Keeping up with the miRNAs: current paradigms of the biogenesis pathway.

For many years we have studied the processes involved in producing miRNAs in plants and the numerous differences from their metazoan counterpart. A well-defined catalytic process, mostly carried out by the RNase III enzyme DICER-LIKE1 (DCL1), it was identified early after the discovery of RNAi and was followed by the isolation of a plethora of miRNA biogenesis cofactors. The production of miRNAs, which later are loaded in ARGONAUTE (AGO) proteins to perform their RNA silencing functions both within the cell and non-cell autonomously, appears to be a highly regulated and dynamic process. Many regulatory events during miRNA biogenesis require the action of specific proteins. However, in recent years, many post-transcriptional modifications, structural features, and coupling with other cellular processing emerged as critical elements controlling the production of miRNA and, thus, a plant's physiology. This review discusses new evidence that has changed the way we understand how miRNAs are produced in plants. We also provide an updated view of the miRNA biogenesis pathways, focusing on the gaps in our knowledge and the most compelling questions that remain open.

Dual function of HYPONASTIC LEAVES 1 during early skotomorphogenic growth in Arabidopsis.

Seeds germinating underground display a specific developmental programme, termed skotomorphogenesis, to ensure survival of the emerging seedlings until they reach the light. They rapidly elongate the hypocotyl and maintain the cotyledons closed, forming a hook with the hypocotyl in order to protect apical meristematic cells from mechanical damage. Such crucial events for the fate of the seedling are tightly regulated and although some transcriptional regulators and phytohormones are known to be implicated in this regulation, we are still far from a complete understanding of these biological processes. Our work provides information on the diverse roles in skotomorphogenesis of the core components of microRNA biogenesis in Arabidopsis, HYL1, DCL1, and SE. We show that hypocotyl elongation is promoted by all these components, probably through the action of specific miRNAs. Hook development also depends on these proteins however, remarkably, HYL1 exerts its role in an opposite way to DCL1 and SE. Interestingly, we found that a specific HYL1 domain involved in protein-protein interaction is required for this function. Genetic evidences also point to the phosphorylation status of HYL1 as important for this function. We propose that HYL1 help maintain the hook closed during early skotomorphogenesis in a microprocessor-independent manner by repressing the activity of HY5, the transcriptional master regulator that triggers light responses. This work uncovers a previously unnoticed link between components of the miRNA biogenesis machinery, the skotomorphogenic growth, and hook development in Arabidopsis.

Alternative use of miRNA-biogenesis co-factors in plants at low temperatures.

Plants use molecular mechanisms to sense temperatures, trigger quick adaptive responses and thereby cope with environmental changes. MicroRNAs (miRNAs) are key regulators of plant development under such conditions. The catalytic action of DICER LIKE 1 (DCL1), in conjunction with HYPONASTIC LEAVES 1 (HYL1) and SERRATE (SE), produces miRNAs from double-stranded RNAs. As plants lack a stable internal temperature to which enzymatic reactions could be optimized during evolution, reactions such as miRNA processing have to be adjusted to fluctuating environmental temperatures. Here, we report that with decreasing ambient temperature, the plant miRNA biogenesis machinery becomes more robust, producing miRNAs even in the absence of the key DCL1 co-factors HYL1 and SE. This reduces the morphological and reproductive defects of se and hyl1 mutants, restoring seed production. Using small RNA-sequencing and bioinformatics analyses, we have identified specific miRNAs that become HYL1/SE independent for their production in response to temperature decrease. We found that the secondary structure of primary miRNAs is key for this temperature recovery. This finding may have evolutionary implications as a potential adaptation-driving mechanism to a changing climate.

A Quick HYL1-Dependent Reactivation of MicroRNA Production Is Required for a Proper Developmental Response after Extended Periods of Light Deprivation.

Light is the most influential environmental stimulus for plant growth. In response to deficient light, plants reprogram their development to adjust their growth in search for a light source. A fine reprogramming of gene expression orchestrates this adaptive trait. Here we show that plants alter microRNA (miRNA) biogenesis in response to light transition. When plants suffer an unusual extended period of light deprivation, the miRNA biogenesis factor HYPONASTIC LEAVES 1 (HYL1) is degraded but an inactive pool of phosphorylated protein remains stable inside the nucleus. Degradation of HYL1 leads to the release of gene silencing, triggering a proper response to dark and shade. Upon light restoration, a quick dephosphorylation of HYL1 leads to the reactivation of miRNA biogenesis and a switch toward a developmental program that maximizes the light uptake. Our findings define a unique and fast regulatory mechanism controlling the plant silencing machinery during plant light response.

Arabidopsis thaliana miRNAs promote embryo pattern formation beginning in the zygote.

miRNAs are essential regulators of cell identity, yet their role in early embryo development in plants remains largely unexplored. To determine the earliest stage at which miRNAs act to promote pattern formation in embryogenesis, we examined a series of mutant alleles in the Arabidopsis thaliana miRNA biogenesis enzymes DICER-LIKE 1 (DCL1), SERRATE (SE), and HYPONASTIC LEAVES 1 (HYL1). Cellular and patterning defects were observed in dcl1, se and hyl1 embryos from the zygote through the globular stage of embryogenesis. To identify miRNAs that are expressed in early embryogenesis, we sequenced mRNAs from globular stage Columbia wild type (wt) and se-1 embryos, and identified transcripts potentially corresponding to 100 miRNA precursors. Considering genome location and transcript increase between wt and se-1, 39 of these MIRNAs are predicted to be bona fide early embryo miRNAs. Among these are conserved miRNAs such as miR156, miR159, miR160, miR161, miR164, miR165, miR166, miR167, miR168, miR171, miR319, miR390 and miR394, as well as miRNAs whose function has never been characterized. Our analysis demonstrates that miRNAs promote pattern formation beginning in the zygote, and provides a comprehensive dataset for functional studies of individual miRNAs in Arabidopsis embryogenesis.

Involvement of microRNA-related regulatory pathways in the glucose-mediated control of Arabidopsis early seedling development.

In plants, sugars such as glucose act as signalling molecules that promote changes in gene expression programmes that impact on growth and development. Recent evidence has revealed the potential importance of controlling mRNA decay in some aspects of glucose-mediated regulatory responses suggesting a role of microRNAs (miRNAs) in these responses. In order to get a better understanding of glucose-mediated development modulation involving miRNA-related regulatory pathways, early seedling development of mutants impaired in miRNA biogenesis (hyl1-2 and dcl1-11) and miRNA activity (ago1-25) was evaluated. All mutants exhibited a glucose hyposensitive phenotype from germination up to seedling establishment, indicating that miRNA regulatory pathways are involved in the glucose-mediated delay of early seedling development. The expression profile of 200 miRNA primary transcripts (pri-miRs) was evaluated by large-scale quantitative real-time PCR profiling, which revealed that 38 pri-miRs were regulated by glucose. For several of them, the corresponding mature miRNAs are known to participate directly or indirectly in plant development, and their accumulation was shown to be co-regulated with the pri-miR by glucose. Furthermore, the expression of several miRNA target genes was found to be deregulated in response to glucose in the miRNA machinery mutants ago1-25, dcl1-11, and hyl1-2. Also, in these mutants, glucose promoted misexpression of genes for the three abscisic acid signalling elements ABI3, ABI4, and ABI5. Thus, miRNA regulatory pathways play a role in the adjustments of growth and development triggered by glucose signalling.