Plant miRNA Biogenesis Research Articles

Widespread changes to the translational landscape in a maize microRNA biogenesis mutant.

MicroRNAs are short, non-coding RNAs that repress gene expression in both plants and animals and have diverse functions related to growth, development, and stress responses. The ribonuclease, DICER-LIKE1 (DCL1) is required for two steps in plant miRNA biogenesis: cleavage of the primary miRNAs (pri-miRNAs) to release a hairpin structure, called the precursor miRNA (pre-miRNA) and cleavage of the pre-miRNA to generate the miRNA/miRNA* duplex. The mature miRNA guides the RNA-induced silencing complex to target RNAs with complementary sequences, resulting in translational repression and/or RNA cleavage of target mRNAs. However, the relative contribution of translational repression versus mRNA degradation by miRNAs remains unknown at the genome-level in crops, especially in maize. The maize fuzzy tassel (fzt) mutant contains a hypomorphic mutation in DCL1 resulting in broad developmental defects. While most miRNAs are reduced in fzt, the levels of miRNA-targeted mRNAs are not dramatically increased, suggesting that translational regulation by miRNAs may be common. To gain insight into the repression mechanism of plant miRNAs, we combined ribosome profiling and RNA-sequencing to globally survey miRNA activities in maize. Our data indicate that translational repression contributes significantly to regulation of most miRNA targets and that approximately one-third of miRNA targets are regulated primarily at the translational level. Surprisingly, ribosomes appear altered in fzt mutants suggesting that DCL1 may also have a role in ribosome biogenesis. Thus, DICER-LIKE1 shapes the translational landscape in plants through both miRNA-dependent and miRNA-independent mechanisms.

AmiR-P3: An AI-based microRNA prediction pipeline in plants.

MicroRNAs (miRNAs) are small noncoding RNAs that play important post-transcriptional regulatory roles in animals and plants. Despite the importance of plant miRNAs, the inherent complexity of miRNA biogenesis in plants hampers the application of standard miRNA prediction tools, which are often optimized for animal sequences. Therefore, computational approaches to predict putative miRNAs (merely) from genomic sequences, regardless of their expression levels or tissue specificity, are of great interest. Here, we present AmiR-P3, a novel ab initio plant miRNA prediction pipeline that leverages the strengths of various utilities for its key computational steps. Users can readily adjust the prediction criteria based on the state-of-the-art biological knowledge of plant miRNA properties. The pipeline starts with finding the potential homologs of the known plant miRNAs in the input sequence(s) and ensures that they do not overlap with protein-coding regions. Then, by computing the secondary structure of the presumed RNA sequence based on the minimum free energy, a deep learning classification model is employed to predict potential pre-miRNA structures. Finally, a set of criteria is used to select the most likely miRNAs from the set of predicted miRNAs. We show that our method yields acceptable predictions in a variety of plant species. AmiR-P3 does not (necessarily) require sequencing reads and/or assembled reference genomes, enabling it to identify conserved and novel putative miRNAs from any genomic or transcriptomic sequence. Therefore, AmiR-P3 is suitable for miRNA prediction even in less-studied plants, as it does not require any prior knowledge of the miRNA repertoire of the organism. AmiR-P3 is provided as a docker container, which is a portable and self-contained software package that can be readily installed and run on any platform and is freely available for non-commercial use from: https://hub.docker.com/r/micrornaproject/amir-p3.

MicroRNA production in Arabidopsis.

In plants, microRNAs (miRNAs) associate with ARGONAUTE (AGO) proteins and act as sequence-specific repressors of target gene expression, at the post-transcriptional level through target transcript cleavage and/or translational inhibition. MiRNAs are mainly transcribed by DNA-dependent RNA polymerase II (POL II) and processed by DICER LIKE1 (DCL1) complex into 21∼22 nucleotide (nt) long. Although the main molecular framework of miRNA biogenesis and modes of action have been established, there are still new requirements continually emerging in the recent years. The studies on the involvement factors in miRNA biogenesis indicate that miRNA biogenesis is not accomplished separately step by step, but is closely linked and dynamically regulated with each other. In this article, we will summarize the current knowledge on miRNA biogenesis, including MIR gene transcription, primary miRNA (pri-miRNA) processing, miRNA AGO1 loading and nuclear export; and miRNA metabolism including methylation, uridylation and turnover. We will describe how miRNAs are produced and how the different steps are regulated. We hope to raise awareness that the linkage between different steps and the subcellular regulation are becoming important for the understanding of plant miRNA biogenesis and modes of action.

