mirEX 2.0 - an integrated environment for expression profiling of plant microRNAs.

BACKGROUND: MicroRNAs are the key post-transcriptional regulators of gene expression in development and stress responses. Thus, precisely quantifying the level of each particular microRNA is of utmost importance when studying the biology of any organism. DESCRIPTION: The mirEX 2.0 web portal ( http://www.combio.pl/mirex ) provides a comprehensive platform for the exploration of microRNA expression data based on quantitative Real Time PCR and NGS sequencing experiments, covering various developmental stages, from wild-type to mutant plants. The portal includes mature and pri-miRNA expression levels detected in three plant species (Arabidopsis thaliana, Hordeum vulgare and Pellia endiviifolia), and in A. thaliana miRNA biogenesis pathway mutants. In total, the database contains information about the expression of 461 miRNAs representing 268 families. The data can be explored through the use of advanced web tools, including (i) a graphical query builder system allowing a combination of any given species, developmental stages and tissues, (ii) a modular presentation of the results in the form of thematic windows, and (iii) a number of user-friendly utilities such as a community-building discussion system and extensive tutorial documentation (e.g., tooltips, exemplary videos and presentations). All data contained within the mirEX 2.0 database can be downloaded for use in further applications in a context-based way from the result windows or from a dedicated web page. CONCLUSIONS: The mirEX 2.0 portal provides the plant research community with easily accessible data and powerful tools for application in multi-conditioned analyses of miRNA expression from important plant species in different biological and developmental backgrounds.

Gene structures and processing of Arabidopsis thaliana HYL1-dependent pri-miRNAs.

Arabidopsis thaliana HYL1 is a nuclear double-stranded RNA-binding protein involved in the maturation of pri-miRNAs. A quantitative real-time PCR platform for parallel quantification of 176 pri-miRNAs was used to reveal strong accumulation of 57 miRNA precursors in the hyl1 mutant that completely lacks HYL1 protein. This approach enabled us for the first time to pinpoint particular members of MIRNA family genes that require HYL1 activity for efficient maturation of their precursors. Moreover, the accumulation of miRNA precursors in the hyl1 mutant gave us the opportunity to carry out 3' and 5' RACE experiments which revealed that some of these precursors are of unexpected length. The alignment of HYL1-dependent miRNA precursors to A. thaliana genomic sequences indicated the presence of introns in 12 out of 20 genes studied. Some of the characterized intron-containing pri-miRNAs undergo alternative splicing such as exon skipping or usage of alternative 5' splice sites suggesting that this process plays a role in the regulation of miRNA biogenesis. In the hyl1 mutant intron-containing pri-miRNAs accumulate alongside spliced pri-miRNAs suggesting the recruitment of HYL1 into the miRNA precursor maturation pathway before their splicing occurs.

[Plant micro RNA biogenesis]. / Biogeneza roslinnych mikro RNA.

Micro RNAs are 21-24 nt long RNA molecules involved in the regulation of gene expression on mRNA level. Mature miRNA molecules associated with the RNA-induced silencing complex (RISC) guide specifically mRNA cleavage or inhibit the translation. In plants selective mRNA degradation in miRNA-dependent pathway is a widespread mechanism that occurs more frequently than translation inhibition. miRNA precursors, known as pri-miRNA, are processed in several steps to produce mature miRNA molecules. There are several plant proteins involved in miRNA processing: DCL1, HYL1, SE, HEN1 and HASTY. The role of these proteins is discussed.

Non-Canonical Processing of Arabidopsis pri-miR319a/b/c Generates Additional microRNAs to Target One RAP2.12 mRNA Isoform.

Arabidopsis miR319a/b/c primary transcripts are unusual due to the presence of a long stem and loop structure containing functional miR319a/b/c molecules. In our experiments carried out using high throughput sequencing (HTS), we have shown that additional microRNAs (miRNAs), miR319a.2/b.2/c.2 are generated from the upper part of the same hairpin structure. We have also found cognate miRNAa.2*/b.2*/c.2* to be present in the HTS results with a considerably lower number of reads. Northern hybridization revealed that miR319b.2 is mainly expressed in 35-day-old plant rosette leaves, as well as in stem and inflorescences of 42- and 53-day-old plants. Moreover, it carries multiple signatures of a functional miRNA, including as follows: (i) its biogenesis is HYL1-dependent; (ii) it is incorporated in a substantial amount into RISC complexes containing AGO1, AGO2, or AGO4 protein; (iii) 24 nt-long species of miR319b.2 have been found in inflorescences to be more abundant than 21 nt miR319b.2 species; (iv) it is present in various ratios to miR319b during plant development, which suggests the existence of a regulatory mechanism responsible for its biogenesis/processing; (v) there is an observed cross-species conservation of the miR319a/b/c stem nucleotide sequence extending beyond mature miRNA region; and (vi) all evidence suggests that intron-containing RAP2.12 mRNA isoform is the target for miR319b.2. All these features prompt us to claim miR319b.2 as a functional miRNA molecule.

The Role of the P1BS Element Containing Promoter-Driven Genes in Pi Transport and Homeostasis in Plants.

Inorganic phosphate (Pi) is an easily accessible form of phosphorus for plants. Plant Pi uptake is usually limited however by slow Pi diffusion through the soil which strongly adsorps phosphate species. Plants have developed mechanisms to increase Pi availability. There are also abiotic (phosphate level) and biotic (e.g., mycorrhizal) factors regulating the expression of Pi-responsive genes. Transcription factors binding to the promoters of Pi-responsive genes activate different pathways of Pi transport, distribution, and homeostasis maintenance. Pi metabolism involves not only functional proteins but also microRNAs and other non-coding RNAs.