Small RNAs in pollen

In plants, each pollen mother cell undergoes two rounds of cell divisions to form a mature pollen grain, which contains a vegetative cell(VC) and two sperm cells(SC). As a companion cell, the VC carries the SCs to an ovule by germinating a pollen tube. In-depth sequencing analyses of mature pollen showed that micro RNAs(mi RNAs) and short interfering RNAs(si RNAs) are present in both the VC and SCs. Additionally, epigenetically-regulated transposable elements(TEs) are reactivated in the VC and these TE m RNAs are further processed into 21-nt epigenetically reactivated si RNA(easiR NA) in SCs, which prevent 24-nt si RNA accumulation and sequester mi RNA loading. Small RNAs are thought to move from the VC to SCs, where they regulate gene expression and reinforce TE silencing. Here, we summarize current knowledge of the biogenesis and function of mi RNAs, si RNAs, and easi RNAs in pollen, emphasizing how these different small RNAs coordinately contribute to sperm cell formation and TE silencing.     

Characterization of unique small RNA populations from rice grain

Small RNAs (approximately 20 to 24 nucleotides) function as naturally occurring molecules critical in developmental pathways in plants and animals. Here we analyze small RNA populations from mature rice grain and seedlings by pyrosequencing. Using a clustering algorithm to locate regions producing small RNAs, we classified hotspots of small RNA generation within the genome. Hotspots here are defined as 1 kb regions within which small RNAs are significantly overproduced relative to the rest of the genome. Hotspots were identified to facilitate characterization of different categories of small RNA regulatory elements. Included in the hotspots, we found known members of 23 miRNA families representing 92 genes, one trans acting siRNA (ta-siRNA) gene, novel siRNA-generating coding genes and phased siRNA generating genes. Interestingly, over 20% of the small RNA population in grain came from a single foldback structure, which generated eight phased 21-nt siRNAs. This is reminiscent of a newly arising miRNA derived from duplication of progenitor genes. Our results provide data identifying distinct populations of small RNAs, including phased small RNAs, in mature grain to facilitate characterization of small regulatory RNA expression in monocot species.     

Expression Profile and Function Analysis of Long Non-coding RNAs in the Infection of Coxsackievirus B3

The roles of lnc RNAs in the infection of enteroviruses have been barely demonstrated. In this study, we used coxsackievirus B3(CVB3), a typical enterovirus, as a model to investigate the expression profiles and functional roles of lnc RNAs in enterovirus infection. We profiled lnc RNAs and m RNA expression in CVB3-infected He La cells by lnc RNA-m RNA integrated microarrays. As a result, 700 differentially expressed lnc RNAs(431 up-regulated and 269 down-regulated) and665 differentially expressed m RNAs(299 up-regulated and 366 down-regulated) were identified in CVB3 infection. Then we performed lnc RNA-m RNA integrated pathway analysis to identify potential functional impacts of the differentially expressed m RNAs, in which lnc RNA-m RNA correlation network was built. According to lnc RNA-m RNA correlation, we found that XLOC-001188, an lnc RNA down-regulated in CVB3 infection, was negatively correlated with NFAT5 m RNA,an anti-CVB3 gene reported previously. This interaction was supported by q PCR detection following si RNA-mediated knockdown of XLOC-001188, which showed an increase of NFAT5 m RNA and a reduction of CVB3 genomic RNA. In addition, we observed that four most significantly altered lnc RNAs, SNHG11, RP11-145 F16.2, RP11-1023 L17.1 and RP11-1021 N1.2 share several common correlated genes critical for CVB3 infection, such as BRE and IRF2 BP1. In all, our studies reveal the alteration of lnc RNA expression in CVB3 infection and its potential influence on CVB3 replication,providing useful information for future studies of enterovirus infection.     

Identification of microRNAs,phasiRNAs and Their Targets in Pineapple

MicroRNAs (miRNAs) are small non-coding RNAs that regulate their target mRNA levels by directing cleavage or repressing its translation. Besides its outstanding nutritional and medicinal significances, pineapple serves as a model for studying genome evolution in cereal crops as well as obligate crassulacean acid metabolism (CAM) photosynthesis. Thus, studying miRNAs in pineapple is critical for better understanding their roles in this plant species. Here we carried out computational and experimental analysis of miRNAs and phased small interfering RNAs (phasiRNAs) in pineapple by analyzing small RNA profiles from flowers, fruits and leaves. The analyses have identified 131 conserved miRNAs that could be grouped into 37 families and 16 novel miRNAs. Three TAS3 loci and forty five 21 nucleotide (nt) PHAS loci, and seventy three 24 nt PHAS loci were also identified. The putative targets of the identified miRNAs and phasiRNAs were predicted. The presented results provide a comprehensive view of small regulatory RNAs and their targets in pineapple.     

