RNA Hairpin Research Articles

Specific Nucleic Acid Chaperone Activity of HIV-1 Nucleocapsid Protein Deduced from Hairpin Unfolding.

RNA and DNA hairpin formation and disruption play key regulatory roles in a variety of cellular processes. The 59-nucleotide transactivation response (TAR) RNA hairpin facilitates the production of full-length transcripts of the HIV-1 genome. Yet the stability of this long, irregular hairpin becomes a liability during reverse transcription as 24 base pairs must be disrupted for strand transfer. Retroviral nucleocapsid (NC) proteins serve as nucleic acid chaperones that have been shown to both destabilize the TAR hairpin and facilitate strand annealing with its complementary DNA sequence. Yet it has remained difficult to elucidate the way NC targets and dramatically destabilizes this hairpin while only weakly affecting the annealed product. In this work, we used optical tweezers to measure the stability of TAR and found that adding NC destabilized the hairpin and simultaneously caused a distinct change in both the height and location of the energy barrier. This data was matched to an energy landscape predicted from a simple theory of definite base pair destabilization. Comparisons revealed the specific binding sites found by NC along the irregular TAR hairpin. Furthermore, specific binding explained both the unusual shift in the transition state and the much weaker effect on the annealed product. These experiments illustrate a general method of energy landscape transformation that exposes important physical insights.

Control of western corn rootworm via RNAi traits in maize: lethal and sublethal effects of Sec23 dsRNA.

RNA interference (RNAi) triggered by maize plants expressing RNA hairpins against specific western corn rootworm (WCR) transcripts have proven to be effective at controlling this pest. To provide robust crop protection, mRNA transcripts targeted by double-stranded RNA must be sensitive to knockdown and encode essential proteins. Using WCR adult feeding assays, we identified Sec23 as a highly lethal RNAi target. Sec23 encodes a coatomer protein, a component of the coat protein (COPII) complex that mediates ER-Golgi transport. The lethality detected in WCR adults was also observed in early instar larvae, the life stage causing most of the crop damage, suggesting that WCR adults can serve as an alternative to larvae for dsRNA screening. Surprisingly, over 85% transcript inhibition resulted in less than 40% protein knockdown, suggesting that complete protein knockdown is not necessary for Sec23 RNAi-mediated mortality. The efficacy of Sec23 dsRNA for rootworm control was confirmed in planta; T0 maize events carrying rootworm Sec23 hairpin transgenes showed high levels of root protection in greenhouse assays. A reduction in larval survival and weight were observed in the offspring of WCR females exposed to Sec23 dsRNA LC25 in diet bioassays. We describe Sec23 as RNAi target for in planta rootworm control. High mortality in exposed adult and larvae and moderate sublethal effects in the offspring of females exposed to Sec23 dsRNA LC25 , suggest the potential for field application of this RNAi trait and the need to factor in responses to sublethal exposure into insect resistance management programs. © 2019 Society of Chemical Industry.

Efficient methods for determining folding free energies in single-molecule pulling experiments

The remarkable accuracy and versatility of single-molecule techniques make new measurements that are not feasible in bulk assays possible. Among these, the precise estimation of folding free energies using fluctuation theorems in nonequilibrium pulling experiments has become a benchmark in modern biophysics. In practice, the use of fluctuation relations to determine free energies requires a thorough evaluation of the usually large energetic contributions caused by the elastic deformation of the different elements of the experimental setup (such as the optical trap, the molecular linkers and the stretched-unfolded polymer). We review and describe how to optimally estimate such elastic energy contributions to extract folding free energies, using DNA and RNA hairpins as model systems pulled by laser optical tweezers. The methodology is generally applicable to other force-spectroscopy techniques and molecular systems.

Structural transitions in the RNA 7SK 5' hairpin and their effect on HEXIM binding.

7SK RNA, as part of the 7SK ribonucleoprotein complex, is crucial to the regulation of transcription by RNA-polymerase II, via its interaction with the positive transcription elongation factor P-TEFb. The interaction is induced by binding of the protein HEXIM to the 5′ hairpin (HP1) of 7SK RNA. Four distinct structural models have been obtained experimentally for HP1. Here, we employ computational methods to investigate the relative stability of these structures, transitions between them, and the effects of mutations on the observed structural ensembles. We further analyse the results with respect to mutational binding assays, and hypothesize a mechanism for HEXIM binding. Our results indicate that the dominant structure in the wild type exhibits a triplet involving the unpaired nucleotide U40 and the base pair A43-U66 in the GAUC/GAUC repeat. This conformation leads to an open major groove with enough potential binding sites for peptide recognition. Sequence mutations of the RNA change the relative stability of the different structural ensembles. Binding affinity is consequently lost if these changes alter the dominant structure.

