3’ uridylation is increasingly recognized as a conserved RNA modification process associated with RNA turnover in eukaryotes. 2’-O-methylation on the 3’ terminal ribose protects micro(mi)RNAs from 3’ truncation and 3’ uridylation in Arabidopsis. Previously, we identified HESO1 as the nucleotidyl transferase that uridylates most unmethylated miRNAs in vivo, but substantial 3’ tailing of miRNAs still remains in heso1 loss-of-function mutants. In this study, we found that among nine other potential nucleotidyl transferases, UTP:RNA URIDYLYLTRANSFERASE 1 (URT1) is the single most predominant nucleotidyl transferase that tails miRNAs. URT1 and HESO1 prefer substrates with different 3’ end nucleotides in vitro and act cooperatively to tail different forms of the same miRNAs in vivo. Moreover, both HESO1 and URT1 exhibit nucleotidyl transferase activity on AGO1-bound miRNAs. Although these enzymes are able to add long tails to AGO1-bound miRNAs, the tailed miRNAs remain associated with AGO1. Moreover, tailing of AGO1-bound miRNA165/6 drastically reduces the slicing activity of AGO1-miR165/6, suggesting that tailing reduces miRNA activity. However, monouridylation of miR171a by URT1 endows the miRNA the ability to trigger the biogenesis of secondary siRNAs. Therefore, 3’ tailing could affect the activities of miRNAs in addition to leading to miRNA degradation. The tailing of RNAs with non-templated uridines, known as uridylation, is often associated with RNA degradation. We previously identified HESO1 as a nucleotidyl transferase that uridylates microRNAs (miRNAs) to lead to their degradation in Arabidopsis. But HESO1 cannot account for all the miRNA uridylation activity in vivo. Here, we have uncovered UTP:RNA URIDYLYLTRANSFERASE 1 (URT1) as another nucleotidyl transferase that uridylates miRNAs. HESO1 and URT1 have different substrate preferences and act cooperatively to tail miRNAs. We show that both enzymes are able to act on ARGONAUTE1 (AGO1)-bound miRNAs and that the tailed miRNAs stay bound by AGO1. We show that URT1-mediated tailing affects the activities of miR165/6 and miR171a differently. This study reveals intricate miRNA uridylation processes as well as functional outcomes of miRNA uridylation.

As key regulators of gene expression in eukaryotes, small RNAs have been characterized in many seed plants, and pathways for their biogenesis, degradation, and action have been defined in model angiosperms. However, both small RNAs themselves and small RNA pathways are not well characterized in other land plants such as lycophytes and ferns, preventing a comprehensive evolutionary perspective on small RNAs in land plants. Using 25 representatives from major lineages of lycophytes and ferns, most of which lack sequenced genomes, we characterized small RNAs and small RNA pathways in these plants. We identified homologs of DICER-LIKE (DCL), ARGONAUTE (AGO), and other genes involved in small RNA pathways, predicted over 2600 conserved microRNA (miRNA) candidates, and performed phylogenetic analyses on small RNA pathways as well as miRNAs. Pathways underlying miRNA biogenesis, degradation, and activity were established in the common ancestor of land plants, but the 24-nucleotide siRNA pathway that guides DNA methylation is incomplete in sister species of seed plants, especially lycophytes. We show that the functional diversification of key gene families such as DCL and AGO as observed in angiosperms occurred early in land plants followed by parallel expansion of the AGO family in ferns and angiosperms. We uncovered a conserved AGO subfamily absent in angiosperms. Our phylogenetic analyses of miRNAs in bryophytes, lycophytes, ferns, and angiosperms refine the time-of-origin for conserved miRNA families as well as small RNA machinery in land plants.

Plants evolved an array of disease resistance genes (R genes) to fight pathogens. In the absence of pathogen infection, NBS-LRR genes, which comprise a major subfamily of R genes, are suppressed by a small RNA cascade involving microRNAs (miRNAs) that trigger the biogenesis of phased siRNAs (phasiRNAs) from R gene transcripts. However, whether or how R genes influence small RNA biogenesis is unknown. In this study, we isolate a mutant with global defects in the biogenesis of miRNAs and phasiRNAs in Arabidopsis thaliana and trace the defects to the over accumulation and nuclear localization of an R protein SNC1. We show that nuclear SNC1 represses the transcription of miRNA and phasiRNA loci, probably through the transcriptional corepressor TPR1. Intriguingly, nuclear SNC1 reduces the accumulation of phasiRNAs from three source R genes and concomitantly, the expression of a majority of the ~170R genes is up-regulated. Taken together, this study suggests an R gene-miRNA-phasiRNA regulatory module that amplifies plant immune responses.

MicroRNAs (miRNAs) are 20–24 nucleotide RNAs that regulate a variety of developmental and metabolic processes. The accumulation of miRNAs in vivo can be controlled at multiple levels. In addition to miRNA biogenesis, mechanisms that lead to RNA degradation, such as 3′ uridylation and 3′ truncation, also affect the steady-state levels of miRNAs. On the other hand, 2’-O-methylation in plant miRNAs protects their 3′ ends from truncation and uridylation. The recent identification of HESO1 as the key enzyme responsible for miRNA uridylation in Arabidopsis was a first step toward a full understanding of the mechanisms underlying miRNA turnover. Analyses of the heso1 mutant predicted the existence of another uridylation activity and a previously unknown nuclease that act on miRNAs. The future identification of these enzymes will enrich our understanding of miRNA turnover.

Because of climate change, crops will experience increasing heat stress. However, the ways in which heat stress affects crop growth and yield at the molecular level remain poorly understood. We generated spatiotemporal mRNA and small RNA transcriptome data, spanning seven tissues at three time points, to investigate the effects of heat stress on vegetative and reproductive development in maize (Zea mays). Among the small RNAs significantly induced by heat stress was a plastid-derived 19-nucleotide small RNA, which is possibly the residual footprint of a pentatricopeptide repeat protein. This suggests that heat stress induces the turnover of certain plastid transcripts. Consistently, genes responsible for photosynthesis in chloroplasts were repressed after heat stress. Analysis also revealed that the abundance of 24-nucletide small interfering RNAs from transposable elements was conspicuously reduced by heat stress in tassels and roots; nearby genes showed a similar expression trend. Finally, specific microRNA and passenger microRNA species were identified, which in other plant species have not before been reported as responsive to heat stress. This study generated an atlas of genome-wide transcriptomic responses to heat stress, revealing several key regulators as potential targets for thermotolerance improvement in maize.