Grass phasiRNAs and male fertility

Recent studies have indicated that a special type of small noncoding RNAs, phased small-interfering RNAs (phasiRNAs) play crucial roles in many cellular processes of plant development. PhasiRNAs are generated from long RNA precursors at intervals of 21 or 24 nt in plants, and they are produced from both protein-coding gene and long noncoding RNA genes. Different from those in eudicots, grass phasiRNAs include a special class of small RNAs that are specifically expressed in reproductive organs. These grass phasiRNAs are associated with gametogenesis, especially with anther development and male fertility. In this review, we summarized current knowledge on these small noncoding RNAs in male germ cells and their possible biological functions and mechanisms in grass species.     

The functional analysis of transiently upregulated miR-101 suggests a “braking” regulatory mechanism during myogenesis

Skeletal muscle differentiation is a highly coordinated process that involves many cellular signaling pathways and micro RNAs(mi RNAs). A group of muscle-specific mi RNAs has been reported to promote myogenesis by suppressing key signaling pathways for cell growth. However, the functional role and regulatory mechanism of most non-muscle-specific mi RNAs with stage-specific changes during differentiation are largely unclear. Here, we describe the functional characterization of mi R-101 a/b, a pair of non-muscle-specific mi RNAs that show the largest change among a group of transiently upregulated mi RNAs during myogenesis in C2 C12 cells. The overexpression of mi R-101 a/b inhibits myoblast differentiation by suppressing the p38/MAPK,Interferon Gamma, and Wnt pathways and enhancing the C/EBP pathway. Mef2 a, a key protein in the p38/MAPK pathway, was identified as a direct target of mi R-101 a/b. Interestingly, we found that the long non-coding RNA(lnc RNA) Malat1, which promotes muscle differentiation, interacts with mi R-101 a/b, and this interaction competes with Mef2 a m RNA to relieve the inhibition of the p38/MAPK pathway during myogenesis. These results uncovered a "braking" role in differentiation of transiently upregulated mi RNAs and provided new insights into the competing endogenous RNA(ce RNA) regulatory mechanism in myoblast differentiation and myogenesis.     

Dynamic and Coordinated Expression Changes of Rice Small RNAs in Response to Xanthomonas oryzae pv.oryzae

Endogenous small RNAs are newly identified players in plant immune responses, yet their roles in rice(Oryza sativa) responding to pathogens are still less understood, especially for pathogens that can cause severe yield losses. We examined the small RNA expression profiles of rice leaves at 2, 6, 12, and 24 hours post infection of Xanthomonas oryzae pv. oryzae(Xoo) virulent strain PXO99, the causal agent of rice bacterial blight disease. Dynamic expression changes of some mi RNAs and trans-acting si RNAs were identified, together with a few novel mi RNA targets, including an RLK gene targeted by osa-mi R159 a.1. Coordinated expression changes were observed among some small RNAs in response to Xoo infection, with small RNAs exhibiting the same expression pattern tended to regulate genes in the same or related signaling pathways, including auxin and GA signaling pathways, nutrition and defense-related pathways. These findings reveal the dynamic and complex roles of small RNAs in rice-Xoo interactions, and identify new targets for regulating plant responses to Xoo.     

Small RNA and Degradome Sequencing Reveal Complex Roles of miRNAs and Their Targets in Developing Wheat Grains

Plant microRNAs (miRNAs) have been shown to play critical roles in plant development. In this study, we employed small RNA combined with degradome sequencing to survey development-related miRNAs and their validated targets during wheat grain development. A total of 186 known miRNAs and 37 novel miRNAs were identified in four small RNA libraries. Moreover, a miRNA-like long hairpin locus was first identified to produce 21~22-nt phased siRNAs that act in trans to cleave target mRNAs. A comparison of the miRNAomes revealed that 55 miRNA families were differentially expressed during the grain development. Predicted and validated targets of these development-related miRNAs are involved in different cellular responses and metabolic processes including cell proliferation, auxin signaling, nutrient metabolism and gene expression. This study provides insight into the complex roles of miRNAs and their targets in regulating wheat grain development.     

Low temperature inhibits RNA silencing-mediated defence by the control of siRNA generation

Temperature dramatically affects plant-virus interactions. Outbreaks of virus diseases are frequently associated with low temperature, while at high temperature viral symptoms are often attenuated (heat masking) and plants rapidly recover from virus diseases. However, the underlying mechanisms of these well-known observations are not yet understood. RNA silencing is a conserved defence system of eukaryotic cells, which operates against molecular parasites including viruses and transgenes. Here we show that at low temperature both virus and transgene triggered RNA silencing are inhibited. Therefore, in cold, plants become more susceptible to viruses, and RNA silencing-based phenotypes of transgenic plants are lost. Consistently, the levels of virus- and transgene-derived small (21-26 nucleotide) interfering (si) RNAs-the central molecules of RNA silencing-mediated defence pathways-are dramatically reduced at low temperature. In contrast, RNA silencing was activated and the amount of siRNAs gradually increased with rising temperature. However, temperature does not influence the accumulation of micro (mi) RNAs, which play a role in developmental regulation, suggesting that the two classes of small (si and mi) RNAs are generated by different nuclease complexes.     

