Long-Range Genomic Enrichment,Sequencing, and Assembly to Determine Unknown Sequences Flanking a Known microRNA

Conserved plant microRNAs (miRNAs) modulate important biological processes but little is known about conserved cis-regulatory elements (CREs) surrounding MIRNA genes. We developed a solution-based targeted genomic enrichment methodology to capture, enrich, and sequence flanking genomic regions surrounding conserved MIRNA genes with a locked-nucleic acid (LNA)-modified, biotinylated probe complementary to the mature miRNA sequence. Genomic DNA bound by the probe is captured by streptavidin-coated magnetic beads, amplified, sequenced and assembled de novo to obtain genomic DNA sequences flanking MIRNA locus of interest. We demonstrate the sensitivity and specificity of this enrichment methodology in Arabidopsis thaliana to enrich targeted regions spanning 10–20 kb surrounding known MIR166 and MIR165 loci. Assembly of the sequencing reads successfully recovered all targeted loci. While further optimization for larger, more complex genomes is needed, this method may enable determination of flanking genomic DNA sequence surrounding a known core (like a conserved mature miRNA) from multiple species that currently don''t have a full genome assembly available.     

An expression atlas of miRNAs in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

MicroRNAs (miRNAs) are small non-coding RNAs that regulate a variety of biological processes. MiRNA expression often exhibits spatial and temporal specificity. However, genome-wide miRNA expression patterns in different organs during development of Arabidopsis thaliana have not yet been systemically investigated. In this study, we sequenced small RNA libraries generated from 27 different organ/tissue types, which cover the entire life cycle of Arabidopsis. Analysis of the sequencing data revealed that most miRNAs are ubiquitously expressed, whereas a small set of miRNAs display highly specific expression patterns. In addition, different miRNA members within the same family have distinct spatial and temporal expression patterns. Moreover, we found that some miRNAs are produced from different arms of their hairpin precursors at different developmental stages. This work provides new insights into the regulation of miRNA biogenesis and a rich resource for future investigation of miRNA functions in Arabidopsis.     

An atypical epigenetic mechanism affects uniparental expression of Pol IV-dependent siRNAs

Background

Small RNAs generated by RNA polymerase IV (Pol IV) are the most abundant class of small RNAs in flowering plants. In Arabidopsis thaliana Pol IV-dependent short interfering (p4-si)RNAs are imprinted and accumulate specifically from maternal chromosomes in the developing seeds. Imprinted expression of protein-coding genes is controlled by differential DNA or histone methylation placed in gametes. To identify epigenetic factors required for maternal-specific expression of p4-siRNAs we analyzed the effect of a series of candidate mutations, including those required for genomic imprinting of protein-coding genes, on uniparental expression of a representative p4-siRNA locus.

Results

Paternal alleles of imprinted genes are marked by DNA or histone methylation placed by DNA METHYLTRANSFERASE 1 or the Polycomb Repressive Complex 2. Here we demonstrate that repression of paternal p4-siRNA expression at locus 08002 is not controlled by either of these mechanisms. Similarly, loss of several chromatin modification enzymes, including a histone acetyltransferase, a histone methyltransferase, and two nucleosome remodeling proteins, does not affect maternal expression of locus 08002. Maternal alleles of imprinted genes are hypomethylated by DEMETER DNA glycosylase, yet expression of p4-siRNAs occurs irrespective of demethylation by DEMETER or related glycosylases.

Conclusions

Differential DNA methylation and other chromatin modifications associated with epigenetic silencing are not required for maternal-specific expression of p4-siRNAs at locus 08002. These data indicate that there is an as yet unknown epigenetic mechanism causing maternal-specific p4-siRNA expression that is distinct from the well-characterized mechanisms associated with DNA methylation or the Polycomb Repressive Complex 2.     

Genome-Wide Identification of Reverse Complementary microRNA Genes in Plants

MicroRNAs (miRNAs) are ∼21-nucleotide small RNAs (sRNAs) with essential regulatory roles in plants. They are generated from stem-loop-structured precursors through two sequential Dicer-like 1 (DCL1)-mediated cleavages. To date, hundreds of plant miRNAs have been uncovered. However, the question, whether the sequences reverse complementary (RC) to the miRNA precursors could form hairpin-like structures and produce sRNA duplexes similar to the miRNA/miRNA* pairs has not been solved yet. Here, we interrogated this possibility in 16 plant species based on sRNA high-throughput sequencing data and secondary structure prediction. A total of 59 RC sequences with great potential to form stem-loop structures and generate miRNA/miRNA*-like duplexes were identified in ten plants, which were named as RC-miRNA precursors. Unlike the canonical miRNAs, only a few cleavage targets of the RC-miRNAs were identified in Arabidopsis (Arabidopsis thaliana) and rice (Oryza sativa), and none in Soybean (Glycine max) based on degradome data. Surprisingly, the genomic regions surrounding some of the RC-miRNA target recognition sites were observed to be specifically methylated in both Arabidopsis and rice. Taken together, we reported a new class of miRNAs, called RC-miRNAs, which were generated from the antisense strands of the miRNA precursors. Based on the results, we speculated that the mature RC-miRNAs might have subtle regulatory activity through target cleavages, but might possess short interfering RNA-like activity by guiding sequence-specific DNA methylation.     