Recent Insights into Plant miRNA Biogenesis: Multiple Layers of miRNA Level Regulation.

MicroRNAs are small RNAs, 20-22 nt long, the main role of which is to downregulate gene expression at the level of mRNAs. MiRNAs are fundamental regulators of plant growth and development in response to internal signals as well as in response to abiotic and biotic factors. Therefore, the deficiency or excess of individual miRNAs is detrimental to particular aspects of a plant's life. In consequence, the miRNA levels must be appropriately adjusted. To obtain proper expression of each miRNA, their biogenesis is controlled at multiple regulatory layers. Here, we addressed processes discovered to influence miRNA steady-state levels, such as MIR transcription, co-transcriptional pri-miRNA processing (including splicing, polyadenylation, microprocessor assembly and activity) and miRNA-encoded peptides synthesis. MiRNA stability, RISC formation and miRNA export out of the nucleus and out of the plant cell also define the levels of miRNAs in various plant tissues. Moreover, we show the evolutionary conservation of miRNA biogenesis core proteins across the plant kingdom.

The plant FYVE domain-containing protein FREE1 associates with microprocessor components to repress miRNA biogenesis.

FYVE domain protein required for endosomal sorting 1 (FREE1), originally identified as a plant-specific component of the endosomal sorting complex required for transport (ESCRT) machinery, plays diverse roles either in endosomal sorting in the cytoplasm or in transcriptional regulation of abscisic acid signaling in the nucleus. However, to date, a role for FREE1 or other ESCRT components in the regulation of plant miRNA biology has not been discovered. Here, we demonstrate a nuclear function of FREE1 as a cofactor in miRNA biogenesis in plants. FREE1 directly interacts with the plant core microprocessor component CPL1 in nuclear bodies and disturbs the association between HYL1, SE and CPL1. Inactivation of FREE1 in the nucleus increases the binding affinity between HYL1, SE, and CPL1 and causes a transition of HYL1 from the inactive hyperphosphorylated version to the active hypophosphorylated form, thereby promoting miRNA biogenesis. Our results suggest that FREE1 has evolved as a negative regulator of miRNA biogenesis and provides evidence for a link between FYVE domain-containing proteins and miRNA biogenesis in plants.

Plant responses to drought stress: microRNAs in action

Drought is common in most regions of the world, and it has a significant impact on plant growth and development. Plants, on the other hand, have evolved their own defense systems to deal with the extreme weather. The reprogramming of gene expression by microRNAs (miRNAs) is one of these defense mechanisms. miRNAs are short noncoding RNAs that have emerged as key post-transcriptional gene regulators in a variety of species. Drought stress modulates the expression of certain miRNAs that are functionally conserved across plant species. These characteristics imply that miRNA-based genetic changes might improve drought resistance in plants. This study highlights current knowledge of plant miRNA biogenesis, regulatory mechanisms and their role in drought stress responses. miRNAs functions and their adaptations by plants during drought stress has also been explained that can be exploited to promote drought-resistance among economically important crops.

R-loops at microRNA encoding loci promote co-transcriptional processing of pri-miRNAs in plants

In most organisms, the maturation of nascent RNAs is coupled to transcription. Unlike in animals, the RNA polymerase II (RNAPII) transcribes microRNA genes (MIRNAs) as long and structurally variable pri-miRNAs in plants. Current evidence suggests that the miRNA biogenesis complex assembly initiates early during the transcription of pri-miRNAs in plants. However, it is unknown whether miRNA processing occurs co-transcriptionally. Here, we used native elongating transcript sequencing data and imaging techniques to demonstrate that plant miRNA biogenesis occurs coupled to transcription. We found that the entire biogenesis occurs co-transcriptionally for pri-miRNAs processed from the loop of the hairpin but requires a second nucleoplasmic step for those processed from the base. Furthermore, we found that co- and post-transcriptional miRNA processing mechanisms co-exist for most miRNAs in a dynamic balance. Notably, we discovered that R-loops, formed near the transcription start site region of MIRNAs, promote co-transcriptional pri-miRNA processing. Furthermore, our results suggest the neofunctionalization of co-transcriptionally processed miRNAs, boosting countless regulatory scenarios.