Trans-species small RNAs move long distances in a parasitic plant complex

Parasitic plants exchange various types of RNAs with their host plants, including mRNA, and small non-coding RNA. Among small non-coding RNAs, miRNA production is known to be induced at the haustorial interface. The induced miRNAs transfer to the host plant and activate secondary siRNA production to silence target genes in the host. In addition to interfacial transfer, long-distance movement of the small RNAs has also been known to mediate signaling and regulate biological processes. In this study, we tested the long-distance movement of trans-species small RNAs in a parasitic-plant complex. Small RNA-Seq was performed using a complex of a stem parasitic plant, Cuscuta campestris, and a host, Arabidopsis thaliana. In the host plant’s parasitized stem, genes involved in the production of secondary siRNA, AtSGS3 and AtRDR6, were upregulated, and 22-nt small RNA was enriched concomitantly, suggesting the activation of secondary siRNA production. Stem-loop RT-PCR and subsequent sequencing experimentally confirmed the mobility of the small RNAs. Trans-species mobile small RNAs were detected in the parasitic interface and also in distant organs. To clarify the mode of long-distance translocation, we examined whether C. campestris-derived small RNA moves long distances in A. thaliana sgs3 and rdr6 mutants or not. Mobility of C. campestris-derived small RNA in sgs3 and rdr6 mutants suggested the occurrence of direct long-distance transport without secondary siRNA production in the recipient plant.     

Pyrosequencing of small non-coding RNAs in HIV-1 infected cells: evidence for the processing of a viral-cellular double-stranded RNA hybrid

Small non-coding RNAs of 18–25 nt in length can regulate gene expression through the RNA interference (RNAi) pathway. To characterize small RNAs in HIV-1-infected cells, we performed linker-ligated cloning followed by high-throughput pyrosequencing. Here, we report the composition of small RNAs in HIV-1 productively infected MT4 T-cells. We identified several HIV-1 small RNA clones and a highly abundant small 18-nt RNA that is antisense to the HIV-1 primer-binding site (PBS). This 18-nt RNA apparently originated from the dsRNA hybrid formed by the HIV-1 PBS and the 3′ end of the human cellular tRNAlys3. It was found to associate with the Ago2 protein, suggesting its possible function in the cellular RNAi machinery for targeting HIV-1.     

Identification and characterization of small RNAs from vernalized<Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

MicroRNAs (miRNAs) and small interfering RNAs (siRNAs) are two major classes of small non-coding RNAs with important roles in the regulation of gene expression, such as mRNA degradation and translational repression, heterochromatin formation, genome defense against transposons and viruses in eukaryotes. MiRNA- and siRNA-directed processes have emerged as a regulatory mechanism for growth and development in both animals and plants. To identify small RNAs that might be involved in vernalization, a process accelerating flowering brought on by a long period of cold, we generated a library of small RNAs from Arabidopsis that had been subject to vernalization. From the analysis of the library, 277 small RNAs were identified. They were distributed throughout all the five chromosomes. While the vast majority of small RNA genes locate on intergenic regions, others locate on repeat-rich regions, centromeric regions, transposon-related genes, and protein-coding genes. Five of them were mapped to convergent overlapping gene pairs. Two-hundred and forty of them were novel endogenous small RNAs that have not been cloned yet from plants grown under normal conditions and other environmental stresses. Seven putative miRNAs were up- or down-regulated by vernalization. In conclusion, many small RNAs were identified from vernalized Arabidopsis and some of these identified small RNAs may play roles in plant responses to vernalization.     

Roles of DCL4 and DCL3b in rice phased small RNA biogenesis

Higher plants have evolved multiple proteins in the RNase III family to produce and regulate different classes of small RNAs with specialized molecular functions. In rice (Oryza sativa), numerous genomic clusters are targeted by one of two microRNAs (miRNAs), miR2118 and miR2275, to produce secondary small interfering RNAs (siRNAs) of either 21 or 24 nucleotides in a phased manner. The biogenesis requirements or the functions of the phased small RNAs are completely unknown. Here we examine the rice Dicer-Like (DCL) family, including OsDCL1, -3a, -3b and -4. By deep sequencing of small RNAs from different tissues of the wild type and osdcl4-1, we revealed that the processing of 21-nucleotide siRNAs, including trans-acting siRNAs (tasiRNA) and over 1000 phased small RNA loci, was largely dependent on OsDCL4. Surprisingly, the processing of 24-nucleotide phased small RNA requires the DCL3 homolog OsDCL3b rather than OsDCL3a, suggesting functional divergence within DCL3 family. RNA ligase-mediated 5' rapid amplification of cDNA ends and parallel analysis of RNA ends (PARE)/degradome analysis confirmed that most of the 21- and 24-nucleotide phased small RNA clusters were initiated from the target sites of miR2118 and miR2275, respectively. Furthermore, the accumulation of the two triggering miRNAs requires OsDCL1 activity. Finally, we show that phased small RNAs are preferentially produced in the male reproductive organs and are likely to be conserved in monocots. Our results revealed significant roles of OsDCL4, OsDCL3b and OsDCL1 in the 21- and 24-nucleotide phased small RNA biogenesis pathway in rice.