Innate, translation-dependent silencing of an invasive transposon in Arabidopsis.

Co-evolution between hosts' and parasites' genomes shapes diverse pathways of acquired immunity based on silencing small (s)RNAs. In plants, sRNAs cause heterochromatinization, sequence degeneration, and, ultimately, loss of autonomy of most transposable elements (TEs). Recognition of newly invasive plant TEs, by contrast, involves an innate antiviral-like silencing response. To investigate this response's activation, we studied the single-copy element EVADÉ (EVD), one of few representatives of the large Ty1/Copia family able to proliferate in Arabidopsis when epigenetically reactivated. In Ty1/Copia elements, a short subgenomic mRNA (shGAG) provides the necessary excess of structural GAG protein over the catalytic components encoded by the full-length genomic flGAG-POL. We show here that the predominant cytosolic distribution of shGAG strongly favors its translation over mostly nuclear flGAG-POL. During this process, an unusually intense ribosomal stalling event coincides with mRNA breakage yielding unconventional 5'OH RNA fragments that evade RNA quality control. The starting point of sRNA production by RNA-DEPENDENT-RNA-POLYMERASE-6 (RDR6), exclusively on shGAG, occurs precisely at this breakage point. This hitherto-unrecognized "translation-dependent silencing" (TdS) is independent of codon usage or GC content and is not observed on TE remnants populating the Arabidopsis genome, consistent with their poor association, if any, with polysomes. We propose that TdS forms a primal defense against EVD de novo invasions that underlies its associated sRNA pattern.

MicroRNA Dynamics and Functions During Arabidopsis Embryogenesis.

MicroRNAs (miRNAs) are short noncoding RNAs that mediate the repression of target transcripts in plants and animals. Although miRNAs are required throughout plant development, relatively little is known regarding their embryonic functions. To systematically characterize embryonic miRNAs in Arabidopsis (Arabidopsis thaliana), we developed or applied high-throughput sequencing-based methods to profile hundreds of miRNAs and associated targets throughout embryogenesis. We discovered dozens of miRNAs that dynamically cleave and repress target transcripts, including 30 that encode transcription factors. Transcriptome analyses indicated that these miRNA:target interactions have profound effects on embryonic gene expression programs. Moreover, we demonstrated that the miRNA-mediated repression of six transcription factors are individually required for proper division patterns of various embryonic cell lineages. These data indicate that the miRNA-directed repression of multiple transcription factors is critically important for the establishment of the plant body plan, and they provide a foundation to further investigate how miRNAs contribute to these initial cellular differentiation events.

NanoPARE: parallel analysis of RNA 5' ends from low-input RNA.

Diverse RNA 5' ends are generated through both transcriptional and post-transcriptional processes. These important modes of gene regulation often vary across cell types and can contribute to the diversification of transcriptomes and thus cellular differentiation. Therefore, the identification of primary and processed 5' ends of RNAs is important for their functional characterization. Methods have been developed to profile either RNA 5' ends from primary transcripts or the products of RNA degradation genome-wide. However, these approaches either require high amounts of starting RNA or are performed in the absence of paired gene-body mRNA-seq data. This limits current efforts in RNA 5' end annotation to whole tissues and can prevent accurate RNA 5' end classification due to biases in the data sets. To enable the accurate identification and precise classification of RNA 5' ends from standard and low-input RNA, we developed a next-generation sequencing-based method called nanoPARE and associated software. By integrating RNA 5' end information from nanoPARE with gene-body mRNA-seq data from the same RNA sample, our method enables the identification of transcription start sites at single-nucleotide resolution from single-cell levels of total RNA, as well as small RNA-mediated cleavage events from at least 10,000-fold less total RNA compared to conventional approaches. NanoPARE can therefore be used to accurately profile transcription start sites, noncapped RNA 5' ends, and small RNA targeting events from individual tissue types. As a proof-of-principle, we utilized nanoPARE to improve Arabidopsis thaliana RNA 5' end annotations and quantify microRNA-mediated cleavage events across five different flower tissues.

Animal Hen1 2'-O-methyltransferases as tools for 3'-terminal functionalization and labelling of single-stranded RNAs.

S-adenosyl-L-methionine-dependent 2'-O-methylati-on of the 3'-terminal nucleotide plays important roles in biogenesis of eukaryotic small non-coding RNAs, such as siRNAs, miRNAs and Piwi-interacting RNAs (piRNAs). Here we demonstrate that, in contrast to Mg2+/Mn2+-dependent plant and bacterial homologues, the Drosophila DmHen1 and human HsHEN1 piRNA methyltransferases require cobalt cations for their enzymatic activity in vitro. We also show for the first time the capacity of the animal Hen1 to catalyse the transfer of a variety of extended chemical groups from synthetic analogues of the AdoMet cofactor onto a wide range (22-80 nt) of single-stranded RNAs permitting their 3'-terminal functionalization and labelling. Moreover, we provide evidence that deletion of a small C-terminal region of the DmHen1 protein further increases its modification efficiency and abolishes a modest 3'-terminal nucleotide bias observed for the full-length protein. Finally, we show that fluorophore-tagged ssRNA molecules are successfully detected in fluorescence resonance energy transfer assays both individually and in a total RNA mixture. The presented DmHen1-assisted RNA labelling provides a solid basis for developing novel chemo-enzymatic approaches for in vitro studies and in vivo monitoring of single-stranded RNA pools.