Profiling of Single-cell-type-specific MicroRNAs in Arabidopsis Roots by Immunoprecipitation of Root Cell-layer-specific GFP-AGO1.

MicroRNAs (miRNA) are small (21-24 nt) non-coding RNAs involved in many biological processes in both plants and animals. The biogenesis of plant miRNAs starts with the transcription of MIRNA (MIR ) genes by RNA polymerase II; then, the primary miRNA transcripts are cleaved by Dicer-like proteins into mature miRNAs, which are then loaded into Argonaute (AGO) proteins to form the effector complex, the miRNA-induced silencing complex (miRISC). In Arabidopsis , some MIR genes are expressed in a tissue-specific manner; however, the spatial patterns of MIR gene expression may not be the same as the spatial distribution of miRISCs due to the non-cell autonomous nature of some miRNAs, making it challenging to characterize the spatial profiles of miRNAs. A previous study utilized protoplasting of green fluorescent protein (GFP) marker transgenic lines followed by fluorescence-activated cell sorting (FACS) to isolate cell-type-specific small RNAs. However, the invasiveness of this approach during the protoplasting and cell sorting may stimulate the expression of stress-related miRNAs. To non-invasively profile cell-type-specific miRNAs, we generated transgenic lines in which root cell layer-specific promoters drive the expression of AGO1 and performed immunoprecipitation to non-invasively isolate cell-layer-specific miRISCs. In this protocol, we provide a detailed description of immunoprecipitation of root cell layer-specific GFP-AGO1 using EN7::GFP-AGO1 and ACL5::GFP-AGO1 transgenic plants, followed by small RNA sequencing to profile single-cell-type-specific miRNAs. This protocol is also suitable to profile cell-type-specific miRISCs in other tissues or organs in plants, such as flowers or leaves. This protocol was validated in: Dev Cell (2022), DOI: 10.1016/j.devcel.2022.03.015 Graphical abstract.

Structural basis of microRNA processing by Dicer-like 1.

MicroRNAs (miRNAs) are short non-coding RNAs that inhibit the expression of target genes by directly binding to their mRNAs. In animals, pri-miRNAs are cleaved by Drosha to generate pre-miRNAs, which are subsequently cleaved by Dicer to generate mature miRNAs. Instead of being cleaved by two different enzymes, both cleavages in plants are performed by Dicer-like 1 (DCL1). With a similar domain architecture as human Dicer, it is mysterious how DCL1 recognizes pri-miRNAs and performs two cleavages sequentially. Here, we report the single-particle cryo-electron microscopy structures of Arabidopsis DCL1 complexed with a pri-miRNA and a pre-miRNA, respectively, in cleavage-competent states. These structures uncover the plasticity of the PAZ domain, which is critical for the recognition of both pri-miRNA and pre-miRNA. These structures suggest that the helicase module serves as an engine that transfers the substrate between two sequential cleavage events. This study lays a foundation for dissecting the regulation mechanism of miRNA biogenesis in plants and provides insights into the dicing state of human Dicer.

Identification of key sequence features required for microRNA biogenesis in plants

MicroRNAs (miRNAs) are endogenous small RNAs of ∼21 nt that regulate multiple biological pathways in multicellular organisms. They derive from longer transcripts that harbor an imperfect stem-loop structure. In plants, the ribonuclease type III DICER-LIKE1 assisted by accessory proteins cleaves the precursor to release the mature miRNA. Numerous studies highlight the role of the precursor secondary structure during plant miRNA biogenesis; however, little is known about the relevance of the precursor sequence. Here, we analyzed the sequence composition of plant miRNA primary transcripts and found specifically located sequence biases. We show that changes in the identity of specific nucleotides can increase or abolish miRNA biogenesis. Most conspicuously, our analysis revealed that the identity of the nucleotides at unpaired positions of the precursor plays a crucial role during miRNA biogenesis in Arabidopsis.