RNA silencing of South African cassava mosaic virus in transgenic cassava expressing AC1/AC4 hp- RNA induces tolerance

Cassava mosaic disease (CMD), caused by geminiviruses, is a major hurdle to cassava production. Due to the heterozygous nature of cassava, breeding for virus resistance is difficult, but cassava has been shown to be a good candidate for genetic engineering using RNA interference (RNAi). T This study reports on the ability of a transgene-derived RNA hairpin, homologous to an overlapping region of the SACMV replication associated protein and putative virus suppressor of silencing protein (AC1/AC4), to confer tolerance in the CMD-susceptible model cassava cultivar 60444. Three of the fourteen transgenic lines expressing SACMV AC1/AC4 hairpin-derived siRNAs showed decreased symptoms and viral loads compared to untransformed control plants. Expression of SACMV AC1/AC4 homologous siRNAs showed that this tolerance is most likely associated with post-transcriptional gene silencing of the virus. This is the first report of targeting the overlapping AC1 and AC4 genes of SACMV conferring CMD tolerance in cassava.

3D structure stability of the HIV-1 TAR RNA in ion solutions: A coarse-grained model study.

As an extremely common structural motif, RNA hairpins with bulge loops [e.g., the human immunodeficiency virus type 1 (HIV-1) transactivation response (TAR) RNA] can play essential roles in normal cellular processes by binding to proteins and small ligands, which could be very dependent on their three-dimensional (3D) structures and stability. Although the structures and conformational dynamics of the HIV-1 TAR RNA have been extensively studied, there are few investigations on the thermodynamic stability of the TAR RNA, especially in ion solutions, and the existing studies also have some divergence on the unfolding process of the RNA. Here, we employed our previously developed coarse-grained model with implicit salt to predict the 3D structure, stability, and unfolding pathway for the HIV-1 TAR RNA over a wide range of ion concentrations. As compared with the extensive experimental/theoretical results, the present model can give reliable predictions on the 3D structure stability of the TAR RNA from the sequence. Based on the predictions, our further comprehensive analyses on the stability of the TAR RNA as well as its variants revealed that the unfolding pathway of an RNA hairpin with a bulge loop is mainly determined by the relative stability between different states (folded state, intermediate state, and unfolded state) and the strength of the coaxial stacking between two stems in folded structures, both of which can be apparently modulated by the ion concentrations as well as the sequences.

ATP1B3 cooperates with BST-2 to promote hepatitis B virus restriction.

Increasing evidence indicates ATP1B3, one of the regulatory subunits of Na+/K+‐ATPase, is involved in numerous viral propagations, such as HIV and EV71. However, the function and mechanism of ATP1B3 on hepatitis B virus (HBV) propagation is unknown. Here, we demonstrated that ATP1B3 overexpression reduced the quantity of hepatitis B surface antigen (HBsAg) and hepatitis B e antigen (HBeAg) in supernatants of HBV expression plasmids cotransfected HepG2 cells. Correspondingly, small interfering RNA and short hairpin RNA mediated ATP1B3 silencing promoted HBsAg and HBeAg expression in the supernatants of HBV expression plasmids transfected HepG2 cells. Mechanically, we reported that ATP1B3 expression could activate nuclear factor‐κB (NF‐κB) pathway by inducing the expression, phosphorylation, and nuclear import of P65 for the first time. And NF‐κB inhibitor (Bay11) impaired the restraint of ATP1B3 on HBV replication. This counteraction effect of Bay11 proved that ATP1B3‐induced NF‐κB activation was crucial for HBV restriction. Accordingly, we observed that anti‐HBV factors interferon‐α (IFN‐α) and interleukin‐6 (IL‐6) production were increased in HepG2 cells after the NF‐κB activation. It suggested that ATP1B3 suppressed HBsAg and HBeAg by NF‐κB/IFN‐α and NF‐κB/IL‐6 axis. Further experiments proved that ATP1B3 overexpression induced anti‐HBV factor BST‐2 expression by NF‐κB/IFN‐α axis in HepG2 cells but not HEK293T cells, and ATP1B3 silencing downregulated BST‐2 messenger RNA level in HepG2 cells. As an HBV restriction factor, BST‐2 cooperated with ATP1B3 to antagonize HBsAg but not HBeAg in HepG2 cells. Our work identified ATP1B3 as a novel candidate of HBV restrictor with unrevealed mechanism and we highlighted it might serve as a potential therapeutic molecule for HBV infection.

