- Eukaryotic small RNA (~20-30 nucleotides) are diverse regulatory molecules that repress gene expression at the transcriptional and post-transcriptional levels, defend hosts against invading viruses and defend genomes against selfish DNA elements. Small RNA populations are studied by high-throughput sequencing of the total small RNA fraction isolated from cells, however, the sequencing depth achieved by next-generation platforms makes genome mapping and analysis computationally intensive with standard methods. Here, methods to generate, parse, map, quantify, standardize and analyze large small RNA data sets are presented. This work demonstrates that small RNA profiling is quantitative and reproducible and that statistical methods can be adapted to facilitate objective comparisons between small RNA and small RNA populations.
Plants RNA silencing systems, including microRNA (miRNA), are important components of complex regulatory networks. Several plant MIRNA gene families and their target gene families are ancient, but over two-thirds of Arabidopsis MIRNA families are species-specific or restricted to the Brassicaceae lineage. In this work, the repertoires of MIRNA in the closely related species A. thaliana, A. lyrata and Capsella rubella were studied. Despite the relatively recent speciation of A. thaliana and A. lyrata ~10 million years ago, at least 13% of the MIRNA from each is species-specific. Additionally, 24–46 Arabidopsis MIRNA families arose after the Arabidopsis–Capsella split ~20 million years ago, supporting a net birth-death rate of 1.2–2.3 MIRNA per million years. These data, and data from other species, suggest that MIRNA are born and lost frequently throughout the evolution of plants. Further, evidence for the recent origin of 32 MIRNA families by duplication events, mostly of protein-coding loci, was demonstrated, but only ~50% of these loci are predicted miRNA targets. Despite the link between MIRNA formation and potential target loci, only 25 young A. thaliana miRNA have verified targets. As a group, young miRNA tend to be expressed weakly, processed imprecisely and lack biologically relevant targets. Additionally, variation between young Arabidopsis miRNA was significantly higher than for ancient miRNA, suggesting that most of the young MIRNA are more likely evolving neutrally. Together, the data presented argue that most young MIRNA are evolutionarily transient.