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.

Systematic Characterization of MicroRNA Processing Modes in Plants With Parallel Amplification of RNA Ends.

In plants, the RNase III-type enzyme Dicer-like 1 (DCL1) processes most microRNAs (miRNAs) from their primary transcripts called pri-miRNAs. Four distinct processing modes (i.e., short base to loop, sequential base to loop, short loop to base, and sequential loop to base) have been characterized in Arabidopsis, mainly by the Specific Parallel Amplification of RNA Ends (SPARE) approach. However, SPARE is a targeted cloning method which requires optimization of cloning efficiency and specificity for each target. PARE (Parallel Amplification of RNA Ends) is an untargeted method per se and is widely used to identify miRNA mediated target slicing events. A major concern with PARE in characterizing miRNA processing modes is the potential contamination of mature miRNAs. Here, we provide a method to estimate miRNA contamination levels and showed that most publicly available PARE libraries have negligible miRNA contamination. Both the numbers and processing modes detected by PARE were similar to those identified by SPARE in Arabidopsis. PARE also determined the processing modes of 36 Arabidopsis miRNAs that were unexplored by SPARE, suggesting that it can complement the SPARE approach. Using publicly available PARE datasets, we identified the processing modes of 36, 91, 90, and 54 miRNAs in maize, rice, soybean, and tomato, respectively, and demonstrated that the processing mode was conserved overall within each miRNA family. Through its power of tracking miRNA processing remnants, PARE also facilitated miRNA characterization and annotation.

Serrate-Associated Protein 1, a splicing-related protein, promotes miRNA biogenesis in Arabidopsis.

MicroRNAs (miRNAs) are essential regulators of gene expression in metazoans and plants. In plants, most miRNAs are generated from primary miRNA transcripts (pri-miRNAs), which are processed by the Dicer-like 1 (DCL1) complex along with accessory proteins. Serrate-Associated Protein 1 (SEAP1), a conserved splicing-related protein, has been studied in human and yeast. However, the functions of SEAP1 in plants remain elusive. Lack of SEAP1 results in embryo lethality and knockdown of SEAP1 by an artificial miRNA (amiRSEAP1 ) causes pleiotropic developmental defects and reduction in miRNA accumulation. SEAP1 associates with the DCL1 complex, and may promote the interaction of the DCL1 complexes with pri-miRNAs. SEAP1 also enhances pri-miRNA accumulation, but does not affect pri-miRNA transcription, suggesting it may indirectly or directly stabilize pri-miRNAs. In addition, SEAP1 affects the splicing of some pri-miRNAs and intron retention of messenger RNAs at global levels. Our findings uncover both conserved and novel functions of SEAP1 in plants. Besides the role as a splicing factor, SEPA1 may promote miRNA biogenesis by positively modulating pri-miRNA splicing, processing and/or stability.

Recent advances in the regulation of plant miRNA biogenesis.

MicroRNAs (miRNAs) are essential non-coding riboregulators of gene expression in plants and animals. In plants, miRNAs guide their effector protein named ARGONAUTE (AGO) to find target RNAs for gene silencing through target RNA cleavage or translational inhibition. miRNAs are derived from primary miRNA transcripts (pri-miRNAs), most of which are transcribed by the DNA-dependent RNA polymerase II. In plants, an RNase III enzyme DICER-LIKE1-containing complex processes pri-miRNAs in the nucleus into miRNAs. To ensure proper function of miRNAs, plants use multiple mechanisms to control miRNA accumulation. On one hand, pri-miRNA levels are controlled through transcription and stability. On the other hand, the activities of the DCL1 complex are regulated by many protein factors at transcriptional, post-transcriptional and post-translational levels. Notably, recent studies reveal that pri-miRNA structure/sequence features and modifications also play important roles in miRNA biogenesis. In this review, we summarize recent progresses on the mechanisms regulating miRNA biogenesis.

AGL15 Controls the Embryogenic Reprogramming of Somatic Cells in Arabidopsis through the Histone Acetylation-Mediated Repression of the miRNA Biogenesis Genes.