FSATOOL: A useful tool to do the conformational sampling and trajectory analysis work for biomolecules.

Reliable conformational sampling and trajectory analysis are always important to the study of the folding or binding mechanisms of biomolecules. Generally, one has to prepare many complicated parameters and follow a lot of steps to obtain the final data. The whole process is too complicated to new users. In this article, we provide a convenient and user-friendly tool that is compatible to AMBER, called fast sampling and analysis tool (FSATOOL). FSATOOL has some useful features. First and the most important, the whole work is extremely simplified into two steps, one is the fast sampling procedure and the other is the trajectory analysis procedure. Second, it contains several powerful sampling methods for the simulation on graphics process unit, including our previous mixing replica exchange molecular dynamics method. The method combines the advantages of the biased and unbiased simulations. Finally, it extracts the dominant transition pathways automatically from the folding network by Markov state model. Users do not need to do the tedious intermediate steps by hand. To illustrate the usage of FSATOOL in practice, we perform one simulation for a RNA hairpin in explicit solvent. All the results are presented. © 2019 Wiley Periodicals, Inc.

High-yield fabrication of DNA and RNA constructs for single molecule force and torque spectroscopy experiments.

Single molecule biophysics experiments have enabled the observation of biomolecules with a great deal of precision in space and time, e.g. nucleic acids mechanical properties and protein–nucleic acids interactions using force and torque spectroscopy techniques. The success of these experiments strongly depends on the capacity of the researcher to design and fabricate complex nucleic acid structures, as the outcome and the yield of the experiment also strongly depend on the high quality and purity of the final construct. Though the molecular biology techniques involved are well known, the fabrication of nucleic acid constructs for single molecule experiments still remains a difficult task. Here, we present new protocols to generate high quality coilable double-stranded DNA and RNA, as well as DNA and RNA hairpins with ∼500–1000 bp long stems. Importantly, we present a new approach based on single-stranded DNA (ssDNA) annealing and we use magnetic tweezers to show that this approach simplifies the fabrication of complex DNA constructs, such as hairpins, and converts more efficiently the input DNA into construct than the standard PCR-digestion-ligation approach. The protocols we describe here enable the design of a large range of nucleic acid construct for single molecule biophysics experiments.

AAV-Mediated Expression of AP-1-Neutralizing RNA Decoy Oligonucleotides Attenuates Transplant Vasculopathy in Mouse Aortic Allografts

Transplant vasculopathy (TV), characterized by obstructive lesions in affected vessels, represents one of the long-term complications of cardiac transplantation. Activation of the transcription factor activator protein-1 (AP-1) is implicated in smooth muscle cell (SMC) phenotypic switch from contractile to synthetic function, increasing the migration and proliferation rate of these cells. We hypothesize that adeno-associated virus (AAV)-mediated delivery of an RNA hairpin AP-1 decoy oligonucleotide (dON) might effectively ameliorate TV severity in a mouse aortic allograft model. Aortic allografts from DBA/2 mice ex vivo transduced with modified AAV9-SLR carrying a targeting peptide within the capsid surface were transplanted into the infrarenal aorta of C57BL/6 mice. Cyclosporine A (10 mg/kg BW) was administered daily. AP-1 dONs were intracellularly expressed in the graft tissue as small hairpin RNA proved by fluorescent in situ hybridization. Explantation after 30 days and histomorphometric evaluation revealed that AP-1 dON treatment significantly reduced intima-to-media ratio by 41.5% (p < 0.05) in the grafts. In addition, expression of adhesion molecules, cytokines, as well as numbers of proliferative SMCs, matrix metalloproteinase-9-positive cells, and inflammatory cell infiltration were significantly decreased in treated aortic grafts. Our findings demonstrate the feasibility, efficacy, and specificity of the anti-AP-1 RNA dON approach for the treatment of allograft vasculopathy in an animal model. Moreover, the AAV-based approach in general provides the possibility to achieve a prolonged delivery of nucleic-acids-based therapeutics in to the blood vessel wall.

