Comparative analyses of super-enhancers reveal conserved elements in vertebrate genomes.

Super-enhancers (SEs) are key transcriptional drivers of cellular, developmental, and disease states in mammals, yet the conservational and regulatory features of these enhancer elements in nonmammalian vertebrates are unknown. To define SEs in zebrafish and enable sequence and functional comparisons to mouse and human SEs, we used genome-wide histone H3 lysine 27 acetylation (H3K27ac) occupancy as a primary SE delineator. Our study determined the set of SEs in pluripotent state cells and adult zebrafish tissues and revealed both similarities and differences between zebrafish and mammalian SEs. Although the total number of SEs was proportional to the genome size, the genomic distribution of zebrafish SEs differed from that of the mammalian SEs. Despite the evolutionary distance separating zebrafish and mammals and the low overall SE sequence conservation, ∼42% of zebrafish SEs were located in close proximity to orthologs that also were associated with SEs in mouse and human. Compared to their nonassociated counterparts, higher sequence conservation was revealed for those SEs that have maintained orthologous gene associations. Functional dissection of two of these SEs identified conserved sequence elements and tissue-specific expression patterns, while chromatin accessibility analyses predicted transcription factors governing the function of pluripotent state zebrafish SEs. Our zebrafish annotations and comparative studies show the extent of SE usage and their conservation across vertebrates, permitting future gene regulatory studies in several tissues.

Plants Encode a General siRNA Suppressor That Is Induced and Suppressed by Viruses.

Small RNAs play essential regulatory roles in genome stability, development, and responses to biotic and abiotic stresses in most eukaryotes. In plants, the RNaseIII enzyme DICER-LIKE1 (DCL1) produces miRNAs, whereas DCL2, DCL3, and DCL4 produce various size classes of siRNAs. Plants also encode RNASE THREE-LIKE (RTL) enzymes that lack DCL-specific domains and whose function is largely unknown. We found that virus infection induces RTL1 expression, suggesting that this enzyme could play a role in plant-virus interaction. To first investigate the biochemical activity of RTL1 independent of virus infection, small RNAs were sequenced from transgenic plants constitutively expressing RTL1. These plants lacked almost all DCL2-, DCL3-, and DCL4-dependent small RNAs, indicating that RTL1 is a general suppressor of plant siRNA pathways. In vivo and in vitro assays revealed that RTL1 prevents siRNA production by cleaving dsRNA prior to DCL2-, DCL3-, and DCL4-processing. The substrate of RTL1 cleavage is likely long-perfect (or near-perfect) dsRNA, consistent with the RTL1-insensitivity of miRNAs, which derive from DCL1-processing of short-imperfect dsRNA. Virus infection induces RTL1 mRNA accumulation, but viral proteins that suppress RNA silencing inhibit RTL1 activity, suggesting that RTL1 has evolved as an inducible antiviral defense that could target dsRNA intermediates of viral replication, but that a broad range of viruses counteract RTL1 using the same protein toolbox used to inhibit antiviral RNA silencing. Together, these results reveal yet another level of complexity in the evolutionary battle between viruses and plant defenses.

In plants, decapping prevents RDR6-dependent production of small interfering RNAs from endogenous mRNAs.

Cytoplasmic degradation of endogenous RNAs is an integral part of RNA quality control (RQC) and often relies on the removal of the 5' cap structure and their subsequent 5' to 3' degradation in cytoplasmic processing (P-)bodies. In parallel, many eukaryotes degrade exogenous and selected endogenous RNAs through post-transcriptional gene silencing (PTGS). In plants, PTGS depends on small interfering (si)RNAs produced after the conversion of single-stranded RNAs to double-stranded RNAs by the cellular RNA-dependent RNA polymerase 6 (RDR6) in cytoplasmic siRNA-bodies. PTGS and RQC compete for transgene-derived RNAs, but it is unknown whether this competition also occurs for endogenous transcripts. We show that the lethality of decapping mutants is suppressed by impairing RDR6 activity. We establish that upon decapping impairment hundreds of endogenous mRNAs give rise to a new class of rqc-siRNAs, that over-accumulate when RQC processes are impaired, a subset of which depending on RDR6 for their production. We observe that P- and siRNA-bodies often are dynamically juxtaposed, potentially allowing for cross-talk of the two machineries. Our results suggest that the decapping of endogenous RNA limits their entry into the PTGS pathway. We anticipate that the rqc-siRNAs identified in decapping mutants represent a subset of a larger ensemble of endogenous siRNAs.