The embryogenic transition of somatic cells requires an extensive reprogramming of the cell transcriptome. Relevantly, the extensive modulation of the genes that have a regulatory function, in particular the genes encoding the transcription factors (TFs) and miRNAs, have been indicated as controlling somatic embryogenesis (SE) that is induced in vitro in the somatic cells of plants. Identifying the regulatory relationships between the TFs and miRNAs during SE induction is of central importance for understanding the complex regulatory interplay that fine-tunes a cell transcriptome during the embryogenic transition. Hence, here, we analysed the regulatory relationships between AGL15 (AGAMOUS-LIKE 15) TF and miR156 in an embryogenic culture of Arabidopsis. Both AGL15 and miR156 control SE induction and AGL15 has been reported to target the MIR156 genes in planta. The results showed that AGL15 contributes to the regulation of miR156 in an embryogenic culture at two levels that involve the activation of the MIR156 transcription and the containment of the abundance of mature miR156 by repressing the miRNA biogenesis genes DCL1 (DICER-LIKE1), SERRATE and HEN1 (HUA-ENHANCER1). To repress the miRNA biogenesis genes AGL15 seems to co-operate with the TOPLESS co-repressors (TPL and TPR1-4), which are components of the SIN3/HDAC silencing complex. The impact of TSA (trichostatin A), an inhibitor of the HDAC histone deacetylases, on the expression of the miRNA biogenesis genes together with the ChIP results implies that histone deacetylation is involved in the AGL15-mediated repression of miRNA processing. The results indicate that HDAC6 and HDAC19 histone deacetylases might co-operate with AGL15 in silencing the complex that controls the abundance of miR156 during embryogenic induction. This study provides new evidence about the histone acetylation-mediated control of the miRNA pathways during the embryogenic reprogramming of plant somatic cells and the essential role of AGL15 in this regulatory mechanism.

Plant microRNAs: Biogenesis, Homeostasis, and Degradation.

MicroRNAs (miRNAs), a class of endogenous, tiny, non-coding RNAs, are master regulators of gene expression among most eukaryotes. Intracellular miRNA abundance is regulated under multiple levels of control including transcription, processing, RNA modification, RNA-induced silencing complex (RISC) assembly, miRNA-target interaction, and turnover. In this review, we summarize our current understanding of the molecular components and mechanisms that influence miRNA biogenesis, homeostasis, and degradation in plants. We also make comparisons with findings from other organisms where necessary.

SERRATE, a miRNA biogenesis factor, affects viroid infection in Nicotiana benthamiana and Nicotiana tabacum.

Viroids are plant infecting, non - coding RNA molecules of economic importance. Potato spindle tuber viroid (PSTVd), the type species of Pospiviroidae family, has been shown to be affected by specific RNA silencing pathways. Dicer like 1 (DCL1), a key player in micro RNA (miRNA) pathway has been previously linked with PSTVd infectivity. In this report we aim to further dissect the interaction between the miRNA pathway and Pospiviroid virulence. We mainly focused on the Zinc-finger protein SERRATE (SE) a co-factor of DCL1 and core component of miRNA pathway. We generated Nicotiana tabacum and Nicotiana benthamiana SE knock-down plants exhibiting considerable miRNA reduction and strong phenotypic abnormalities. PSTVd infection of SE suppressed plants resulted in a significant viroid reduction, especially at the initial infection stages. This positive correlation between SE levels and viroid infectivity underlines its role in PSTVd life cycle and reveals the importance of the miRNA pathway upon viroid infection.

Regulation of Plant Microprocessor Function in Shaping microRNA Landscape.

