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.

6-Color Crystal Digital PCR 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 PCR 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.

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.

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.

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.

LncRNAs in vertebrates: advances and challenges.

Beyond the handful of classic and well-characterized long noncoding RNAs (lncRNAs), more recently, hundreds of thousands of lncRNAs have been identified in multiple species including bacteria, plants and vertebrates, and the number of newly annotated lncRNAs continues to increase as more transcriptomes are analyzed. In vertebrates, the expression of many lncRNAs is highly regulated, displaying discrete temporal and spatial expression patterns, suggesting roles in a wide range of developmental processes and setting them apart from classic housekeeping ncRNAs. In addition, the deregulation of a subset of these lncRNAs has been linked to the development of several diseases, including cancers, as well as developmental anomalies.

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.

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.

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.

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.

A novel fry1 allele reveals the existence of a mutant phenotype unrelated to 5'->3' exoribonuclease (XRN) activities in Arabidopsis thaliana roots.

Background: Mutations in the FRY1/SAL1 Arabidopsis locus are highly pleiotropic, affecting drought tolerance, leaf shape and root growth. FRY1 encodes a nucleotide phosphatase that in vitro has inositol polyphosphate 1-phosphatase and 3',(2'),5'-bisphosphate nucleotide phosphatase activities. It is not clear which activity mediates each of the diverse biological functions of FRY1 in planta.

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.

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.

Criteria for annotation of plant MicroRNAs.

MicroRNAs (miRNAs) are approximately 21 nucleotide noncoding RNAs produced by Dicer-catalyzed excision from stem-loop precursors. Many plant miRNAs play critical roles in development, nutrient homeostasis, abiotic stress responses, and pathogen responses via interactions with specific target mRNAs. miRNAs are not the only Dicer-derived small RNAs produced by plants: A substantial amount of the total small RNA abundance and an overwhelming amount of small RNA sequence diversity is contributed by distinct classes of 21- to 24-nucleotide short interfering RNAs.

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.

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.

Arabidopsis FIERY1, XRN2, and XRN3 are endogenous RNA silencing suppressors.

The eukaryotic defense response posttranscriptional gene silencing (PTGS) is directed by short-interfering RNAs and thwarts invading nucleic acids via the RNA slicing activity of conserved ARGONAUTE (AGO) proteins. PTGS can be counteracted by exogenous or endogenous suppressors, including the cytoplasmic exoribonuclease XRN4, which also degrades microRNA (miRNA)-guided mRNA cleavage products but does not play an obvious role in development. Here, we show that the nuclear exoribonucleases XRN2 and XRN3 are endogenous PTGS suppressors.

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.

AGO1 homeostasis entails coexpression of MIR168 and AGO1 and preferential stabilization of miR168 by AGO1.

Arabidopsis ARGONAUTE1 (AGO1) encodes the RNA slicer enzyme of the microRNA (miRNA) pathway and is regulated by miR168-programmed, AGO1-catalyzed mRNA cleavage. Here, we describe two additional regulatory processes required for AGO1 homeostasis: transcriptional coregulation of MIR168 and AGO1 genes, and posttranscriptional stabilization of miR168 by AGO1. Disrupting any of these regulatory processes by using mutations or transgenes disturbs a proper functioning of the miRNA pathway.

Ectopic DICER-LIKE1 expression in P1/HC-Pro Arabidopsis rescues phenotypic anomalies but not defects in microRNA and silencing pathways.

Expression of the viral silencing suppressor P1/HC-Pro in plants causes severe developmental anomalies accompanied by defects in both short interfering RNA (siRNA) and microRNA (miRNA) pathways. P1/HC-Pro transgenic lines fail to accumulate the siRNAs that mediate RNA silencing and are impaired in both miRNA processing and function, accumulating abnormally high levels of miRNA/miRNA* processing intermediates as well as miRNA target messages. Both miRNA and RNA silencing pathways require participation of DICER-LIKE (DCL) ribonuclease III-like enzymes.

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).

MicroRNA-directed regulation of Arabidopsis AUXIN RESPONSE FACTOR17 is essential for proper development and modulates expression of early auxin response genes.

The phytohormone auxin plays critical roles during plant growth, many of which are mediated by the auxin response transcription factor (ARF) family. MicroRNAs (miRNAs), endogenous 21-nucleotide riboregulators, target several mRNAs implicated in auxin responses. miR160 targets ARF10, ARF16, and ARF17, three of the 23 Arabidopsis thaliana ARF genes.

Endogenous trans-acting siRNAs regulate the accumulation of Arabidopsis mRNAs.

Here we describe a set of endogenous short interfering RNAs (siRNAs) in Arabidopsis, some of which direct the cleavage of endogenous mRNAs. These siRNAs correspond to both sense and antisense strands of a noncoding RNA (At2g27400) that apparently is converted to double-stranded RNA and then processed in 21 nt increments. These siRNAs differ from previously described regulatory small RNAs in two respects.