Cytoplasmic and nuclear quality control and turnover of single-stranded RNA modulate post-transcriptional gene silencing in plants.

Eukaryotic RNA quality control (RQC) uses both endonucleolytic and exonucleolytic degradation to eliminate dysfunctional RNAs. In addition, endogenous and exogenous RNAs are degraded through post-transcriptional gene silencing (PTGS), which is triggered by the production of double-stranded (ds)RNAs and proceeds through short-interfering (si)RNA-directed ARGONAUTE-mediated endonucleolytic cleavage. Compromising cytoplasmic or nuclear 5'-3' exoribonuclease function enhances sense-transgene (S)-PTGS in Arabidopsis, suggesting that these pathways compete for similar RNA substrates. Here, we show that impairing nonsense-mediated decay, deadenylation or exosome activity enhanced S-PTGS, which requires host RNA-dependent RNA polymerase 6 (RDR6/SGS2/SDE1) and SUPPRESSOR OF GENE SILENCING 3 (SGS3) for the transformation of single-stranded RNA into dsRNA to trigger PTGS. However, these RQC mutations had no effect on inverted-repeat-PTGS, which directly produces hairpin dsRNA through transcription. Moreover, we show that these RQC factors are nuclear and cytoplasmic and are found in two RNA degradation foci in the cytoplasm: siRNA-bodies and processing-bodies. We propose a model of single-stranded RNA tug-of-war between RQC and S-PTGS that ensures the correct partitioning of RNA substrates among these RNA degradation pathways.

Functions of microRNAs and related small RNAs in plants.

MicroRNAs (miRNAs) and short interfering RNAs (siRNAs), 20- to 27-nt in length, are essential regulatory molecules that act as sequence-specific guides in several processes in most eukaryotes (with the notable exception of the yeast Saccharomyces cerevisiae). These processes include DNA elimination, heterochromatin assembly, mRNA cleavage and translational repression. This review focuses on the regulatory roles of plant miRNAs during development, in the adaptive response to stresses and in the miRNA pathway itself. This review also covers the regulatory roles of two classes of endogenous plant siRNAs, ta-siRNAs and nat-siRNAs, which participate in post-transcriptional control of gene expression.

Chromatin meets the cell cycle.

The cell cycle is one of the most comprehensively studied biological processes, due primarily to its significance in growth and development, and its deregulation in many human disorders. Studies using a diverse set of model organisms, including yeast, worms, flies, frogs, mammals, and plants, have greatly expanded our knowledge of the cell cycle and have contributed to the universally accepted view of how the basic cell cycle machinery is regulated. In addition to the oscillating activity of various cyclin-dependent kinase (CDK)-cyclin complexes, a plethora of proteins affecting various aspects of chromatin dynamics has been shown to be essential for cell proliferation during plant development. Furthermore, it was reported recently that core cell cycle regulators control gene expression by modifying histone patterns. This review focuses on the intimate relationship between the cell cycle and chromatin. It describes the dynamics and functions of chromatin structures throughout cell cycle progression and discusses the role of heterochromatin as a barrier against re-replication and endoreduplication. It also proposes that core plant cell cycle regulators control gene expression in a manner similar to that described in mammals. At present, our challenge in plants is to define the complete set of effectors and actors that coordinate cell cycle progression and chromatin structure and to understand better the functional interplay between these two processes.

The miRNA pathway limits AGO1 availability during siRNA-mediated PTGS defense against exogenous RNA.

