Plant genomes encode various small RNAs that function in distinct, yet overlapping, genetic and epigenetic silencing pathways. However, the abundance and diversity of small-RNA classes varies among plant species, suggesting coevolution between environmental adaptations and gene-silencing mechanisms. Biogenesis of small RNAs in plants is well understood, but we are just beginning to uncover their intricate regulation and activity. Here, we discuss the biogenesis of plant small RNAs, such as microRNAs, secondary siRNAs and heterochromatic siRNAs, and their diverse cellular and developmental functions, including in reproductive transitions, genomic imprinting and paramutation. We also discuss the diversification of small-RNA-directed silencing pathways through the expansion of RNA-dependent RNA polymerases, DICER proteins and ARGONAUTE proteins.

/pdf/the-expanding-world-of-small-rnas-in-plants-1ouxzc3mtq.pdf

The expanding world of small RNAs in plants

MicroRNAs (miRNAs) represent an important class of small non-coding RNAs (sRNAs) that regulate gene expression by targeting messenger RNAs. However, assigning miRNAs to their regulatory target genes remains technically challenging. Recently, high-throughput CLIP-Seq and degradome sequencing (Degradome-Seq) methods have been applied to identify the sites of Argonaute interaction and miRNA cleavage sites, respectively. In this study, we introduce a novel database, starBase (sRNA target Base), which we have developed to facilitate the comprehensive exploration of miRNA–target interaction maps from CLIP-Seq and Degradome-Seq data. The current version includes high-throughput sequencing data generated from 21 CLIP-Seq and 10 DegradomeSeq experiments from six organisms. By analyzing millions of mapped CLIP-Seq and Degradome-Seq reads, we identified � 1 million Ago-binding clusters and � 2 million cleaved target clusters in animals and plants, respectively. Analyses of these clusters, and of target sites predicted by 6 miRNA target prediction programs, resulted in our identification of approximately 400 000 and approximately 66 000 miRNA-target regulatory relationships from CLIP-Seq and Degradome-Seq data, respectively. Furthermore, two web servers were provided to discover novel miRNA target sites from CLIP-Seq and Degradome-Seq data. Our web implementation supports diverse query types and exploration of common targets, gene ontologies and pathways. The starBase is available at http://starbase.sysu .edu.cn/.

/pdf/starbase-a-database-for-exploring-microrna-mrna-interaction-3efa3k1418.pdf

starBase: a database for exploring microRNA–mRNA interaction maps from Argonaute CLIP-Seq and Degradome-Seq data

MicroRNAs (miRNAs) are endogenous ∼23 nt RNAs that play important gene-regulatory roles in animals and plants by pairing to the mRNAs of protein-coding genes to direct their posttranscriptional repression. This review outlines the current understanding of miRNA target recognition in animals and discusses the widespread impact of miRNAs on both the expression and evolution of protein-coding genes.

MicroRNAs: Target Recognition and Regulatory Functions

https://www.cell.com/molecular-cell/pdf/S1097-2765(10)00242-X.pdf

DNA Methylation Mediated by a MicroRNA Pathway

In mammals, cadmium is widely considered as a non-genotoxic carcinogen acting through a methylation-dependent epigenetic mechanism. Here, the effects of Cd treatment on the DNA methylation patten are examined together with its effect on chromatin reconfiguration in Posidonia oceanica. DNA methylation level and pattern were analysed in actively growing organs, under short- (6 h) and long- (2 d or 4 d) term and low (10 mM) and high (50 mM) doses of Cd, through a Methylation-Sensitive Amplification Polymorphism technique and an immunocytological approach, respectively. The expression of one member of the CHROMOMETHYLASE (CMT) family, a DNA methyltransferase, was also assessed by qRT-PCR. Nuclear chromatin ultrastructure was investigated by transmission electron microscopy. Cd treatment induced a DNA hypermethylation, as well as an up-regulation of CMT, indicating that de novo methylation did indeed occur. Moreover, a high dose of Cd led to a progressive heterochromatinization of interphase nuclei and apoptotic figures were also observed after long-term treatment. The data demonstrate that Cd perturbs the DNA methylation status through the involvement of a specific methyltransferase. Such changes are linked to nuclear chromatin reconfiguration likely to establish a new balance of expressed/repressed chromatin. Overall, the data show an epigenetic basis to the mechanism underlying Cd toxicity in plants.