MicroRNAs are small molecules (∼21 nucleotides long) that are key regulators of gene expression. They originate from long stem-loop RNAs as a product of cleavage by a protein complex called Microprocessor. The core components of the plant Microprocessor are the RNase type III enzyme Dicer-Like 1 (DCL1), the zinc finger protein Serrate (SE), and the double-stranded RNA binding protein Hyponastic Leaves 1 (HYL1). Microprocessor assembly and its processing of microRNA precursors have been reported to occur in discrete nuclear bodies called Dicing bodies. The accessibility of and modifications to Microprocessor components affect microRNA levels and may have dramatic consequences in plant development. Currently, numerous lines of evidence indicate that plant Microprocessor activity is tightly regulated. The cellular localization of HYL1 is dependent on a specific KETCH1 importin, and the E3 ubiquitin ligase COP1 indirectly protects HYL1 from degradation in a light-dependent manner. Furthermore, proper localization of HYL1 in Dicing bodies is regulated by MOS2. On the other hand, the Dicing body localization of DCL1 is regulated by NOT2b, which also interacts with SE in the nucleus. Post-translational modifications are substantial factors that contribute to protein functional diversity and provide a fine-tuning system for the regulation of protein activity. The phosphorylation status of HYL1 is crucial for its activity/stability and is a result of the interplay between kinases (MPK3 and SnRK2) and phosphatases (CPL1 and PP4). Additionally, MPK3 and SnRK2 are known to phosphorylate SE. Several other proteins (e.g., TGH, CDF2, SIC, and RCF3) that interact with Microprocessor have been found to influence its RNA-binding and processing activities. In this minireview, recent findings on the various modes of Microprocessor activity regulation are discussed.

Discovering Structural Motifs in miRNA Precursors from the Viridiplantae Kingdom.

A small non-coding molecule of microRNA (19⁻24 nt) controls almost every biological process, including cellular and physiological, of various organisms' lives. The amount of microRNA (miRNA) produced within an organism is highly correlated to the organism's key processes, and determines whether the system works properly or not. A crucial factor in plant biogenesis of miRNA is the Dicer Like 1 (DCL1) enzyme. Its responsibility is to perform the cleavages in the miRNA maturation process. Despite everything we already know about the last phase of plant miRNA creation, recognition of miRNA by DCL1 in pre-miRNA structures of plants remains an enigma. Herein, we present a bioinformatic procedure we have followed to discover structure patterns that could guide DCL1 to perform a cleavage in front of or behind an miRNA:miRNA* duplex. The patterns in the closest vicinity of microRNA are searched, within pre-miRNA sequences, as well as secondary and tertiary structures. The dataset consists of structures of plant pre-miRNA from the Viridiplantae kingdom. The results confirm our previous observations based on Arabidopsis thaliana precursor analysis. Hereby, our hypothesis was tested on pre-miRNAs, collected from the miRBase database to show secondary structure patterns of small symmetric internal loops 1-1 and 2-2 at a 1⁻10 nt distance from the miRNA:miRNA* duplex.

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.

1H, 13C and 15N resonance assignments of human DCL-1 (CD302) extracellular domain.

DCL-1 (CD302) is a single-pass type one transmembrane protein which is predominantly expressed on myeloid cell lines. It possess the ability of endocytosis and is assumed to play a role in cell adhesion and migration. It has been also connected to several illnesses but more on the level of mRNA than on the protein expression level. More interestingly it is alternatively expressed in the form of a fusion protein with another single-pass type one transmembrane protein DEC205 (CD205) which is normally involved in antigen-uptake and endocytosis. The fusion protein has been assigned to have altered function compared to the wild type proteins. We have performed NMR structural analysis of the 16.2 kDa extracellular domain of DCL-1 to get a better insight onto this molecule. We have been able to assign nearly 97 % of resonance frequencies for the (15)N and (13)C labeled recombinant protein. The assignments have been deposited into Biological Magnetic Resonance Data Bank under the accession number 25802.

Recovery of dicer-like 1-late flowering phenotype by miR172 expressed by the noncanonical DCL4-dependent biogenesis pathway.

