Yijun Qi

Criteria for Annotation of Plant MicroRNAs

MicroRNAs (miRNAs) are ∼21 nucleotide noncoding RNAs produced by Dicer-catalyzed excision from stem-loop precursors. Many plant miRNAs play critical roles in development, nutrient homeostasis, abiotic stress responses, and pathogen responses via interactions with specific target mRNAs. miRNAs are not the only Dicer-derived small RNAs produced by plants: A substantial amount of the total small RNA abundance and an overwhelming amount of small RNA sequence diversity is contributed by distinct classes of 21- to 24-nucleotide short interfering RNAs. This fact, coupled with the rapidly increasing rate of plant small RNA discovery, demands an increased rigor in miRNA annotations. Herein, we update the specific criteria required for the annotation of plant miRNAs, including experimental and computational data, as well as refinements to standard nomenclature.

Distinct catalytic and non-catalytic roles of ARGONAUTE4 in RNA-directed DNA methylation

DNA methylation has important functions in stable, transcriptional gene silencing, immobilization of transposable elements and genome organization. In Arabidopsis, DNA methylation can be induced by double-stranded RNA through the RNA interference (RNAi) pathway, a response known as RNA-directed DNA methylation. This requires a specialized set of RNAi components, including ARGONAUTE4 (AGO4). Here we show that AGO4 binds to small RNAs including small interfering RNAs (siRNAs) originating from transposable and repetitive elements, and cleaves target RNA transcripts. Single mutations in the Asp-Asp-His catalytic motif of AGO4 do not affect siRNA-binding activity but abolish its catalytic potential. siRNA accumulation and non-CpG DNA methylation at some loci require the catalytic activity of AGO4, whereas others are less dependent on this activity. Our results are consistent with a model in which AGO4 can function at target loci through two distinct and separable mechanisms. First, AGO4 can recruit components that signal DNA methylation in a manner independent of its catalytic activity. Second, AGO4 catalytic activity can be crucial for the generation of secondary siRNAs that reinforce its repressive effects.

Global Epigenetic and Transcriptional Trends among Two Rice Subspecies and Their Reciprocal Hybrids

This work examines the molecular basis of heterosis by comprehensively describing the epigenetic modifications and transcriptional output, including both mRNA and small RNAs, of two rice subspecies and their reciprocal hybrids. The behavior of transcriptomes and epigenomes in hybrids of heterotic parents is of fundamental interest. Here, we report highly integrated maps of the epigenome, mRNA, and small RNA transcriptomes of two rice (Oryza sativa) subspecies and their reciprocal hybrids. We found that gene activity was correlated with DNA methylation and both active and repressive histone modifications in transcribed regions. Differential epigenetic modifications correlated with changes in transcript levels among hybrids and parental lines. Distinct patterns in gene expression and epigenetic modifications in reciprocal hybrids were observed. Through analyses of single nucleotide polymorphisms from our sequence data, we observed a high correlation of allelic bias of epigenetic modifications or gene expression in reciprocal hybrids with their differences in the parental lines. The abundance of distinct small RNA size classes differed between the parents, and more small RNAs were downregulated than upregulated in the reciprocal hybrids. Together, our data reveal a comprehensive overview of transcriptional and epigenetic trends in heterotic rice crosses and provide a useful resource for the rice community.

A Role for Small RNAs in DNA Double-Strand Break Repair

Eukaryotes have evolved complex mechanisms to repair DNA double-strand breaks (DSBs) through coordinated actions of protein sensors, transducers, and effectors. Here we show that ∼21-nucleotide small RNAs are produced from the sequences in the vicinity of DSB sites in Arabidopsis and in human cells. We refer to these as diRNAs for DSB-induced small RNAs. In Arabidopsis, the biogenesis of diRNAs requires the PI3 kinase ATR, RNA polymerase IV (Pol IV), and Dicer-like proteins. Mutations in these proteins as well as in Pol V cause significant reduction in DSB repair efficiency. In Arabidopsis, diRNAs are recruited by Argonaute 2 (AGO2) to mediate DSB repair. Knock down of Dicer or Ago2 in human cells reduces DSB repair. Our findings reveal a conserved function for small RNAs in the DSB repair pathway. We propose that diRNAs may function as guide molecules directing chromatin modifications or the recruitment of protein complexes to DSB sites to facilitate repair.