Genetic framework for flowering-time regulation by ambient temperature-responsive miRNAs in Arabidopsis

Flowering is the primary trait affected by ambient temperature changes. Plant microRNAs (miRNAs) are small non-coding RNAs playing an important regulatory role in plant development. In this study, to elucidate the mechanism of flowering-time regulation by small RNAs, we identified six ambient temperature-responsive miRNAs (miR156, miR163, miR169, miR172, miR398 and miR399) in Arabidopsis via miRNA microarray and northern hybridization analyses. We also determined the expression profile of 120 unique miRNA loci in response to ambient temperature changes by miRNA northern hybridization analysis. The expression of the ambient temperature-responsive miRNAs and their target genes was largely anticorrelated at two different temperatures (16 and 23°C). Interestingly, a lesion in short vegetative phase (SVP), a key regulator within the thermosensory pathway, caused alteration in the expression of miR172 and a subset of its target genes, providing a link between a thermosensory pathway gene and miR172. The miR172-overexpressing plants showed a temperature-independent early flowering phenotype, suggesting that modulation of miR172 expression leads to temperature insensitivity. Taken together, our results suggest a genetic framework for flowering-time regulation by ambient temperature-responsive miRNAs under non-stress temperature conditions.     

The evolution of plant microRNAs: insights from a basal eudicot sacred lotus

microRNAs (miRNAs) are important noncoding small RNAs that regulate mRNAs in eukaryotes. However, under which circumstances different miRNAs/miRNA families exhibit different evolutionary trajectories in plants remains unclear. In this study, we sequenced the small RNAs and degradome from a basal eudicot, sacred lotus (Nelumbo nucifera or lotus), to identify miRNAs and their targets. Combining with public miRNAs, we predicted 57 pre‐eudicot miRNA families from different evolutionary stages. We found that miRNA families featuring older age, higher copy and target number tend to show lower propensity for miRNA family loss (PGL) and stronger signature of purifying selection during divergence of temperate and tropical lotus. Further analyses of lotus genome revealed that there is an association between loss of miRNA families in descendent plants and in duplicated genomes. Gene dosage balance is crucial in maintaining those preferentially retained MIRNA duplicates by imposing stronger purifying selection. However, these factors and selection influencing miRNA family evolution are not applicable to the putative MIRNA‐likes. Additionally, the MIRNAs participating in lotus pollen–pistil interaction, a conserved process in angiosperms, also have a strong signature of purifying selection. Functionally, sequence divergence in MIRNAs escalates expression divergence of their target genes between temperate and tropical lotus during rhizome and leaf growth. Overall, our study unravels several important factors and selection that determine the miRNA family distribution in plants and duplicated genomes, and provides evidence for functional impact of MIRNA sequence evolution.     

Double-stranded RNA binding proteins DRB2 and DRB4 have an antagonistic impact on polymerase IV-dependent siRNA levels in Arabidopsis

Biogenesis of the vast majority of plant siRNAs depends on the activity of the plant-specific RNA polymerase IV (PolIV) enzyme. As part of the RNA-dependent DNA methylation (RdDM) process, PolIV-dependent siRNAs (p4-siRNAs) are loaded onto an ARGONAUTE4-containing complex and guide de novo DNA methyltransferases to target loci. Here we show that the double-stranded RNA binding proteins DRB2 and DRB4 are required for proper accumulation of p4-siRNAs. In flowers, loss of DRB2 results in increased accumulation of p4-siRNAs but not ta-siRNAs, inverted repeat (IR)-derived siRNAs, or miRNA. Loss of DRB2 does not impair uniparental expression of p4-dependent siRNAs in developing endosperm, indicating that p4-siRNA increased accumulation is not the result of the activation of the polIV pathway in the male gametophyte. In contrast to drb2, drb4 mutants exhibit reduced p4-siRNA levels, but the extent of this reduction is variable, according to the nature and size of the p4-siRNAs. Loss of DRB4 also leads to a spectacular increase of p4-independent IR-derived 24-nt siRNAs, suggesting a reallocation of factors from p4-dependent to p4-independent siRNA pathways in drb4. Opposite effects of drb2 and drb4 mutations on the accumulation of p4-siRNAs were also observed in vegetative tissues. Moreover, transgenic plants overexpressing DRB2 mimicked drb4 mutants at the morphological and molecular levels, confirming the antagonistic roles of DRB2 and DRB4.     

siRNAs from miRNA sites mediate DNA methylation of target genes

Arabidopsis microRNA (miRNA) genes (MIR) give rise to 20- to 22-nt miRNAs that are generated predominantly by the type III endoribonuclease Dicer-like 1 (DCL1) but do not require any RNA-dependent RNA Polymerases (RDRs) or RNA Polymerase IV (Pol IV). Here, we identify a novel class of non-conserved MIR genes that give rise to two small RNA species, a 20- to 22-nt species and a 23- to 27-nt species, at the same site. Genetic analysis using small RNA pathway mutants reveals that the 20- to 22-nt small RNAs are typical miRNAs generated by DCL1 and are associated with Argonaute 1 (AGO1). In contrast, the accumulation of the 23- to 27-nt small RNAs from the miRNA-generating sites is dependent on DCL3, RDR2 and Pol IV, components of the typical heterochromatic small interfering RNA (hc-siRNA) pathway. We further demonstrate that these MIR-derived siRNAs associate with AGO4 and direct DNA methylation at some of their target loci in trans. In addition, we find that at the miRNA-generating sites, some conserved canonical MIR genes also produce siRNAs, which also induce DNA methylation at some of their target sites. Our systematic examination of published small RNA deep sequencing datasets of rice and moss suggests that this type of dual functional MIRs exist broadly in plants.