In plants, most microRNAs (miRNAs) and several endogenous small interfering RNAs (siRNAs) bind to ARGONAUTE1 (AGO1) to regulate the expression of endogenous genes through post-transcriptional gene silencing (PTGS). AGO1 also participates in a siRNA-mediated PTGS defense response that thwarts exogenous RNA deriving from viruses and transgenes. Here, we reveal that plants supporting transgene PTGS exhibit increased levels of AGO1 protein. Moreover, increasing AGO1 levels either by mutating miRNA pathway components or, more specifically, by impairing miR168-directed regulation of AGO1 mRNA leads to increased PTGS efficiency, indicating that the miRNA pathway dampens the efficiency of PTGS, likely by limiting the availability of AGO1. We propose that during the transgene PTGS initiation phase, transgene siRNAs and endogenous siRNAs and miRNA compete to bind to AGO1, leading to a transient reduction in AGO1-miR168 complexes and a decline in AGO1 mRNA cleavage. The concomitant increase in AGO1 protein levels would facilitate the formation of AGO1-transgene siRNA complexes and the entry into the PTGS amplification phase. We suggest that the miRNA pathway imposes an important limitation on PTGS efficiency, which could help protect endogenous mRNAs from being routinely targeted by PTGS.

Fast and Accurate Multiplex Identification and Quantification of Seven Genetically Modified Soybean Lines Using Six-Color Digital PCR.

The proliferation of genetically modified organisms (GMOs) presents challenges to GMO testing laboratories and policymakers. Traditional methods, like quantitative real-time PCR (qPCR), face limitations in quantifying the increasing number of GMOs in a single sample. Digital PCR (dPCR), specifically multiplexing, offers a solution by enabling simultaneous quantification of multiple GMO targets. This study explores the use of the Naica six-color Crystal dPCR platform for quantifying five GM soybean lines within a single six-plex assay. Two four-color assays were also developed for added flexibility. These assays demonstrated high specificity, sensitivity (limit of detection or LOD < 25 copies per reaction) and precision (bias to an estimated copy number concentration <15%). Additionally, two approaches for the optimization of data analysis were implemented. By applying a limit-of-blank (LOB) correction, the limit of quantification (LOQ) and LOD could be more precisely determined. Pooling of reactions additionally lowered the LOD, with a two- to eight-fold increase in sensitivity. Real-life samples from routine testing were used to confirm the assays' applicability for quantifying GM soybean lines in complex samples. This study showcases the potential of the six-color Crystal dPCR platform to revolutionize GMO testing, facilitating comprehensive analysis of GMOs in complex samples.

Redundant and specific roles of the ARGONAUTE proteins AGO1 and ZLL in development and small RNA-directed gene silencing.

The Arabidopsis ARGONAUTE1 (AGO1) and ZWILLE/PINHEAD/AGO10 (ZLL) proteins act in the miRNA and siRNA pathways and are essential for multiple processes in development. Here, we analyze what determines common and specific function of both proteins. Analysis of ago1 mutants with partially compromised AGO1 activity revealed that loss of ZLL function re-establishes both siRNA and miRNA pathways for a subset of AGO1 target genes. Loss of ZLL function in ago1 mutants led to increased AGO1 protein levels, whereas AGO1 mRNA levels were unchanged, implicating ZLL as a negative regulator of AGO1 at the protein level. Since ZLL, unlike AGO1, is not subjected to small RNA-mediated repression itself, this cross regulation has the potential to adjust RNA silencing activity independent of feedback dynamics. Although AGO1 is expressed in a broader pattern than ZLL, expression of AGO1 from the ZLL promoter restored transgene PTGS and most developmental defects of ago1, whereas ZLL rescued only a few AGO1 functions when expressed from the AGO1 promoter, suggesting that the specific functions of AGO1 and ZLL are mainly determined by their protein sequence. Protein domain swapping experiments revealed that the PAZ domain, which in AGO1 is involved in binding small RNAs, is interchangeable between both proteins, suggesting that this common small RNA-binding domain contributes to redundant functions. By contrast, the conserved MID and PIWI domains, which are involved in 5'-end small RNA selectivity and mRNA cleavage, and the non-conserved N-terminal domain, to which no function has been assigned, provide specificity to AGO1 and ZLL protein function.

