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Small RNAs regulate key physiological functions in land plants. Small RNAs can be divided into two categories: microRNAs (miRNAs) and short interfering RNAs (siRNAs); siRNAs are further subdivided into transposon/repetitive region-localized heterochromatic siRNAs and phased siRNAs (phasiRNAs). PhasiRNAs are produced from the miRNA-mediated cleavage of a Pol II RNA transcript; the miRNA cleavage site provides a defined starting point from which phasiRNAs are produced in a distinctly phased pattern. 21-22 nucleotide (nt)-dominated phasiRNA-producing loci (PHAS) are well represented in all land plants to date. In contrast, 24 nt-dominated PHAS loci are known to be encoded only in monocots and are generally restricted to male reproductive tissues. Currently, only one miRNA (miR2275) is known to trigger the production of these 24 nt-dominated PHAS loci. In this study, we use stringent methodologies in order to examine whether or not 24 nt-dominated PHAS loci also exist in Arabidopsis thaliana. We find that highly expressed heterochromatic siRNAs were consistently misidentified as 24 nt-dominated PHAS loci using multiple PHAS-detecting algorithms. We also find that MIR2275 is not found in A. thaliana, and it seems to have been lost in the last common ancestor of Brassicales. Altogether, our research highlights the potential issues with widely used PHAS-detecting algorithms which may lead to false positives when trying to annotate new PHAS, especially 24 nt-dominated loci.
Pubmed ID: 31245701
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Central online repository for microRNA nomenclature, sequence data, annotation and target prediction.Collection of published miRNA sequences and annotation.
View all literature mentionsSoftware ultrafast memory efficient tool for aligning sequencing reads. Bowtie is short read aligner.
View all literature mentionsA software tool developed to process and analyze small RNA-seq data with respect to a reference genome, and output a comprehensive and informative annotation of all discovered small RNA genes. ShortStack discovers small RNA ''clusters'' de novo, based on user-set thresholds, and annotates clusters with respect to small RNA size, orientation, and repetitiveness. ShortStack also discovers and annotates MIRNA genes, and other Hairpin-associated small RNA genes. In addition, ShortStack includes a robust method to detect genes producing small RNAs in a phased manner. It outputs a descriptive table of all results, useful genome browser tracks, a table describing the results of the hairpin / MIRNA analysis for each cluster, and detailed text-based alignments of all MIRNAs and hairpin-associated clusters. It can also be run in ''count'' mode, to quantify a set of input loci with genomic coordinates determined a priori by the user. ShortStack is a perl program. Besides perl, ShortStack also requires samtools and the RNALfold and RNAeval programs from the Vienna RNA Package to execute. When used to control the alignment of small RNA data to a reference genome, ShortStack also requires bowtie and bowtie-build. Finally, for optimal results, ShortStack uses a file of inverted repeats produced by the EMBOSS application einverted.
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