Genome-Wide and Organ-Specific Landscapes of Epigenetic Modifications and Their Relationships to mRNA and Small RNA Transcriptomes in Maize

Maize (Zea mays) has an exceptionally complex genome with a rich history in both epigenetics and evolution. We report genomic landscapes of representative epigenetic modifications and their relationships to mRNA and small RNA (smRNA) transcriptomes in maize shoots and roots. The epigenetic patterns differed dramatically between genes and transposable elements, and two repressive marks (H3K27me3 and DNA methylation) were usually mutually exclusive. We found an organ-specific distribution of canonical microRNAs (miRNAs) and endogenous small interfering RNAs (siRNAs), indicative of their tissue-specific biogenesis. Furthermore, we observed that a decreasing level of mop1 led to a concomitant decrease of 24-nucleotide siRNAs relative to 21-nucleotide miRNAs in a tissue-specific manner. A group of 22-nucleotide siRNAs may originate from long-hairpin double-stranded RNAs and preferentially target gene-coding regions. Additionally, a class of miRNA-like smRNAs, whose putative precursors can form short hairpins, potentially targets genes in trans. In summary, our data provide a critical analysis of the maize epigenome and its relationships to mRNA and smRNA transcriptomes.Epigenetic modifications and small RNAs play an important role in gene regulation. Here, we discuss results of our Solexa/Illumina 1G sequencing-based survey of DNA methylation, activating and repressive histone modifications, small RNAs and mRNA in the maize genome. We analyze tissue-specific epigenetic patterns, discuss antagonistic relationships between repressive epigenetic marks and highlight synergistic relationships between activating histone modifications. We discuss our observation that small RNAs show a tissue-specific distribution in maize. Whereas 24-nucleotide long small interfering RNAs (siRNAs) accumulated preferentially in shoots, 21-nucleotide long micro RNAs (miRNAs) were the most abundant group in roots, which follows the transcript level of mop1. Furthermore, we discuss the possibility that a novel class of 22-nucleotide siRNAs might originate from long double-stranded RNAs in an RNA-dependent RNA polymerase (RdRP)-independent manner. This supports the intriguing possibility that maize possesses at least two distinct pathways to generate siRNAs, one of which relies on RdRP and a second one that might be RdRP-independent.

Rice MicroRNA Effector Complexes and Targets

MicroRNAs (miRNAs) are small silencing RNAs with regulatory roles in gene expression. miRNAs interact with Argonaute (AGO) proteins to form effector complexes that cleave target mRNAs or repress translation. Rice (Oryza sativa) encodes four AGO1 homologs (AGO1a, AGO1b, AGO1c, and AGO1d). We used RNA interference (RNAi) to knock down the four AGO1s. The RNAi lines displayed pleiotropic developmental phenotypes and had increased accumulation of miRNA targets. AGO1a, AGO1b, and AGO1c complexes were purified and further characterized. The three AGO1s all have a strong preference for binding small RNAs (sRNAs) with 5′ U and have Slicer activity. We cataloged the sRNAs in each AGO1 complex by deep sequencing and found that all three AGO1s predominantly bound known miRNAs. Most of the miRNAs were evenly distributed in the three AGO1 complexes, suggesting a redundant role for the AGO1s. Intriguingly, a subset of miRNAs were specifically incorporated into or excluded from one of the AGO1s, suggesting functional specialization among the AGO1s. Furthermore, we identified rice miRNA targets at a global level. The validated targets include transcription factors that control major stages of development and also genes involved in a variety of physiological processes, indicating a broad regulatory role for miRNAs in rice.

Identification of MicroRNAs Involved in Pathogen-Associated Molecular Pattern-Triggered Plant Innate Immunity

Pathogen-associated molecular patterns (PAMPs) trigger plant defenses when perceived by surface-localized immune receptors. PAMP-triggered immunity (PTI) plays a vital role in the resistance of plants to numerous potential pathogens. MicroRNA (miRNA) biogenesis is known to be important for PTI, but miRNA species involved in this process have not been fully explored. Here we show that the Arabidopsis (Arabidopsis thaliana) miRNA effector protein, Argonaute1 (AGO1), is required for a number of PTI responses including PAMP-induced callose deposition, gene expression, and seedling growth inhibition. Deep sequencing of AGO1-bound small RNAs led to the identification of a number of miRNAs that are up- or down-regulated by flg22, a well-studied PAMP. Overexpression of selected miRNAs in stable transgenic plants demonstrated that miR160a positively regulate PAMP-induced callose deposition, whereas miR398b and miR773 negatively regulate PAMP-induced callose deposition and disease resistance to bacteria, suggesting a complexity of the miRNA regulation in plant innate immunity.