Detailed insight into the dynamics of the initial phases of de novo RNA-directed DNA methylation in plant cells

BackgroundMethylation of cytosines is an evolutionarily conserved epigenetic mark that is essential for the control of chromatin activity in many taxa. It acts mainly repressively, causing transcriptional gene silencing. In plants, de novo DNA methylation is established mainly by RNA-directed DNA-methylation pathway. Even though the protein machinery involved is relatively well-described, the course of the initial phases remains covert.ResultsWe show the first detailed description of de novo DNA-methylation dynamics. Since prevalent plant model systems do not provide the possibility to collect homogenously responding material in time series with short intervals, we developed a convenient system based on tobacco BY-2 cell lines with inducible production of siRNAs (from an RNA hairpin) guiding the methylation machinery to the CaMV 35S promoter controlling GFP reporter. These lines responded very synchronously, and a high level of promoter-specific siRNAs triggered rapid promoter methylation with the first increase observed already 12 h after the induction. The previous presence of CG methylation in the promoter did not affect the methylation dynamics. The individual cytosine contexts reacted differently. CHH methylation peaked at about 80% in 2 days and then declined, whereas CG and CHG methylation needed more time with CHG reaching practically 100% after 10 days. Spreading of methylation was only minimal outside the target region in accordance with the absence of transitive siRNAs. The low and stable proportion of 24-nt siRNAs suggested that Pol IV was not involved in the initial phases.ConclusionsOur results show that de novo DNA methylation is a rapid process initiated practically immediately with the appearance of promoter-specific siRNAs and independently of the prior presence of methylcytosines at the target locus. The methylation was precisely targeted, and its dynamics varied depending on the cytosine sequence context. The progressively increasing methylation resulted in a smooth, gradual inhibition of the promoter activity, which was entirely suppressed in 2 days.

Incorporating G-C Pair-Recognizing Guanidinium into PNAs for Sequence and Structure Specific Recognition of dsRNAs over dsDNAs and ssRNAs.

Recognition of RNAs under physiological conditions is important for the development of chemical probes and therapeutic ligands. Nucleobase-modified dsRNA-binding PNAs (dbPNAs) are promising for the recognition of dsRNAs in a sequence and structure specific manner under near-physiological conditions. Guanidinium is often present in proteins and small molecules for the recognition of G bases in nucleic acids, in cell-penetrating carriers, and in bioactive drug molecules, which might be due to the fact that guanidinium is amphiphilic and has unique hydrogen bonding and stacking properties. We hypothesized that a simple guanidinium moiety can be directly incorporated into PNAs to facilitate enhanced molecular recognition of G-C pairs in dsRNAs and improved bioactivity. We grafted a guanidinium moiety directly into a PNA monomer (designated as R) using a two-carbon linker as guided by computational modeling studies. The synthetic scheme of the PNA R monomer is relatively simple compared to that of the previously reported L monomer. We incorporated the R residue into various dbPNAs for binding studies. dbPNAs incorporated with R residues are excellent in sequence specifically recognizing G-C pairs in dsRNAs over dsDNA and ssRNAs. We demonstrated that the R residue is compatible with unmodified T and C and previously developed modified L and Q residues in dbPNAs for targeting model dsRNAs, the influenza A viral panhandle duplex structure, and the HIV-1 frameshift site RNA hairpin. Furthermore, R residues enhance the cellular uptake of PNAs.

Structure-guided design of immunomodulatory RNAs specifically targeting the cytoplasmic viral RNA sensor RIG-I.

The cytoplasmic immune sensor RIG-I detects viral RNA and initiates an antiviral immune response upon activation. It has become a potential target for vaccination and immunotherapies. To develop the smallest but potent immunomodulatory RNA (immRNAs) species, we performed structure-guided RNA design and used biochemical, structural, and cell-based methods to select and characterize the immRNAs. We demonstrated that inserting guanosine at position 9 to the 10mer RNA hairpin (3p10LG9) activates RIG-I more robustly than the parental RNA. 3p10LG9 interacts strongly with the RIG-I helicase-CTD RNA sensing module and disrupts the auto-inhibitory interaction between the HEL2i and CARDs domains. We further showed that 3p10LA9 has a stronger cellular activity than 3p10LG9. Collectively, purine insertion at position 9 of the immRNA species triggered more robust activation of RIG-1.