ARGONAUTE 1 homeostasis invokes the coordinate action of the microRNA and siRNA pathways.

ARGONAUTE 1 (AGO1) slices endogenous messenger RNAs (mRNAs) during both microRNA (miRNA)- and short interfering RNA (siRNA)-guided post-transcriptional silencing. We have previously reported that AGO1 homeostasis is maintained through the repressive action of miR168 on AGO1 mRNA and the stabilizing effect of AGO1 protein on miR168, but siRNA-mediated AGO1 regulation has not been reported. Here, we show that AGO1-derived siRNAs trigger RNA DEPENDENT RNA POLYMERASE 6 (RDR6)-, SUPPRESSOR OF GENE SILENCING 3 (SGS3)- and SILENCING DEFECTIVE 5 (SDE5)-dependent AGO1 silencing, which also requires DICER-LIKE 2 (DCL2) and DCL4. By varying the efficacy of miR168-guided AGO1 mRNA cleavage, we show that siRNA-mediated AGO1 silencing depends on correct miRNA targeting, pointing to coordinated regulatory actions of the miRNA and siRNA pathways during the maintenance of AGO1 homeostasis. Finally, our results reveal that dcl2, dcl3 and dcl4 mutations similarly affect post-transcriptional gene silencing (PTGS) mediated by a sense transgene and PTGS mediated by inverted repeats, validating the branched pathway model proposed previously.

6-Color Crystal Digital PCRTM for the High-Plex Detection of EGFR Mutations in Non-Small Cell Lung Cancer.

The profiling of EGFR mutations, the most common genetic alterations in non-small cell lung cancer (NSCLC) predictive of targeted therapy efficacy, is crucial to anticipate the patient response to EGFR tyrosine kinase inhibitors. Here, we introduce the naica® system for 6-color Crystal Digital PCRTM and describe in detail a standardized workflow for the multiplexed, single-assay detection of the 19 most prevalent sensitizing and resistance EGFR mutations in both tumor and circulating tumor DNA (ctDNA) samples. Two major advantages of the 6-color multiplexing system over current digital PCR systems are the rapid time to results, and the large quantity of mutational information obtained per patient sample, rendering the 6-color system highly cost effective. The 6-color Crystal Digital PCRTM technology enables highly sensitive and efficient therapeutic monitoring through liquid biopsy, resulting in the early detection of treatment resistance. While the assay presented here specifically addresses EGFR mutation status monitoring in NSCLC patients, 6-color Crystal Digital PCRTM assays are flexible and evolutive in design. As such, 6-color detection assays can be optimized to monitor mutations associated with a range of cancers and other genetic diseases, as well as to detect genetic changes beyond the oncology and human health domains.

MicroRNA maturation and action--the expanding roles of ARGONAUTEs.

MicroRNAs are endogenously produced 21-nt riboregulators that associate with ARGONAUTE (AGO) proteins to direct mRNA cleavage or repress translation of complementary RNAs. In addition to protein-coding gene repression, miRNA-directed regulation of non-protein-coding transcripts can incite production of trans-acting siRNA (tasiRNA) populations that themselves direct mRNA repression. Arabidopsis encodes 10 AGO proteins among which, AGO1, AGO7, and AGO10 have been implicated in miRNA-guided gene repression in vivo. Recent work has shown that AGO proteins discriminate their associated small RNA populations on the basis of size and 5'-terminal nucleotide identity, extending the roles of AGO proteins beyond small RNA action. Our expanding appreciation of miRNA-directed regulation during plant development and stress adaptations has placed miRNAs at the forefront of plant biology.

MicroRNA-directed regulation: to cleave or not to cleave.