MicroRNA biogenesis in plant

MicroRNAs (miRNAs), a type of endogenous non-coding small RNA, with a length of 20 to 24 nt, represses their target gene expression post-transcriptionally. There are a growing number of studies have discovered that miRNAs are found in animals and plants. Plant miRNAs are involved in growth and development, and they play an important role in physiological and biochemical regulation. This review mainly focuses on components of different miRNA processing pathways and how miRNA effectors inhibit the expression of target genes in plants. We discuss not only the various pathways affecting miRNA biogenesis, but also the different forms of miRNA effector pathways. Previous studies found that there have been some primary advances in the formation of miRNAs, we hope to provide a comprehensive introduction to the biogenesis of plant miRNAs.

A conserved sequence signature is essential for robust plant miRNA biogenesis.

Micro (mi)RNAs are 20–22nt long non-coding RNA molecules involved in post-transcriptional silencing of targets having high base-pair complementarity. Plant miRNAs are processed from long Pol II-transcripts with specific stem-loop structures by Dicer-like (DCL) 1 protein. Although there were reports indicating how a specific region is selected for miRNA biogenesis, molecular details were unclear. Here, we show that the presence of specific GC-rich sequence signature within miRNA/miRNA* region is required for the precise miRNA biogenesis. The involvement of GC-rich signatures in precise processing and abundance of miRNAs was confirmed through detailed molecular and functional analysis. Consistent with the presence of the miRNA-specific GC signature, target RNAs of miRNAs also possess conserved complementary sequence signatures in their miRNA binding motifs. The selection of these GC signatures was dependent on an RNA binding protein partner of DCL1 named HYL1. Finally, we demonstrate a direct application of this discovery for enhancing the abundance and efficiency of artificial miRNAs that are popular in plant functional genomic studies.

Plant microRNAs: biogenesis, gene silencing, web-based analysis tools and their use as molecular markers.

MicroRNAs (miRNAs) are tiny (20-24 nt bp) regulatory non-protein-coding RNA molecules that have been extensively characterized and found important for many physiological and developmental processes. The miss-expression of miRNAs leads to various defects in plants. MicroRNAs repress gene expression by directing mRNA degradation or translational arrest. Several proteins such as PP43A, HYL1, DCL, HST are indispensable role players in promoting miRNA biogenesis in plants. During miRNA biogenesis, lariat RNAs are produced as by-products of pre-mRNA splicing which have a negative role in regulation of miRNA homeostasis. By acting as a decoy and by sequestering to the dicing complex, lariat RNA can prevent the processing of miRNAs. A number of bioinformatic tools with different methodologies are available to identify and validate miRNAs and their targets. Many miRNAs have been reported in different crops for different traits; however, no reports are available on their use in plant breeding. Recently, researchers have developed trait specific miRNA-based molecular markers (miRNA-SSRs/SNP) for many quantitative traits in different plant species. In the future, these molecular markers can be used for plant breeding programs. In this review, a comprehensive up-to-date information is provided on the bioinformatic tools used for analysis of plant miRNAs and their targets, the number of miRNAs, their biogenesis, gene silencing mechanism and miRNA-based molecular markers.

MicroRNAs and Their Regulatory Roles in Plant-Environment Interactions.

MicroRNAs (miRNAs) are 20-24 nucleotide noncoding RNAs abundant in plants and animals. The biogenesis of plant miRNAs involves transcription of miRNA genes, processing of primary miRNA transcripts by DICER-LIKE proteins into mature miRNAs, and loading of mature miRNAs into ARGONAUTE proteins to form miRNA-induced silencing complex (miRISC). By targeting complementary sequences, miRISC negatively regulates gene expression, thereby coordinating plant development and plant-environment interactions. In this review, we present and discuss recent updates on the mechanisms and regulation of miRNA biogenesis, miRISC assembly and actions as well as the regulatory roles of miRNAs in plant developmental plasticity, abiotic/biotic responses, and symbiotic/parasitic interactions. Finally, we suggest future directions for plant miRNA research.

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.

Efficiency and precision of microRNA biogenesis modes in plants.