Functional mapping of the plant small RNA methyltransferase: HEN1 physically interacts with HYL1 and DICER-LIKE 1 proteins.

Methylation of 3'-terminal nucleotides of miRNA/miRNA* is part of miRNAs biogenesis in plants but is not found in animals. In Arabidopsis thaliana this reaction is carried out by a multidomain AdoMet-dependent 2'-O-methyltransferase HEN1. Using deletion and structure-guided mutational analysis, we show that the double-stranded RNA-binding domains R(1) and R(2) of HEN1 make significant but uneven contributions to substrate RNA binding, and map residues in each domain responsible for this function. Using GST pull-down assays and yeast two-hybrid analysis we demonstrate direct HEN1 interactions, mediated by its FK506-binding protein-like domain and R(2) domain, with the microRNA biogenesis protein HYL1. Furthermore, we find that HEN1 forms a complex with DICER-LIKE 1 (DCL1) ribonuclease, another key protein involved in miRNA biogenesis machinery. In contrast, no direct interaction is detectable between HEN1 and SERRATE. On the basis of these findings, we propose a mechanism of plant miRNA maturation which involves binding of the HEN1 methyltransferase to the DCL1•HYL1•miRNA complex excluding the SERRATE protein.

Oligonucleotide-Addressed Covalent 3'-Terminal Derivatization of Small RNA Strands for Enrichment and Visualization.

The HEN1 RNA 2'-O-methyltransferase plays important roles in the biogenesis of small non-coding RNAs in plants and proved a valuable tool for selective transfer of functional groups from cofactor analogues onto miRNA and siRNA duplexes in vitro. Herein, we demonstrate the versatile HEN1-mediated methylation and alkylation of small RNA strands in heteroduplexes with a range of complementary synthetic DNA oligonucleotides carrying user-defined moieties such as internal or 3'-terminal extensions or chemical reporter groups. The observed DNA-guided covalent functionalization of RNA broadens our understanding of the substrate specificity of HEN1 and paves the way for the development of novel chemo-enzymatic tools with potential applications in miRNomics, synthetic biology, and nanomedicine.

Selective covalent labeling of miRNA and siRNA duplexes using HEN1 methyltransferase.

MicroRNAs regulate gene expression in numerous biological pathways and are typically methylated at their 3'-termini in plants but not in animals. Here we show that the HEN1 RNA 2'-O-methyltransferase from Arabidopsis thaliana catalyzes the transfer of extended propargylic moieties from synthetic AdoMet cofactor analogs to duplex miRNAs or siRNAs. The presented approach permits selective and efficient covalent labeling of small RNA duplexes with a variety of functional or reporter groups for their enrichment and analysis.

Mechanistic insights into small RNA recognition and modification by the HEN1 methyltransferase.

The HEN1 methyltransferase from Arabidopsis thaliana modifies the 3'-terminal nucleotides of small regulatory RNAs. Although it is one of the best characterized members of the 2'-O-methyltransferase family, many aspects of its interactions with the cofactor and substrate RNA remained unresolved. To better understand the substrate interactions and contributions of individual steps during HEN1 catalysis, we studied the binding and methylation kinetics of the enzyme using a series of unmethylated, hemimethylated and doubly methylated miRNA and siRNA substrates. The present study shows that HEN1 specifically binds double-stranded unmethylated or hemimethylated miR173/miR173* substrates with a subnanomolar affinity in a cofactor-dependent manner. Kinetic studies under single turnover and pre-steady state conditions in combination with isotope partitioning analysis showed that the binary HEN1-miRNA/miRNA* complex is catalytically competent; however, successive methylation of the two strands in a RNA duplex occurs in a non-processive (distributive) manner. We also find that the observed moderate methylation strand preference is largely exerted at the RNA-binding step and is fairly independent of the nature of the 3'-terminal nucleobase, but shows some dependency on proximal nucleotide mispairs. The results of the present study thus provide novel insights into the mechanism of RNA recognition and modification by a representative small RNA 2'-O-methyltransferase.

Kinetic and functional analysis of the small RNA methyltransferase HEN1: the catalytic domain is essential for preferential modification of duplex RNA.

The HEN1 RNA methyltransferase from Arabidopsis thaliana catalyzes S-adenosyl-L-methionine (AdoMet)-dependent 2'-O-methylation at the 3'-termini of small double-stranded RNAs and is a crucial factor in the biogenesis of plant small noncoding RNAs, such as miRNAs or siRNAs. We performed functional and kinetic studies of the full-length HEN1 methyltransferase and its truncated form comprising the C-terminal part of the protein (residues 666-942) with a variety of model RNA substrates. Kinetic parameters obtained with natural RNA substrates indicate that HEN1 is highly catalytically efficient in the absence of any supplementary proteins. We find that the enzyme modifies individual strands in succession leading to complete methylation of an RNA duplex. The rates of methyl group transfer to individual strands of hemimethylated substrates under single turnover conditions are comparable with the multiple turnover rate under steady-state conditions, suggesting that release of reaction products is not a rate-limiting event in the reaction cycle. The truncated protein, which includes conserved motifs characteristic for AdoMet binding, efficiently modifies double-stranded RNA substrates in vitro; however, in contrast to the full-length methyltransferase, it shows weaker interactions with both substrates and is sensitive to base mispairing in the first and second positions of the RNA duplex. Our findings suggest an important role for the N-terminal domains in stabilizing the catalytic complex and indicate that major structural determinants required for selective recognition and methylation of RNA duplexes reside in the C-terminal domain.