植物RNA沉默的分子机制研究进展

RNA沉默（RNA silencing）是真核生物中的一种抵抗外源遗传因子（病毒、转座子或转基因）及调控基凶表达的防御机制。参与植物RNA沉默的酶及蛋白质主要包括6种RNA依赖的RNA聚合酶、4种Dicer-like（DCL）核酸内切酶和10种Argonautes蛋白。植物中4条RNA沉默途径分别由微小RNA（miRNAs）和3种小干扰RNA（siRNAs）介导,包括反式作用siRNAs（ta－siRNAs）、天然反义siRNAs（natsiRNAs）和异染色质siRNAs（hc-siRNAs）。在植物RNA沉默的系统性传播中,由DCL4或DCL2将dsRNAs裁剪为次级SiRNAS,以放大RNA沉默信号和增强沉默效应。     

MicroRNAs: something important between the genes

Non-coding small endogenous RNAs, of 21-24 nucleotides in length, have recently emerged as important regulators of gene expression in both plants and animals. At least three categories of small RNAs exist in plants: short interfering RNAs (siRNAs) deriving from viruses or transgenes and mediating virus resistance or transgene silencing via RNA degradation; siRNAs deriving from transposons or transgene promoters and controlling transposon and transgene silencing probably via chromatin changes; and microRNAs (miRNAs) deriving from intergenic regions of the genome and regulating the expression of endogenous genes either by mRNA cleavage or translational repression. The disruption of miRNA-mediated regulation causes developmental abnormalities in plants, demonstrating that miRNAs play an important role in the regulation of developmental decisions.     

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.     

Diversity of endogenous small non-coding RNAs in Oryza sativa

Small non-coding RNAs play important roles in regulating cell functions by controlling mRNA turnover and translational repression in eukaryotic cells. Here we isolated 162 endogenous small RNA molecules from Oryza sativa, which ranged from 16 to 35 nt in length. Further analysis indicated that they represented a diversity of small RNA molecules, including 17 microRNAs (miRNAs), 30 tiny non-coding RNAs (tncRNAs) and 20 repeat-associated small interfering RNAs (rasiRNAs). Among 17 miRNAs, 13 were novel miRNA candidates and their potential targets were important regulatory genes in the rice genome. We also found that a cluster of small RNAs, including many rasiRNAs, matched to a nuclear DNA fragment that evolutionarily derived from chloroplast. These results demonstrate clearly the existence of distinct types of small RNAs in rice and further suggest that small RNAs may control gene regulation through diverse mechanisms.     

Identification and characterization of endogenous small interfering RNAs from rice

RNA silencing-mediated small interfering RNAs (siRNAs) and microRNAs (miRNAs) have diverse natural roles, ranging from regulation of gene expression and heterochromatin formation to genome defense against transposons and viruses. Unlike miRNAs, endogenous siRNAs are generally not conserved between species; consequently, their identification requires experimental approaches. Thus far, endogenous siRNAs have not been reported from rice, which is a model species for monocotyledonous plants. We identified a large set of putative endogenous siRNAs from root, shoot and inflorescence small RNA cDNA libraries of rice. Most of these siRNAs are from intergenic regions, although a substantial proportion (22%) originates from the introns and exons of protein-coding genes. Northern and RT–PCR analysis revealed that the expression of some of the siRNAs is tissue specific or developmental stage specific. A total of 25 transposons and 21 protein-coding genes were predicted to be cis-targets of some of the siRNAs. Based on sequence homology, we also predicted 111 putative trans-targets for 44 of the siRNAs. Interestingly, ~46% of the predicted trans-targets are transposable elements, which suggests that endogenous siRNAs may play an important role in the suppression of transposon proliferation. Using RNA ligase-mediated-5′ rapid amplification of cDNA end assays, we validated three of the predicted targets and provided evidence for both cis- and trans-silencing of target genes by siRNAs-guided mRNA cleavage.     

Artificial microRNA-mediated virus resistance in plants

RNA silencing in plants is a natural defense system against foreign genetic elements including viruses. This natural antiviral mechanism has been adopted to develop virus-resistant plants through expression of virus-derived double-stranded RNAs or hairpin RNAs, which in turn are processed into small interfering RNAs (siRNAs) by the host's RNA silencing machinery. While these virus-specific siRNAs were shown to be a hallmark of the acquired virus resistance, the functionality of another set of the RNA silencing-related small RNAs, microRNAs (miRNAs), in engineering plant virus resistance has not been extensively explored. Here we show that expression of an artificial miRNA, targeting sequences encoding the silencing suppressor 2b of Cucumber mosaic virus (CMV), can efficiently inhibit 2b gene expression and protein suppressor function in transient expression assays and confer on transgenic tobacco plants effective resistance to CMV infection. Moreover, the resistance level conferred by the transgenic miRNA is well correlated to the miRNA expression level. Comparison of the anti-CMV effect of the artificial miRNA to that of a short hairpin RNA-derived small RNA targeting the same site revealed that the miRNA approach is superior to the approach using short hairpin RNA both in transient assays and in transgenic plants. Together, our data demonstrate that expression of virus-specific artificial miRNAs is an effective and predictable new approach to engineering resistance to CMV and, possibly, to other plant viruses as well.     