Many evolutionarily conserved microRNAs (miRNAs) in plants regulate transcription factors with key functions in development. Hence, mutations in the core components of the miRNA biogenesis machinery cause strong growth defects. An essential aspect of miRNA biogenesis is the precise excision of the small RNA from its precursor. In plants, miRNA precursors are largely variable in size and shape and can be processed by different modes. Here, we optimized an approach to detect processing intermediates during miRNA biogenesis. We characterized a miRNA whose processing is triggered by a terminal branched loop. Plant miRNA processing can be initiated by internal bubbles, small terminal loops or branched loops followed by dsRNA segments of 15–17 bp. Interestingly, precision and efficiency vary with the processing modes. Despite the various potential structural determinants present in a single a miRNA precursor, DCL1 is mostly guided by a predominant structural region in each precursor in wild-type plants. However, our studies in fiery1, hyl1 and se mutants revealed the existence of cleavage signatures consistent with the recognition of alternative processing determinants. The results provide a general view of the mechanisms underlying the specificity of miRNA biogenesis in plants.

Conformational sampling of the intrinsically disordered dsRBD-1 domain from Arabidopsis thaliana DCL1.

DCL1 is the ribonuclease that carries out miRNA biogenesis in plants. Substrate pri-miRNA recognition by DCL1 requires two double stranded RNA binding domains located at the C-terminus of the protein. We have previously shown that the first of these domains, DCL1-A, is intrinsically disordered and folds upon binding pri-miRNA. Integrating NMR and SAXS data, we study here the conformational landscape of free DCL1-A through an ensemble description. Our results reveal that secondary structure elements, corresponding to the folded form of the protein, are transiently populated in the unbound state. The conformation of one of the dsRNA binding regions in the free protein shows that, at a local level, RNA recognition proceeds through a conformational selection mechanism. We further explored the stability of the preformed structural elements via temperature and urea destabilization. The C-terminal helix is halfway on the folding pathway in free DCL1-A, constituting a potential nucleation site for the final folding of the protein. In contrast, the N-terminal helix adopts stable non-native structures that could hinder the correct folding of the protein in the absence of RNA. This description of the unfolded form allows us to understand details of the mechanism of binding-induced folding of the protein.

Regulation of Plant miRNA Biogenesis

Regulation of Plant miRNA Biogenesis

The Protein Phosphatase 4 and SMEK1 Complex Dephosphorylates HYL1 to Promote miRNA Biogenesis by Antagonizing the MAPK Cascade in Arabidopsis

Phosphorylation plays an essential role in microRNA (miRNA) processing by regulating co-factors of the miRNA biogenesis machinery. HYL1 (Hyponastic Leaves 1), a core co-factor in plant miRNA biogenesis, is a short-lived phosphoprotein. However, the precise balance and regulatory mechanism of the stability and phosphorylation of HYL1 remain unclear. Here, we show that a highly conserved PP4 (Protein Phosphatase 4) and SMEK1 (Suppressor ofMEK 1) complex dephosphorylates HYL1 to promote miRNA biogenesis, by antagonizing the MAPK cascade in Arabidopsis. The smek1 mutants exhibit defective miRNA biogenesis due to accelerated degradation of HYL1. SMEK1 stabilizes HYL1 in adual manner: SMEK1, as a suppressor, inhibits MAPK activation to attenuate HYL1 phosphorylation; SMEK1 assembles a functional PP4 to target HYL1 for dephosphorylation. Moreover, the protein level of SMEK1 is increased in response to abscisic acid. Our results provide insights into the delicate balance between a protein kinase and a phosphatase during miRNA biogenesis.

Evolutionary Footprints Reveal Insights into Plant MicroRNA Biogenesis.

MicroRNAs (miRNAs) are endogenous small RNAs that recognize target sequences by base complementarity and play a role in the regulation of target gene expression. They are processed from longer precursor molecules that harbor a fold-back structure. Plant miRNA precursors are quite variable in size and shape, and are recognized by the processing machinery in different ways. However, ancient miRNAs and their binding sites in target genes are conserved during evolution. Here, we designed a strategy to systematically analyze MIRNAs from different species generating a graphical representation of the conservation of the primary sequence and secondary structure. We found that plant MIRNAs have evolutionary footprints that go beyond the small RNA sequence itself, yet their location along the precursor depends on the specific MIRNA We show that these conserved regions correspond to structural determinants recognized during the biogenesis of plant miRNAs. Furthermore, we found that the members of the miR166 family have unusual conservation patterns and demonstrated that the recognition of these precursors in vivo differs from other known miRNAs. Our results describe a link between the evolutionary conservation of plant MIRNAs and the mechanisms underlying the biogenesis of these small RNAs and show that the MIRNA pattern of conservation can be used to infer the mode of miRNA biogenesis.