Exploring the Potential of Small Molecule-Based Therapeutic Approaches for Targeting Trinucleotide Repeat Disorders.

In recent years, neurological disorders have globally become a leading cause of disability and death. Neurological disorders are very common in both high- and low-income countries, and the number of patients is predicted to increase in the coming decades. Disorders caused by the expanded trinucleotide repeats (CAG, CGG, CCG, CTG, CUG, GAA, and GCN) in the genome, also described as trinucleotide repeat expansion disorders (TREDs), comprise of the major class of neurological diseases. Various TREDs have different modes of pathogenesis, but the severity and time of onset of disease depends on the trinucleotide repeat numbers. Numerous therapeutic strategies, including symptomatic treatment, blockage of mutant protein synthesis, targeting the toxic protein aggregates and degradation of RNA transcripts have been developed for the treatment of these diseases. However, various limitations to these therapeutic strategies have been reported, and therefore, researchers are exploring different avenues of therapeutics development. One of the recent developments include targeting the expanded repeats with small molecules. Small molecule binds with the secondary/tertiary structure of RNA (like bulges, loops, and hairpins) irrespective of its sequences. Altogether, small molecule-based therapeutics may have the advantage over others to be able to overcome the hurdles of the blood-brain barrier, poor absorption, and allergic reactions. In this review, we have summarized various TREDs and envisage the potential of small molecule-based therapeutics for targeting these hitherto incurable neurological disorders.

In silico structure analysis of alphaviral RNA genomes shows diversity in the evasion of IFIT1-mediated innate immunity

The IFIT (interferon-induced proteins with tetratricopeptide repeats) family constitutes a major arm of the antiviral function of type I interferon (IFN). Human IFIT1, the earliest discovered member of this family, inhibits several viruses of positivestrand RNA genome. IFIT1 specifically recognizes single-stranded RNAwith canonical 7-methylguanylate cap at the 50 end (Cap0), and inhibits their translation by competing with eIF4E (eukaryotic initiation factor 4E), an essential factor for 50Cap recognition. Recently, a novel viral mechanism of IFIT1 suppression was reported, in which an RNA hairpin in the 50 untranslated region (50UTR) of the viral genome prevented recognition by IFIT1 and enhanced virus growth. Here, I have analyzed the in silico predicted structures in the 50UTR of the genomes of the Alphaviruses, a large group of enveloped RNA virus with positive-sense single-stranded genome. The results uncovered a large ensemble of RNA secondary structures of diverse size and shape in the different viruses, which showed little correspondence to the phylogeny of the viruses. Unexpectedly, the 50UTR of several viral genomes in this family did not fold into any structure, suggesting either their extreme sensitivity to IFIT1 or the existence of alternative viral mechanisms of subverting IFIT1 function.

The RNA hairpin binder TRIM71 modulates alternative splicing by repressing MBNL1.

TRIM71/LIN-41, a phylogenetically conserved regulator of development, controls stem cell fates. Mammalian TRIM71 exhibits both RNA-binding and protein ubiquitylation activities, but the functional contribution of either activity and relevant primary targets remain poorly understood. Here, we demonstrate that TRIM71 shapes the transcriptome of mouse embryonic stem cells (mESCs) predominantly through its RNA-binding activity. We reveal that TRIM71 binds targets through 3' untranslated region (UTR) hairpin motifs and that it acts predominantly by target degradation. TRIM71 mutations implicated in etiogenesis of human congenital hydrocephalus impair target silencing. We identify a set of primary targets consistently regulated in various human and mouse cell lines, including MBNL1 (Muscleblind-like protein 1). MBNL1 promotes cell differentiation through regulation of alternative splicing, and we demonstrate that TRIM71 promotes embryonic splicing patterns through MBNL1 repression. Hence, repression of MBNL1-dependent alternative splicing may contribute to TRIM71's function in regulating stem cell fates.

Deprivation of Muscleblind-Like Proteins Causes Deficits in Cortical Neuron Distribution and Morphological Changes in Dendritic Spines and Postsynaptic Densities.