Genome-Wide Analysis of DNA Methylation and Gene Expression Changes in Two Arabidopsis Ecotypes and Their Reciprocal Hybrids

This work analyzed DNA methylation and gene expression changes in two Arabidopsis ecotypes and their reciprocal hybrids and found that increased DNA methylation in both hybrid genomes, possibly directed by the RdDM pathway, may play a role in heterosis. Heterosis is a fundamental biological phenomenon characterized by the superior performance of a hybrid over its parents in many traits, but the underlying molecular basis remains elusive. To investigate whether DNA methylation plays a role in heterosis, we compared at single-base-pair resolution the DNA methylomes of Arabidopsis thaliana Landsberg erecta and C24 parental lines and their reciprocal F1 hybrids that exhibited heterosis. Both hybrids displayed increased DNA methylation across their entire genomes, especially in transposable elements. Interestingly, increased methylation of the hybrid genomes predominantly occurred in regions that were differentially methylated in the two parents and covered by small RNAs, implying that the RNA-directed DNA methylation (RdDM) pathway may direct DNA methylation in hybrids. In addition, we found that 77 genes sensitive to methylome remodeling were transcriptionally repressed in both reciprocal hybrids, including genes involved in flavonoid biosynthesis and two circadian oscillator genes CIRCADIAN CLOCK ASSOCIATED1 and LATE ELONGATED HYPOCOTYL. Moreover, growth vigor of F1 hybrids was compromised by treatment with an agent that demethylates DNA and by abolishing production of functional small RNAs due to mutations in Arabidopsis RNA methyltransferase HUA ENHANCER1. Together, our data suggest that genome-wide remodeling of DNA methylation directed by the RdDM pathway may play a role in heterosis.

A Structured Viroid RNA Serves as a Substrate for Dicer-Like Cleavage To Produce Biologically Active Small RNAs but Is Resistant to RNA-Induced Silencing Complex-Mediated Degradation

ABSTRACT RNA silencing is a potent means of antiviral defense in plants and animals. A hallmark of this defense response is the production of 21- to 24-nucleotide viral small RNAs via mechanisms that remain to be fully understood. Many viruses encode suppressors of RNA silencing, and some viral RNAs function directly as silencing suppressors as counterdefense. The occurrence of viroid-specific small RNAs in infected plants suggests that viroids can trigger RNA silencing in a host, raising the question of how these noncoding and unencapsidated RNAs survive cellular RNA-silencing systems. We address this question by characterizing the production of small RNAs of Potato spindle tuber viroid (srPSTVds) and investigating how PSTVd responds to RNA silencing. Our molecular and biochemical studies provide evidence that srPSTVds were derived mostly from the secondary structure of viroid RNAs. Replication of PSTVd was resistant to RNA silencing, although the srPSTVds were biologically active in guiding RNA-induced silencing complex (RISC)-mediated cleavage, as shown with a sensor system. Further analyses showed that without possessing or triggering silencing suppressor activities, the PSTVd secondary structure played a critical role in resistance to RISC-mediated cleavage. These findings support the hypothesis that some infectious RNAs may have evolved specific secondary structures as an effective means to evade RNA silencing in addition to encoding silencing suppressor activities. Our results should have important implications in further studies on RNA-based mechanisms of host-pathogen interactions and the biological constraints that shape the evolution of infectious RNA structures.

Gene silencing by artificial microRNAs in Chlamydomonas

Chlamydomonas reinhardtii is a unicellular green alga. It is a model system for studying functions of the chloroplast, basal body and flagella. The completion of the Chlamydomonas genome sequence makes it possible to use reverse genetic approaches in this organism. Chlamydomonas contains a set of endogenous microRNAs (miRNAs) that down-regulate their target gene expression through mRNA cleavage. Here we developed an artificial miRNA-based strategy to knock down gene expression in Chlamydomonas. Using an endogenous Chlamydomonas miRNA precursor as the backbone, we constructed two artificial miRNAs (amiRNAs) targeting the MAA7 and RBCS1/2 genes, respectively. When overexpressed, these two amiRNAs could cleave their respective targets precisely at the predicted sites, resulting in greatly decreased accumulation of MAA7 and RBCS1/2 transcripts and expected mutant phenotypes. We further showed that the two amiRNAs could be produced simultaneously from a dimeric amiRNA precursor. We anticipate that the amiRNA technology developed in this study will be very useful in assessing the functions of individual genes and in genome-wide approaches.