MicroRNAs (miRNAs) act as down-regulators of gene expression, and play a dominant role in eukaryote development. In Arabidopsis thaliana, DICER-LIKE 1 (DCL1) is the main processor in miRNA biogenesis, and dcl1 mutants show various developmental defects at the early stage of embryogenesis or at gamete formation. However, miRNAs responsible for the respective developmental stages of the dcl1 defects have not been identified. Here, we developed a DCL1-independent miRNA expression system using the unique DCL4-dependent miRNA, miR839. By replacing the mature sequence in the miR839 precursor sequence with that of miR172, one of the most widely conserved miRNAs in angiosperms, we succeeded in expressing miR172 from a chimeric miR839 precursor in dcl1-7 plants and observed the repression of miR172 target gene expression. In parallel, the DCL4-dependent miR172 expression rescued the late flowering phenotype of dcl1-7 by acceleration of flowering. We established the DCL1-independent miRNA expression system, and revealed that the reduction of miR172 expression is responsible for the dcl1-7 late flowering phenotype.

Long non-coding RNAs: a novel endogenous source for the generation of Dicer-like 1-dependent small RNAs in Arabidopsis thaliana.

The biological relevance of long non-coding RNAs (lncRNAs) is emerging. Whether the lncRNAs could form structured precursors for small RNAs (sRNAs) production remains elusive. Here, 172 713 DCL1 (Dicer-like 1)-dependent sRNAs were identified in Arabidopsis. Except for the sRNAs mapped onto the microRNA precursors, the remaining ones led us to investigate their originations. Intriguingly, 65 006 sRNAs found their loci on 5891 lncRNAs. These sRNAs were sent to AGO (Argonaute) enrichment analysis. As a result, 1264 sRNAs were enriched in AGO1, which were then subjected to target prediction. Based on degradome sequencing data, 109 transcripts were validated to be targeted by 96 sRNAs. Besides, 44 lncRNAs were targeted by 23 sRNAs. To further support the origination of the DCL1-dependent sRNAs from lncRNAs, we searched for the degradome-based cleavage signals at either ends of the sRNA loci, which were supposed to be produced during DCL1-mediated processing of the long-stem structures. As a result, 63 612 loci were supported by degradome signatures. Among these loci, 6606 reside within the dsRNA-seq (double-stranded RNA sequencing) read-covered regions of 100 nt or longer. These regions were subjected to secondary structure prediction. And, 43 regions were identified to be capable of forming highly complementary long-stem structures. We proposed that these local long-stem structures could be recognized by DCL1 for cropping, thus serving as the sRNA precursors. We hope that our study could inspire more research efforts to study on the biological roles of the lncRNAs in plants.

Computational identification of microRNAs in the strawberry (Fragaria x ananassa) genome sequence and validation of their precise sequences by miR-RACE.

In plants, microRNAs (miRNAs) play significant roles in post-transcriptional gene regulation and have been found to control many genes involved in different biological and metabolic processes. Extensive studies were carried out to discover miRNAs and analyze their functions in model plant species, such as in Arabidopsis and rice that have been reported. In this research, we used bioinformatics to predict microRNAs in an important strawberry rootstock cultivar to discover and validate precise sequences of microRNAs in strawberry. By adopting a range of filtering criteria, we obtained 59 potential miRNAs belonging to 40 miRNA families from the Fragaria vesca genome. Using two specific 5' and 3' miRNA RACE PCR reactions and a sequence-directed cloning method, we accurately determined 34 precise sequences of candidate miRNAs, while six other sequences exhibited some minor divergence in their termini nucleotides, and 19 miRNAs that could not be cloned owing to expression abundance may be too low or these mirRNAs predicted could not be existing in strawberry. Potential target genes were further predicted for the miRNAs above. The expression of the 16 miRNAs unreported and having exact sequences and their targets by experiment could be detected in different tissues of strawberry ranging from roots, stems, leaves, flowers and fruits by qRT-PCR and some of them showed differential expression in various tissues. The functional analysis of 16 miRNAs and their targets was carried out. Finally, we conclude that there are 34 mirRNAs in strawberry and their targets play vital roles not only in growth and development, but also in diverse physiological processes. These results show that regulatory miRNAs exist in agronomically important strawberry and might have an important function in strawberry growth and development.