/pdf/in-posidonia-oceanica-cadmium-induces-changes-in-dna-3cu7uj83mr.pdf

In Posidonia oceanica cadmium induces changes in DNA methylation and chromatin patterning

MicroRNAs (miRNAs) and small interfering RNAs (siRNAs) regulate gene expression in eukaryotes. Plant miRNAs modulate their targets mainly via messenger RNA (mRNA) cleavage. Small RNA (sRNA) targets have been extensively investigated in Arabidopsis using computational prediction, experimental validation, and degradome sequencing. However, small RNA targets are largely unknown in rice (Oryza sativa). Here, we report global identification of small RNA targets using high throughput degradome sequencing in the rice indica cultivar 93–11 (Oryza sativa L. ssp. indica). One hundred and seventy-seven transcripts targeted by a total of 87 unique miRNAs were identified. Of targets for the conserved miRNAs between Arabidopsis and rice, transcription factors comprise around 70% (58 in 82), indicating that these miRNAs act as masters of gene regulatory nodes in rice. In contrast, non-conserved miRNAs targeted diverse genes which provide more complex regulatory networks. In addition, 5 AUXIN RESPONSE FACTORs (ARFs) cleaved by the TAS3 derived ta-siRNAs were also detected. A total of 40 sRNA targets were further validated via RNA ligasemediated 5’ rapid amplification of cDNA ends (RLM 5’-RACE). Our degradome results present a detailed sRNA-target interaction atlas, which provides a guide for the study of the roles of sRNAs and their targets in rice.

Degradome sequencing reveals endogenous small RNA targets in rice ( Oryza sativa L. ssp. indica )

MicroRNAs and small interfering RNAs (siRNAs) are two classes of small regulatory RNAs derived from different types of precursors and processed by distinct Dicer or Dicer-like (DCL) proteins. During evolution, four Arabidopsis thaliana DCLs and six rice (Oryza sativa) DCLs (Os DCLs) appear to have acquired specialized functions. The Arabidopsis DCLs are well characterized, but those in rice remain largely unstudied. Here, we show that both knockdown and loss of function of rice DCL4, the homolog of Arabidopsis DCL4, lead to vegetative growth abnormalities and severe developmental defects in spikelet identity. These phenotypic alterations appear to be distinct from those observed in Arabidopsis dcl4 mutants, which exhibit accelerated vegetative phase change. The difference in phenotype between rice and Arabidopsis dcl4 mutants suggests that siRNA processing by DCL4 has a broader role in rice development than in Arabidopsis. Biochemical and genetic analyses indicate that Os DCL4 is the major Dicer responsible for the 21-nucleotide siRNAs associated with inverted repeat transgenes and for trans-acting siRNA (ta-siRNA) from the endogenous TRANS-ACTING siRNA3 (TAS3) gene. We show that the biogenesis mechanism of TAS3 ta-siRNA is conserved but that putative direct targets of Os DCL4 appear to be differentially regulated between monocots and dicots. Our results reveal a critical role of Os DCL4-mediated ta-siRNA biogenesis in rice development.

http://plantbiol.genetics.ac.cn/the_xue_lab/publication/dicer.pdf

Oryza sativa Dicer-like4 Reveals a Key Role for Small Interfering RNA Silencing in Plant Development

Higher plants have evolved multiple RNA-dependent RNA polymerases (RDRs), which work with Dicer-like (DCL) proteins to produce different classes of small RNAs with specialized molecular functions. Here we report that OsRDR6, the rice (Oryza sativa L.) homolog of Arabidopsis RDR6, acts in the biogenesis of various types and sizes of small RNAs. We isolated a rice osrdr6-1 mutant, which was temperature sensitive and showed spikelet defects. This mutant displays reduced accumulation of tasiR-ARFs, the conserved trans-acting siRNAs (tasiRNAs) derived from the TAS3 locus, and ectopic expression of tasiR-ARF target genes, the Auxin Response Factors (including ARF2 and ARF3/ETTIN). The loss of tasiR-mediated repression of ARFs in osrdr6-1 can explain its morphological defects, as expression of two non-targeted ARF3 gene constructs (ARF3muts) in a wild-type background mimics the osrdr6 and osdcl4-1 mutant phenotypes. Small RNA high-throughput sequencing also reveals that besides tasiRNAs, 21-nucleotide (nt) phased small RNAs are also largely dependent on OsRDR6. Unexpectedly, we found that osrdr6-1 has a strong impact on the accumulation of 24-nt phased small RNAs, but not on unphased ones. Our work uncovers the key roles of OsRDR6 in small RNA biogenesis and directly illustrates the crucial functions of tasiR-ARFs in rice development.

https://onlinelibrary.wiley.com/doi/pdf/10.1111/j.1365-313X.2012.05001.x

Rice RNA-dependent RNA polymerase 6 acts in small RNA biogenesis and spikelet development.

The invention relates to regulation and control of small molecular ribonucleic acid (RNA) on an auxin response factor (ARF) gene. By the regulation and control, the morphological development of plant leaves and floral organs is changed to produce novel flower varieties. An auxin response factor mutation (ARFm) gene obtained by mutating Identification loci of the small molecular RNA is constructed on an expression vector to transform target plants, so that transgenic plants which have curled leaf shapes and large stretching amplitude of the floral organs and are more ornamental are obtained.

Zhiyu Chen

Papers

Degradome sequencing reveals endogenous small RNA targets in rice ( Oryza sativa L. ssp. indica )

Oryza sativa Dicer-like4 Reveals a Key Role for Small Interfering RNA Silencing in Plant Development

Rice RNA-dependent RNA polymerase 6 acts in small RNA biogenesis and spikelet development.

Regulation and control of small molecular ribonucleic acid (RNA) on auxin response factor (ARF) gene and application thereof in production