify a novel class of non-conserved MIR genes that give rise to two small RNA species, a 20- to 22-nt species and a 23- to 27-nt species, at the same site. Genetic analysis using small RNA pathway mutants reveals that the 20- to 22-nt small RNAs are typical miRNAs generated by DCL1 and are associated with Argonaute 1 (AGO1). In contrast, the accumulation of the 23- to 27-nt small RNAs from the miRNA-generating sites is dependent on DCL3, RDR2 and Pol IV, components of the typical heterochromatic small interfering RNA (hc-siRNA) pathway. We further demonstrate that these MIR-derived siRNAs associate with AGO4 and direct DNA methylation at some of their target loci in trans. In addition, we find that at the miRNA-generating sites, some conserved canonical MIR genes also produce siRNAs, which also induce DNA methylation at some of their target sites. Our systematic examination of published small RNA deep sequencing datasets of rice and moss suggests that this type of dual functional MIRs exist broadly in plants.     

Apple ring rot-responsive putative microRNAs revealed by high-throughput sequencing in Malus × domestica Borkh.

MicroRNAs (miRNAs) are small non-coding RNAs, which silence target mRNA via cleavage or translational inhibition to function in regulating gene expression. MiRNAs act as important regulators of plant development and stress response. For understanding the role of miRNAs responsive to apple ring rot stress, we identified disease-responsive miRNAs using high-throughput sequencing in Malus × domestica Borkh.. Four small RNA libraries were constructed from two control strains in M. domestica, crabapple (CKHu) and Fuji Naga-fu No. 6 (CKFu), and two disease stress strains, crabapple (DSHu) and Fuji Naga-fu No. 6 (DSFu). A total of 59 miRNA families were identified and five miRNAs might be responsive to apple ring rot infection and validated via qRT-PCR. Furthermore, we predicted 76 target genes which were regulated by conserved miRNAs potentially. Our study demonstrated that miRNAs was responsive to apple ring rot infection and may have important implications on apple disease resistance.     

The Fascinating World of RNA Interference

Micro- and short-interfering RNAs represent small RNA family that are recognized as critical regulatory species across the eukaryotes. Recent high-throughput sequencing have revealed two more hidden players of the cellular small RNA pool. Reported in mammals and Caenorhabditis elegans respectively, these new small RNAs are named piwi-interacting RNAs (piRNAs) and 21U-RNAs. Moreover, small RNAs including miRNAs have been identified in unicellular alga Chlamydomonas reinhardtii, redefining the earlier concept of multi-cellularity restricted presence of these molecules. The discovery of these species of small RNAs has allowed us to understand better the usage of genome and the number of genes present but also have complicated the situation in terms of biochemical attributes and functional genesis of these molecules. Nonetheless, these new pools of knowledge have opened up avenues for unraveling the finer details of the small RNA mediated pathways.     

Genome-wide identification of cold-responsive and new microRNAs in Populus tomentosa by high-throughput sequencing

MicroRNAs (miRNAs) are small, non-coding RNAs that regulate the expression of target mRNAs in plant growth, development, abiotic stress responses, and pathogen responses. Cold stress is one of the most common abiotic factors affecting plants, and it adversely affects plant growth, development, and spatial distribution. To understand the roles of miRNAs under cold stress in Populus tomentosa, we constructed two small RNA libraries from plantlets treated or not with cold conditions (4 °C for 8 h). High-throughput sequencing of the two libraries identified 144 conserved miRNAs belonging to 33 miRNA families and 29 new miRNAs (as well as their corresponding miRNA¹s) belonging to 23 miRNA families. Differential expression analysis showed that 21 miRNAs were down-regulated and nine miRNAs were up-regulated in response to cold stress. Among them, 19 cold-responsive miRNAs, two new miRNAs and their corresponding miRNA¹s were validated by qRT-PCR. A total of 101 target genes of the new miRNAs were predicted using a bioinformatics approach. These target genes are involved in growth and resistance to various stresses. The results demonstrated that Populus miRNAs play critical roles in the cold stress response.     

Small RNAs in the animal gonad: Guarding genomes and guiding development

Germ cells must safeguard, apportion, package, and deliver their genomes with exquisite precision to ensure proper reproduction and embryonic development. Classical genetic approaches have identified many genes controlling animal germ cell development, but only recently have some of these genes been linked to the RNA interference (RNAi) pathway, a gene silencing mechanism centered on small regulatory RNAs. Germ cells contain microRNAs (miRNAs), endogenous siRNAs (endo-siRNAs), and Piwi-interacting RNAs (piRNAs); these are bound by members of the Piwi/Argonaute protein family. piwi genes were known to specify germ cell development, but we now understand that mutations disrupting germline development can also affect small RNA accumulation. Small RNA studies in germ cells have revealed a surprising diversity of regulatory mechanisms and a unifying function for germline genes in controlling the spread of transposable elements. Future challenges will be to understand the production of germline small RNAs and to identify the full breadth of gene regulation by these RNAs. Progress in this area will likely impact biomedical goals of manipulating stem cells and preventing diseases caused by the transposition of mobile DNA elements.     

Small RNA Profiling in Dengue Virus 2-Infected Aedes Mosquito Cells Reveals Viral piRNAs and Novel Host miRNAs

In Aedes mosquitoes, infections with arthropod-borne viruses (arboviruses) trigger or modulate the expression of various classes of viral and host-derived small RNAs, including small interfering RNAs (siRNAs), PIWI interacting RNAs (piRNAs), and microRNAs (miRNAs). Viral siRNAs are at the core of the antiviral RNA interference machinery, one of the key pathways that limit virus replication in invertebrates. Besides siRNAs, Aedes mosquitoes and cells derived from these insects produce arbovirus-derived piRNAs, the best studied examples being viruses from the Togaviridae or Bunyaviridae families. Host miRNAs modulate the expression of a large number of genes and their levels may change in response to viral infections. In addition, some viruses, mostly with a DNA genome, express their own miRNAs to regulate host and viral gene expression. Here, we perform a comprehensive analysis of both viral and host-derived small RNAs in Aedes aegypti Aag2 cells infected with dengue virus 2 (DENV), a member of the Flaviviridae family. Aag2 cells are competent in producing all three types of small RNAs and provide a powerful tool to explore the crosstalk between arboviral infection and the distinct RNA silencing pathways. Interestingly, besides the well-characterized DENV-derived siRNAs, a specific population of viral piRNAs was identified in infected Aag2 cells. Knockdown of Piwi5, Ago3 and, to a lesser extent, Piwi6 results in reduction of vpiRNA levels, providing the first genetic evidence that Aedes PIWI proteins produce DENV-derived small RNAs. In contrast, we do not find convincing evidence for the production of virus-derived miRNAs. Neither do we find that host miRNA expression is strongly changed upon DENV2 infection. Finally, our deep-sequencing analyses detect 30 novel Aedes miRNAs, complementing the repertoire of regulatory small RNAs in this important vector species.