shRNA Transcribed by RNA Pol II Promoter Induce RNA Interference in Mammalian Cell

RNA interference is a powerful tool for gene functional analysis in mammals. Permanent gene suppression can be achieved by siRNAs as stem-loop precursors transcribed from RNA Pol III promoter such as H1 and U6 based on vector. This approach, however, has a major limitation: inhibition can not be controlled in a time or tissue specific manner because the RNA Pol III promoter is not time or tissue specific. To overcome these limitations, we designed a strategy that allows synthesis of small hairpin RNAs in a GFP-fused form mediated by RNA Pol II promoter CMV to efficiently and specifically knock down expression of both exogenous and endogenous genes in mammalian cells. As assayed by both fluorescence observing and quantitative RT-PCR, the protein and mRNA products of exogenous gene RFP were efficiently and specifically inhibited; quantitative RT-PCR and western blotting results respectively demonstrated that endogenous lamin B2 mRNA and protein was suppressed without global down-regulation of protein synthesis. Furthermore, GFP-fused shRNA efficacy for RNAi is dependent on target position based on this vector system. This method may provide a novel approach for the application of RNAi technology in suppressing gene expression in mammalian system. Jing Yuan, Xiaobo Wang and Ning Li - These authors contributed equally to this work.     

An inside job for siRNAs

Among the three main categories of small silencing RNAs in insects and mammals-siRNAs, miRNAs, and piRNAs-siRNAs were thought to arise primarily from exogenous sources, whereas miRNAs and piRNAs arise from endogenous loci. Recent work in flies and mice reveals several classes of endogenous siRNAs (endo-siRNAs) that contribute to functions previously reserved for miRNAs and piRNAs, including gene regulation and transposon suppression.     

MicroRNAs in Amoebozoa: Deep sequencing of the small RNA population in the social amoeba Dictyostelium discoideum reveals developmentally regulated microRNAs

The RNA interference machinery has served as a guardian of eukaryotic genomes since the divergence from prokaryotes. Although the basic components have a shared origin, silencing pathways directed by small RNAs have evolved in diverse directions in different eukaryotic lineages. Micro (mi)RNAs regulate protein-coding genes and play vital roles in plants and animals, but less is known about their functions in other organisms. Here, we report, for the first time, deep sequencing of small RNAs from the social amoeba Dictyostelium discoideum. RNA from growing single-cell amoebae as well as from two multicellular developmental stages was sequenced. Computational analyses combined with experimental data reveal the expression of miRNAs, several of them exhibiting distinct expression patterns during development. To our knowledge, this is the first report of miRNAs in the Amoebozoa supergroup. We also show that overexpressed miRNA precursors generate miRNAs and, in most cases, miRNA* sequences, whose biogenesis is dependent on the Dicer-like protein DrnB, further supporting the presence of miRNAs in D. discoideum. In addition, we find miRNAs processed from hairpin structures originating from an intron as well as from a class of repetitive elements. We believe that these repetitive elements are sources for newly evolved miRNAs.     

Small RNAs in development - insights from plants

microRNAs (miRNAs) and small interfering RNAs (siRNAs), which constitute two major classes of endogenous small RNAs in plants, impact a multitude of developmental and physiological processes by imparting sequence specificity to gene and genome regulation. Although lacking the third major class of small RNAs found in animals, Piwi-interacting RNAs (piRNAs), plants have expanded their repertoire of endogenous siRNAs, some of which fulfill similar molecular and developmental functions as piRNAs in animals. Research on plant miRNAs and siRNAs has contributed invaluable insights into small RNA biology, thanks to the highly conserved molecular logic behind the biogenesis and actions of small RNAs. Here, I review progress in the plant small RNA field in the past two years, with an emphasis on recent findings related to plant development. I do not recount the numerous developmental processes regulated by small RNAs; instead, I focus on major principles that have been derived from recent studies and draw parallels, when applicable, between plants and animals.     

SRNAome and degradome sequencing analysis reveals specific regulation of sRNA in response to chilling injury in tomato fruit

Plant genomes encode diverse small RNA classes that function in distinct gene‐silencing pathways. To elucidate the intricate regulation of microRNAs (miRNAs) and endogenous small‐interfering RNAs (siRNAs) in response to chilling injury in tomato fruit, the deep sequencing and bioinformatic methods were combined to decipher the small RNAs landscape in the control and chilling‐injured groups. Except for the known miRNAs and ta‐siRNAs, 85 novel miRNAs and 5 ta‐siRNAs members belonging to 3 TAS families (TAS5, TAS9 and TAS10) were identified, 34 putative phased small RNAs and 740 cis/trans‐natural antisense small‐interfering RNAs (nat‐siRNAs) were also found in our results which enriched the tomato small RNAs repository. A large number of genes targeted by those miRNAs and siRNAs were predicted to be involved in the chilling injury responsive process and five of them were verified via degradome sequencing. Based on the above results, a regulatory model that comprehensively reveals the relationships between the small RNAs and their targets was set up. This work provides a foundation for further study of the regulation of miRNAs and siRNAs in the plant in response to chilling injury.