Gene expression is regulated by transcriptional and post-transcriptional pathways, which are crucial for optimizing gene output and for coordinating cellular programs. MicroRNAs (miRNAs) regulate gene expression networks necessary for proper development, cell viability and stress responses. In plants and animals, 20-24-nt miRNAs direct cleavage and translational repression of partially complementary mRNA target transcripts, through conserved ARGONAUTE proteins. In plants, certain miRNAs indirectly regulate developmental programs by instigating the production of small interfering RNAs (siRNAs). In addition, non-cleavable plant miRNA targets sequester miRNAs, thus regulating miRNA availability. This review summarizes the complexities and diversity of plant miRNA-directed gene regulatory mechanisms and highlights the use of miRNAs for the specific knockdown of gene expression in plants.

Author Correction: In-cell identification and measurement of RNA-protein interactions.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

In-cell identification and measurement of RNA-protein interactions.

Regulatory RNAs exert their cellular functions through RNA-binding proteins (RBPs). Identifying RNA-protein interactions is therefore key for a molecular understanding of regulatory RNAs. To date, RNA-bound proteins have been identified primarily through RNA purification followed by mass spectrometry. Here, we develop incPRINT (in cell protein-RNA interaction), a high-throughput method to identify in-cell RNA-protein interactions revealed by quantifiable luminescence. Applying incPRINT to long noncoding RNAs (lncRNAs), we identify RBPs specifically interacting with the lncRNA Firre and three functionally distinct regions of the lncRNA Xist. incPRINT confirms previously known lncRNA-protein interactions and identifies additional interactions that had evaded detection with other approaches. Importantly, the majority of the incPRINT-defined interactions are specific to individual functional regions of the large Xist transcript. Thus, we present an RNA-centric method that enables reliable identification of RNA-region-specific RBPs and is applicable to any RNA of interest.

Partially redundant functions of Arabidopsis DICER-like enzymes and a role for DCL4 in producing trans-acting siRNAs.

Arabidopsis encodes four DICER-like (DCL) proteins. DCL1 produces miRNAs, DCL2 produces some virus-derived siRNAs, and DCL3 produces endogenous RDR2-dependent siRNAs, but the role of DCL4 is unknown. We show that DCL4 is the primary processor of endogenous RDR6-dependent trans-acting siRNAs (tasiRNAs). Molecular and phenotypic analyses of all dcl double mutants also revealed partially compensatory functions among DCL proteins. In the absence of DCL4, some RDR6-dependent siRNAs were produced by DCL2 and DCL3, and in the absence of DCL3, some RDR2-dependent siRNAs were produced by DCL2 and DCL4. Consistent with partial redundancies, dcl2 and dcl3 mutants developed normally, whereas dcl4 and dcl3 dcl4 mutants had weak and severe rdr6 phenotypes, respectively, and increased tasiRNA target mRNA accumulation. After three generations, dcl3 dcl4 and dcl2 dcl3 mutants exhibited stochastic developmental phenotypes, some of which were lethal, likely owing to the accumulated loss of heterochromatic siRNA-directed marks. dcl1 dcl3 and dcl1 dcl4, but not dcl1 dcl2 mutants, had phenotypes more severe than dcl1 mutants, consistent with DCL1, DCL3, and DCL4 acting as the primary processors of the three respective classes of endogenous silencing RNAs and DCL2 acting to produce viral-derived siRNAs and as an alternative DCL for endogenous siRNA production.

MicroRNA degradation by a conserved target RNA regulates animal behavior.

microRNAs (miRNAs) repress target transcripts through partial complementarity. By contrast, highly complementary miRNA-binding sites within viral and artificially engineered transcripts induce miRNA degradation in vitro and in cell lines. Here, we show that a genome-encoded transcript harboring a near-perfect and deeply conserved miRNA-binding site for miR-29 controls zebrafish and mouse behavior. This transcript originated in basal vertebrates as a long noncoding RNA (lncRNA) and evolved to the protein-coding gene NREP in mammals, where the miR-29-binding site is located within the 3' UTR. We show that the near-perfect miRNA site selectively triggers miR-29b destabilization through 3' trimming and restricts its spatial expression in the cerebellum. Genetic disruption of the miR-29 site within mouse Nrep results in ectopic expression of cerebellar miR-29b and impaired coordination and motor learning. Thus, we demonstrate an endogenous target-RNA-directed miRNA degradation event and its requirement for animal behavior.

