Arabidopsis lyrata Small RNAs: Transient MIRNA and Small Interfering RNA Loci within the Arabidopsis Genus

Twenty-one-nucleotide microRNAs (miRNAs) and 24-nucleotide Pol IV-dependent small interfering RNAs (p4-siRNAs) are the most abundant types of small RNAs in angiosperms. Some miRNAs are well conserved among different plant lineages, whereas others are less conserved, and it is not clear whether less-conserved miRNAs have the same functionality as the well conserved ones. p4-siRNAs are broadly produced in the Arabidopsis genome, sometimes from active hot spot loci, but it is unknown whether individual p4-siRNA hot spots are retained as hot spots between plant species. In this study, we compare small RNAs in two closely related species (Arabidopsis thaliana and Arabidopsis lyrata) and find that less-conserved miRNAs have high rates of divergence in MIRNA hairpin structures, mature miRNA sequences, and target-complementary sites in the other species. The fidelity of miRNA biogenesis from many less-conserved MIRNA hairpins frequently deteriorates in the sister species relative to the species of first discovery. We also observe that p4-siRNA occupied loci have a slight tendency to be retained as p4-siRNA loci between species, but the most active A. lyrata p4-siRNA hot spots are generally not syntenic to the most active p4-siRNA hot spots of A. thaliana. Altogether, our findings indicate that many MIRNAs and most p4-siRNA hot spots are rapidly changing and evolutionarily transient within the Arabidopsis genus.     

Sliced microRNA targets and precise loop-first processing of MIR319 hairpins revealed by analysis of the Physcomitrella patens degradome

Expression profiling of the 5′ ends of uncapped mRNAs (“degradome” sequencing) can be used to empirically catalog microRNA (miRNA) targets, to probe patterns of miRNA hairpin processing, to examine mRNA decay, and to analyze accumulation of endogenous short interfering RNA (siRNA) precursors. We sequenced and analyzed the degradome of the moss Physcomitrella patens, an important model system for functional genomic analyses in plant evolution. A total of 52 target mRNAs of 27 different Physcomitrella miRNA families were identified. Many targets of both more conserved and less conserved miRNA families encoded putative regulatory proteins. Remnants of MIRNA hairpin processing also populated the degradome data and indicated an unusual “loop-first” mode of precise processing for the MIR319 gene family. Precise loop-first processing was confirmed for native Physcomitrella, rice, and Arabidopsis MIR319 hairpins, as well as an Arabidopsis artificial MIRNA (aMIRNA) based upon a MIR319 backbone. MIR319 is thus a conserved exception to the general rule of loop-last processing of MIRNA hairpins. Loop-first MIR319 processing may contribute to the high efficacy of a widely used MIR319-based strategy for aMIRNA production in plants.     

Redundant and specific roles of individual MIR172 genes in plant development

Evolutionarily conserved microRNAs (miRNAs) usually have high copy numbers in the genome. The redundant and specific roles of each member of a multimember miRNA gene family are poorly understood. Previous studies have shown that the miR156-SPL-miR172 axis constitutes a signaling cascade in regulating plant developmental transitions. Here, we report the feasibility and utility of CRISPR-Cas9 technology to investigate the functions of all 5 MIR172 family members in Arabidopsis. We show that an Arabidopsis plant devoid of miR172 is viable, although it displays pleiotropic morphological defects. MIR172 family members exhibit distinct expression pattern and exert functional specificity in regulating meristem size, trichome initiation, stem elongation, shoot branching, and floral competence. In particular, we find that the miR156-SPL-miR172 cascade is bifurcated into specific flowering responses by matching pairs of coexpressed SPL and MIR172 genes in different tissues. Our results thus highlight the spatiotemporal changes in gene expression that underlie evolutionary novelties of a miRNA gene family in nature. The expansion of MIR172 genes in the Arabidopsis genome provides molecular substrates for the integration of diverse floral inductive cues, which ensures that plants flower at the optimal time to maximize seed yields.

This study uses CRISPR-Cas9 technology to investigate the functions of all five miR172 genes in Arabidopsis, finding that miRNA172 family members exhibit distinct expression pattern and exert functional specificity in regulating meristem size, trichome initiation, stem elongation, shoot branching and floral competence.     

