Zhixin Xie

Genetic and Functional Diversification of Small RNA Pathways in Plants

Multicellular eukaryotes produce small RNA molecules (approximately 21–24 nucleotides) of two general types, microRNA (miRNA) and short interfering RNA (siRNA). They collectively function as sequence-specific guides to silence or regulate genes, transposons, and viruses and to modify chromatin and genome structure. Formation or activity of small RNAs requires factors belonging to gene families that encode DICER (or DICER-LIKE [DCL]) and ARGONAUTE proteins and, in the case of some siRNAs, RNA-dependent RNA polymerase (RDR) proteins. Unlike many animals, plants encode multiple DCL and RDR proteins. Using a series of insertion mutants of Arabidopsis thaliana, unique functions for three DCL proteins in miRNA (DCL1), endogenous siRNA (DCL3), and viral siRNA (DCL2) biogenesis were identified. One RDR protein (RDR2) was required for all endogenous siRNAs analyzed. The loss of endogenous siRNA in dcl3 and rdr2 mutants was associated with loss of heterochromatic marks and increased transcript accumulation at some loci. Defects in siRNA-generation activity in response to turnip crinkle virus in dcl2 mutant plants correlated with increased virus susceptibility. We conclude that proliferation and diversification of DCL and RDR genes during evolution of plants contributed to specialization of small RNA-directed pathways for development, chromatin structure, and defense.

P1/HC-Pro, a Viral Suppressor of RNA Silencing, Interferes with Arabidopsis Development and miRNA Function

The molecular basis for virus-induced disease in plants has been a long-standing mystery. Infection of Arabidopsis by Turnip mosaic virus (TuMV) induces a number of developmental defects in vegetative and reproductive organs. We found that these defects, many of which resemble those in miRNA-deficient dicer-like1 (dcl1) mutants, were due to the TuMV-encoded RNA-silencing suppressor, P1/HC-Pro. Suppression of RNA silencing is a counterdefensive mechanism that enables systemic infection by TuMV. The suppressor interfered with the activity of miR171 (also known as miRNA39), which directs cleavage of several mRNAs coding for Scarecrow-like transcription factors, by inhibiting miR171-guided nucleolytic function. Out of ten other mRNAs that were validated as miRNA-guided cleavage targets, eight accumulated to elevated levels in the presence of P1/HC-Pro. The basis for TuMV- and other virus-induced disease in plants may be explained, at least partly, by interference with miRNA-controlled developmental pathways that share components with the antiviral RNA-silencing pathway.

Evolution of microRNA genes by inverted duplication of target gene sequences in Arabidopsis thaliana.

MicroRNAs (miRNAs) in plants and animals function as post-transcriptional regulators of target genes, many of which are involved in multicellular development. miRNAs guide effector complexes to target mRNAs through base-pair complementarity, facilitating site-specific cleavage or translational repression. Biogenesis of miRNAs involves nucleolytic processing of a precursor transcript with extensive foldback structure. Here, we provide evidence that genes encoding miRNAs in plants originated by inverted duplication of target gene sequences. Several recently evolved genes encoding miRNAs in Arabidopsis thaliana and other small RNA–generating loci possess the hallmarks of inverted duplication events that formed the arms on each side of their respective foldback precursors. We propose a model for miRNA evolution that suggests a mechanism for de novo generation of new miRNA genes with unique target specificities.

Expression of Arabidopsis MIRNA Genes

MicroRNAs (miRNAs) are approximately 21-nucleotide noncoding RNAs that regulate target transcripts in plants and animals. In addition to miRNAs, plants contain several classes of endogenous small interfering RNAs (siRNAs) involved in target gene regulation and epigenetic silencing. Small RNA libraries were constructed from wild-type Arabidopsis (Arabidopsis thaliana) and mutant plants (rdr2 and dcl3) that were genetically enriched for miRNAs, and a computational procedure was developed to identify candidate miRNAs. Thirty-eight distinct miRNAs corresponding to 22 families were represented in the libraries. Using a 5′ rapid amplification of cDNA ends procedure, the transcription start sites for 63 miRNA primary transcripts from 52 MIRNA loci (99 loci tested) were mapped, revealing features consistent with an RNA polymerase II mechanism of transcription. Ten loci (19%) yielded transcripts from multiple start sites. A canonical TATA box motif was identified upstream of the major start site at 45 (86%) of the mapped MIRNA loci. The 5′-mapping data were combined with miRNA cloning and 3′-PCR data to definitively validate expression of at least 73 MIRNA genes. These data provide a molecular basis to explore regulatory mechanisms of miRNA expression in plants.