Global and local perturbation of the tomato microRNA pathway by a trans-activated DICER-LIKE 1 mutant.

DICER-like 1 (DCL1) is a major player in microRNA (miRNA) biogenesis and accordingly, its few known loss-of-function mutants are either lethal or display arrested development. Consequently, generation of dcl1 mutants by reverse genetics and functional analysis of DCL1 in late-developing organs are challenging. Here, these challenges were resolved through the unique use of trans-activated RNA interference. Global, as well as organ-specific tomato DCL1 (SlDCL1) silencing was induced by crossing the generated responder line (OP:SlDCL1IR) with the appropriate driver line. Constitutive trans-activation knocked down SlDCL1 levels by ~95%, resulting in severe abnormalities including post-germination growth arrest accompanied by decreased miRNA and 21-nucleotide small RNA levels, but prominently elevated levels of 22-nucleotide small RNAs. The increase in the 22-nucleotide small RNAs was correlated with specific up-regulation of SlDCL2b and SlDCL2d, which are probably involved in their biogenesis. Leaf- and flower-specific OP:SlDCL1IR trans-activation inhibited blade outgrowth, induced premature bud senescence and produced pale petals, respectively, emphasizing the importance of SlDCL1-dependent small RNAs in these processes. Together, these results establish OP:SlDCL1IR as an efficient tool for analysing processes regulated by SlDCL1-mediated gene regulation in tomato.

Profiling microRNAs and their targets in an important fleshy fruit: tomato (Solanum lycopersicum).

Tomato (Solanum lycopersicum) is an important and the most useful plant based diet. It is widely used for its antioxidant property. Presently, only two digits, tomato microRNAs (miRNAs) are reported in miRBase: a miRNA database. This study is aimed to profile and characterize more miRNAs and their targets in tomato. A comprehensive comparative genomic approach is applied and a total of 109 new miRNAs belonging to 106 families are identified and characterized from the tomato expressed sequence tags (ESTs). All these potential miRNAs are profiled for the first time in tomato. The profiled miRNAs are also observed with stable stem-loop structures (Precursor-miRNAs), whose length ranges from 45 to 329 nucleotides (nt) with an average of 125 nt. The mature miRNAs are found in the stem of pre-miRNAs and their length ranges from 19 to 24 nt with an average of 21 nt. Furthermore, twelve miRNAs are randomly selected and experimentally validated through RT-PCR. A total of 406 putative targets are also predicted for the newly 109 tomato miRNAs. These targets are involved in structural protein, metabolism, transcription factor, growth & development, stress related, signaling pathways, storage proteins and other vital processes. Some important proteins like; 9-cisepoxycarotenoid dioxygenase (NCED), transcription factor MYB, ATP-binding cassette transporters, terpen synthase, 14-3-3 and TIR-NBS proteins are also predicted as putative targets for tomato miRNAs. These findings improve a baseline data of miRNAs and their targets in tomato. This baseline data can be utilized to fine tune this important fleshy fruit for nutritional & antioxidant properties and also under biotic & abiotic stresses.

Uncovering DCL1-dependent small RNA loci on plant genomes: a structure-based approach.