Identification of Nitrogen Starvation-Responsive MicroRNAs in Arabidopsis thaliana

microRNAs (miRNAs) are a class of negative regulators that take part in many processes such as growth and development, stress responses, and metabolism in plants. Recently, miRNAs were shown to function in plant nutrient metabolism. Moreover, several miRNAs were identified in the response to nitrogen (N) deficiency. To investigate the functions of other miRNAs in N deficiency, deep sequencing technology was used to detect the expression of small RNAs under N-sufficient and -deficient conditions. The results showed that members from the same miRNA families displayed differential expression in response to N deficiency. Upon N starvation, the expression of miR169, miR171, miR395, miR397, miR398, miR399, miR408, miR827, and miR857 was repressed, whereas those of miR160, miR780, miR826, miR842, and miR846 were induced. miR826, a newly identified N-starvation-induced miRNA, was found to target the AOP2 gene. Among these N-starvation-responsive miRNAs, several were involved in cross-talk among responses to different nutrient (N, P, S, Cu) deficiencies. miR160, miR167, and miR171 could be responsible for the development of Arabidopsis root systems under N-starvation conditions. In addition, twenty novel miRNAs were identified and nine of them were significantly responsive to N-starvation. This study represents comprehensive expression profiling of N-starvation-responsive miRNAs and advances our understanding of the regulation of N homeostasis mediated by miRNAs.     

Recovery of dicer-like 1-late flowering phenotype by miR172 expressed by the noncanonical DCL4-dependent biogenesis pathway

MicroRNAs (miRNAs) act as down-regulators of gene expression, and play a dominant role in eukaryote development. In Arabidopsis thaliana, DICER-LIKE 1 (DCL1) is the main processor in miRNA biogenesis, and dcl1 mutants show various developmental defects at the early stage of embryogenesis or at gamete formation. However, miRNAs responsible for the respective developmental stages of the dcl1 defects have not been identified. Here, we developed a DCL1-independent miRNA expression system using the unique DCL4-dependent miRNA, miR839. By replacing the mature sequence in the miR839 precursor sequence with that of miR172, one of the most widely conserved miRNAs in angiosperms, we succeeded in expressing miR172 from a chimeric miR839 precursor in dcl1-7 plants and observed the repression of miR172 target gene expression. In parallel, the DCL4-dependent miR172 expression rescued the late flowering phenotype of dcl1-7 by acceleration of flowering. We established the DCL1-independent miRNA expression system, and revealed that the reduction of miR172 expression is responsible for the dcl1-7 late flowering phenotype.     

Expansion of MIR482/2118 by a class‐II transposable element in cotton

Some plant microRNA (miRNA) families contain multiple members generating identical or highly similar mature miRNA variants. Mechanisms underlying the expansion of miRNA families remain elusive, although tandem and/or segmental duplications have been proposed. In this study of two tetraploid cottons, Gossypium hirsutum and Gossypium barbadense, and their extant diploid progenitors, Gossypium arboreum and Gossypium raimondii, we investigated the gain and loss of members of the miR482/2118 superfamily, which modulates the expression of nucleotide‐binding site leucine‐rich repeat (NBS‐LRR) disease resistance genes. We found significant expansion of MIR482/2118d in G. barbadense, G. hirsutum and G. raimondii, but not in G. arboreum. Several newly expanded MIR482/2118d loci have mutated to produce different miR482/2118 variants with altered target‐gene specificity. Based on detailed analysis of sequences flanking these MIR482/2118 loci, we found that this expansion of MIR482/2118d and its derivatives resulted from an initial capture of an MIR482/2118d by a class‐II DNA transposable element (TE) in G. raimondii prior to the tetraploidization event, followed by transposition to new genomic locations in G. barbadense, G. hirsutum and G. raimondii. The ‘GosTE’ involved in the capture and proliferation of MIR482/2118d and its derivatives belongs to the PIF/Harbinger superfamily, generating a 3‐bp target site duplication upon insertion at new locations. All orthologous MIR482/2118 loci in the two diploids were retained in the two tetraploids, but mutation(s) in miR482/2118 were observed across all four species as well as in different cultivars of both G. barbadense and G. hirsutum, suggesting a dynamic co‐evolution of miR482/2118 and its NBS‐LRR targets. Our results provide fresh insights into the mechanisms contributing to MIRNA proliferation and enrich our knowledge on TEs.     

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.