Negative Feedback Regulation of Dicer-Like1 in Arabidopsis by microRNA-Guided mRNA Degradation

Formation of microRNA (miRNA) requires an RNaseIII domain-containing protein, termed DICER-1 in animals and DICER-LIKE1 (DCL1) in plants, to catalyze processing of an RNA precursor with a fold-back structure. Loss-of-function dcl1 mutants of Arabidopsis have low levels of miRNA and exhibit a range of developmental phenotypes in vegetative, reproductive, and embryonic tissues. In this paper, we show that DCL1 mRNA occurs in multiple forms, including truncated molecules that result from aberrant pre-mRNA processing. Both full-length and truncated forms accumulated to relatively low levels in plants containing a functional DCL1 gene. However, in dcl1 mutant plants, dcl1 RNA forms accumulated to levels several-fold higher than those in DCL1 plants. Elevated levels of DCL1 RNAs were also detected in miRNA-defective hen1 mutant plants and in plants expressing a virus-encoded suppressor of RNA silencing (P1/HC-Pro), which inhibits miRNA-guided degradation of target mRNAs. A miRNA (miR162) target sequence was predicted near the middle of DCL1 mRNA, and a DCL1-derived RNA with the properties of a miR162-guided cleavage product was identified and mapped. These results indicate that DCL1 mRNA is subject to negative feedback regulation through the activity of a miRNA.

An important role of an inducible RNA-dependent RNA polymerase in plant antiviral defense

Plants contain RNA-dependent RNA polymerase (RdRP) activities that synthesize short cRNAs by using cellular or viral RNAs as templates. During studies of salicylic acid (SA)-induced resistance to viral pathogens, we recently found that the activity of a tobacco RdRP was increased in virus-infected or SA-treated plants. Biologically active SA analogs capable of activating plant defense response also induced the RdRP activity, whereas biologically inactive analogs did not. A tobacco RdRP gene, NtRDRP1, was isolated and found to be induced both by virus infection and by treatment with SA or its biologically active analogs. Tobacco lines deficient in the inducible RDRP activity were obtained by expressing antisense RNA for the NtRDRP1 gene in transgenic plants. When infected by tobacco mosaic virus, these transgenic plants accumulated significantly higher levels of viral RNA and developed more severe disease symptoms than wild-type plants. After infection by a strain of potato virus X that does not spread in wild-type tobacco plants, the transgenic NtRDRP1 antisense plants accumulated virus and developed symptoms not only locally in inoculated leaves but also systemically in upper uninoculated leaves. These results strongly suggest that inducible RdRP activity plays an important role in plant antiviral defense.

Diverse small RNA-directed silencing pathways in plants

Small silencing RNAs of 21- to 24-nucleotide (nt) in length are essential regulatory components expressed in most eukaryotic organisms. These regulatory small RNAs are produced through pathways that involve several evolutionarily conserved protein families, including DICER (DCR) or DICER-LIKE (DCL), ARGONAUTE (AGO), and RNA-DEPENDENT RNA POLYMERASE (RDR). Plants possess multiple functional DCL, RDR, and AGO proteins. Genetic analyses in the model plant Arabidopsis thaliana have revealed multiple small RNA pathways, each utilizing a distinct set of RNA silencing factors. In this short review, mainly based on the work done on A. thaliana, we give a brief overview on the multiple small RNA-directed silencing pathways in plants, which includes the biogenesis and function of microRNAs (miRNAs), trans-acting siRNAs (ta-siRNAs), natural cis-antisense transcripts-associated siRNAs (nat-siRNAs), and heterochromatic siRNAs.

Expression of microRNAs and its regulation in plants

MicroRNAs (miRNAs) have emerged as an essential regulatory component in plants. Many of the known miRNAs are evolutionarily conserved across diverse plant species and function in the regulatory control of fundamentally important biological processes such as developmental timing, patterning, and response to environmental changes. Expression of miRNAs in plants involves transcription from MIRNA loci by RNA polymerase II (pol II), multi-step processing of the primary transcripts by the DICER-LIKE1 (DCL1) complex, and formation of effector complexes consisting of mature miRNAs and ARGONAUTE (AGO) family proteins. In this short review, we present the most recent advances in our understanding of the molecular machinery as well as the regulatory mechanisms involved in the expression of miRNAs in plants.

Small RNAs in Plants

Small RNAs associated with RNA silencing have emerged as an essential regulatory component in eukaryotes. Although their widespread existence was revealed only a decade ago, remarkable progress has been made toward our understanding of their biogenesis and cellular function. In plants, the small RNA-mediated regulatory mechanisms are involved in many important biological processes including developmental timing, pattern formation, epigenetic silencing of transposable elements, response to environmental stress, and defense against invading pathogens. Emerging evidence also indicates the involvement of small RNAs in epigenetic reprogramming associated with germ cell and embryo development during sexual reproduction. In this chapter, we provide an overview on the conserved molecular machinery that has evolved to give rise to microRNAs (miRNAs) and several distinct classes of small interfering RNAs (siRNAs) in plants, including heterochromatin-associated siRNAs (hc-siRNAs), trans-acting siRNAs (ta-siRNAs), and natural cis-antisense transcripts-associated siRNAs (nat-siRNAs). These are followed by a description on the cellular function and regulatory targets for each class of these endogenous small RNAs. While the focus of the book is on miRNAs, it is our hope that this chapter will serve as a brief introduction to the plant small RNA world.