In plants, Dicer-like 1 (DCL1)-mediated two-step cleavages are essential for the processing of microRNA (miRNA) gene products. Interestingly, DCL1 has been indicated to be involved in the production of many small RNAs (sRNAs) that cannot be classified as canonical miRNAs. However, genomic and functional information on the non-miRNA, DCL1-dependent sRNAs is still limited. Here, we propose a secondary structure-based approach for identification of the precursors containing novel DCL1-dependent sRNA loci. To demonstrate the utility of the workflow: first, 5898 DCL1-dependent sRNAs of 20-24 nucleotides were identified from the sRNA high-throughput sequencing data sets prepared from rice DCL1 RNA interference transgenic lines. Those perfectly mapped to the rice pre-miRNAs (precursor microRNAs) were removed. The remaining 5795 sRNAs were then mapped onto the rice genome, obtaining 30 902 perfectly matched loci belonging to 2310 sRNAs. A total of 4631 clusters of sRNA loci were defined for secondary structure prediction by using RNAfold. The prediction results generated by two algorithms, namely MFE (minimum free energy) and centroid, were manually compared to identify the conserved long-stem structures containing DCL1-dependent sRNA loci. For the purpose of a case study, a portion of the prediction results was screened manually. As a result, 60 clusters displayed great potential for forming featured long-stem structures for the generation of DCL1-dependent sRNAs. Together, the results indicate that the proposed workflow is applicable for the identification of novel DCL1-dependent sRNA loci on plant genomes.

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.

miRNAs mediate SnRK1-dependent energy signaling in Arabidopsis.

The SnRK1 protein kinase, the plant ortholog of mammalian AMPK and yeast Snf1, is activated by the energy depletion caused by adverse environmental conditions. Upon activation, SnRK1 triggers extensive transcriptional changes to restore homeostasis and promote stress tolerance and survival partly through the inhibition of anabolism and the activation of catabolism. Despite the identification of a few bZIP transcription factors as downstream effectors, the mechanisms underlying gene regulation, and in particular gene repression by SnRK1, remain mostly unknown. microRNAs (miRNAs) are 20-24 nt RNAs that regulate gene expression post-transcriptionally by driving the cleavage and/or translation attenuation of complementary mRNA targets. In addition to their role in plant development, mounting evidence implicates miRNAs in the response to environmental stress. Given the involvement of miRNAs in stress responses and the fact that some of the SnRK1-regulated genes are miRNA targets, we postulated that miRNAs drive part of the transcriptional reprogramming triggered by SnRK1. By comparing the transcriptional response to energy deprivation between WT and dcl1-9, a mutant deficient in miRNA biogenesis, we identified 831 starvation genes misregulated in the dcl1-9 mutant, out of which 155 are validated or predicted miRNA targets. Functional clustering analysis revealed that the main cellular processes potentially co-regulated by SnRK1 and miRNAs are translation and organelle function and uncover TCP transcription factors as one of the most highly enriched functional clusters. TCP repression during energy deprivation was impaired in miR319 knockdown (MIM319) plants, demonstrating the involvement of miR319 in the stress-dependent regulation of TCPs. Altogether, our data indicates that miRNAs are components of the SnRK1 signaling cascade contributing to the regulation of specific mRNA targets and possibly tuning down particular cellular processes during the stress response.

Crystal structure of Arabidopsis thaliana Dawdle forkhead-associated domain reveals a conserved phospho-threonine recognition cleft for dicer-like 1 binding.

Dawdle (DDL) is a microRNA processing protein essential for the development of Arabidopsis. DDL contains a putative nuclear localization signal at its amino-terminus and forkhead-associated (FHA) domain at the carboxyl-terminus. Here, we report the crystal structure of the FHA domain of Arabidopsis Dawdle, determined by multiple-wavelength anomalous dispersion method at 1.7-Å resolution. DDL FHA structure displays a seven-stranded ß-sandwich architecture that contains a unique structural motif comprising two long anti-parallel strands. Strikingly, crystal packing of the DDL FHA domain reveals that a glutamate residue from the symmetry-related DDL FHA domain, a structural mimic of the phospho-threonine, is specifically recognized by the structurally conserved phospho-threonine binding cleft. Consistently with the structural observations, co-immuno-precipitation experiments performed in Nicotiana benthamiana show that the DDL FHA domain co-immuno-precipitates with DCL1 fragments containing the predicted pThr+3(Ile/Val/Leu/Asp) motif. Taken together, we count the recognition of the target residue by the canonical binding cleft of the DDL FHA domain as the key molecular event to instate FHA domain-mediated protein-protein interaction in plant miRNA processing.