MicroRNA regulation of NAC-domain targets is required for proper formation and separation of adjacent embryonic, vegetative, and floral organs.

BACKGROUND: MicroRNAs (miRNAs) are approximately 21 nucleotide (nt) RNAs that regulate gene expression in plants and animals. Most known plant miRNAs target transcription factors that influence cell fate determination, and biological functions of miRNA-directed regulation have been reported for four of 15 known microRNA gene families: miR172, miR159, miR165, and miR168. Here, we identify a developmental role for miR164-directed regulation of NAC-domain genes, which encode a family of transcription factors that includes CUP-SHAPED COTYLEDON1 (CUC1) and CUC2. RESULTS: Expression of a miR164-resistant version of CUC1 mRNA from the CUC1 promoter causes alterations in Arabidopsis embryonic, vegetative, and floral development, including cotyledon orientation defects, reduction of rosette leaf petioles, dramatically misshapen rosette leaves, one to four extra petals, and one or two missing sepals. Reciprocally, constitutive overexpression of miR164 recapitulates cuc1 cuc2 double mutant phenotypes, including cotyledon and floral organ fusions. miR164 overexpression also leads to phenotypes not previously observed in cuc1 cuc2 mutants, including leaf and stem fusions. These likely reflect the misregulation of other NAC-domain mRNAs, including NAC1, At5g07680, and At5g61430, for which miR164-directed cleavage products were detected. CONCLUSIONS: These results demonstrate that miR164-directed regulation of CUC1 is necessary for normal embryonic, vegetative, and floral development. They also show that proper miR164 dosage or localization is required for separation of adjacent embryonic, vegetative, and floral organs, thus implicating miR164 as a common regulatory component of the molecular circuitry that controls the separation of different developing organs and thereby exposes a posttranscriptional layer of NAC-domain gene regulation during plant development.

The amplicon-plus system for high-level expression of transgenes in plants.

Many biotechnological applications require high-level expression of transgenes in plants. One strategy to achieve this goal was the production of potato virus X (PVX) "amplicon" lines: transgenic lines that encode a replicating RNA virus vector carrying a gene of interest. The idea was that transcription of the amplicon transgene would initiate viral RNA replication and gene expression, resulting in very high levels of the gene product of interest. This approach failed, however, because every amplicon transgene, in both tobacco and Arabidopsis thaliana, was subject to post-transcriptional gene silencing (PTGS). In PTGS, the transgene is transcribed but the transcripts fail to accumulate as a result of sequence-specific targeting and destruction. Even though the amplicon locus is silenced, the level of beta-glucuronidase (GUS) activity in a PVX/GUS line is similar to that in some transgenic lines expressing GUS from a conventional (not silenced) GUS locus. This result suggested that the very high levels of expression originally envisioned for amplicons could be achieved if PTGS could be overcome and if the resulting plants did not suffer from severe viral disease. Here we report that high-level transgene expression can be achieved by pairing the amplicon approach with the use of a viral suppressor of PTGS, tobacco etch virus (TEV) helper component proteinase (HC-Pro). Leaves of mature tobacco plants co-expressing HC-Pro and a PVX/GUS amplicon accumulate GUS to approximately 3% of total protein. Moreover, high-level expression occurs without viral symptoms and, when HC-Pro is expressed from a mutant transgene, without detrimental developmental phenotypes.

MicroRNAs: something important between the genes.

Non-coding small endogenous RNAs, of 21-24 nucleotides in length, have recently emerged as important regulators of gene expression in both plants and animals. At least three categories of small RNAs exist in plants: short interfering RNAs (siRNAs) deriving from viruses or transgenes and mediating virus resistance or transgene silencing via RNA degradation; siRNAs deriving from transposons or transgene promoters and controlling transposon and transgene silencing probably via chromatin changes; and microRNAs (miRNAs) deriving from intergenic regions of the genome and regulating the expression of endogenous genes either by mRNA cleavage or translational repression. The disruption of miRNA-mediated regulation causes developmental abnormalities in plants, demonstrating that miRNAs play an important role in the regulation of developmental decisions.