Myotonic dystrophy (Dystrophia Myotonica; DM) is the most common adult-onset muscular dystrophy and its brain symptoms seriously affect patients’ quality of life. It is caused by extended (CTG)n expansions at 3′-UTR of DMPK gene (DM type 1, DM1) or (CCTG)n repeats in the intron 1 of CNBP gene (DM type 2, DM2) and the sequestration of Muscleblind-like (MBNL) family proteins by transcribed (CUG)n RNA hairpin is the main pathogenic mechanism for DM. The MBNL proteins are splicing factors regulating posttranscriptional RNA during development. Previously, Mbnl knockout (KO) mouse lines showed molecular and phenotypic evidence that recapitulate DM brains, however, detailed morphological study has not yet been accomplished. In our studies, control (Mbnl1+/+; Mbnl2cond/cond; Nestin-Cre−/−), Mbnl2 conditional KO (2KO, Mbnl1+/+; Mbnl2cond/cond; Nestin-Cre+/−) and Mbnl1/2 double KO (DKO, Mbnl1ΔE3/ΔE3; Mbnl2cond/cond; Nestin-Cre+/−) mice were generated by crossing three individual lines. Immunohistochemistry for evaluating density and distribution of cortical neurons; Golgi staining for depicting the dendrites/dendritic spines; and electron microscopy for analyzing postsynaptic ultrastructure were performed. We found distributional defects in cortical neurons, reduction in dendritic complexity, immature dendritic spines and alterations of postsynaptic densities (PSDs) in the mutants. In conclusion, loss of function of Mbnl1/2 caused fundamental defects affecting neuronal distribution, dendritic morphology and postsynaptic architectures that are reminiscent of predominantly immature and fetal phenotypes in DM patients.

Design and development of a high temperature stable sweet protein base on monellin

Abstract Monellin, one kind of most sweet proteins, could be used instead of carbohydrates in the diabetic diet and pharmaceuticals. However, the critical problem interferes with monellin usage in the food or pharmaceutical industry was its unstable at high temperatures. Here, we describe a novel method to increase the thermostability of monellin. Inspired by the high Tm value in RNA hairpin containing structure, the beta-hairpin was added between the two polypeptide chains of monellin instead of Gly-Phe dipeptide linker. Three kinds of beta-hairpin libraries were constructed and selected. Remarkably, the Tm has been increased from 54.7℃ of monellin with Gly-Phe dipeptide linker to 79.5℃, 89.1℃ and 89.4℃ of selected three mutants. And combined with E24Q/Y80R mutation, the mutated E24Q/Y80R hairpin monellin Tm was as high as 96.1℃. It was soluble even heated in boiling water for 10 min, and sweetness recovered after cooling. This new hairpin monellin may show remarkable potential for further sweeteners.

Long noncoding RNA FLRL2 alleviated nonalcoholic fatty liver disease through Arntl-Sirt1 pathway.

Nonalcoholic fatty liver disease (NAFLD), which has an unknown pathogenesis and lacks a curative treatment, is becoming more prevalent. A previous long noncoding RNA (lncRNA) profiling analysis revealed a potential role for fatty liver-related lncRNA 2 (FLRL2) in the pathogenesis of NAFLD. To further understand the role of FLRL2 in NAFLD and explore its therapeutic value, both in vivo and in vitro NAFLD models were constructed. Small interfering RNA and small hairpin RNA interference and adenovirus transfection were adopted to manipulate the expressions of FLRL2, aryl-hydrocarbon receptor nuclear translocator-like (Arntl), and sirtuin 1 (Sirt1) expression. Steatosis was evaluated through histologic staining with hematoxylin and eosin and oil red O and also by quantitative triglyceride measurements. FLRL2 is a widely distributed nuclear lncRNA that is down-regulated in NAFLD. Overexpression of FLRL2 resolved steatosis, lipogenesis, inflammation, and endoplasmic reticulum (ER) stress in NAFLD, and down-regulation of FLRL2 resulted in the opposite effects. Sequence analysis demonstrated that FLRL2 was located in the intronic region of the Arntl gene, and a luciferase assay showed transcriptional activation of the Arntl gene upon FLRL2 overexpression. A similar expression pattern and synergistic effect of Arntl manipulation was observed in NAFLD in vitro. Inhibition of Arntl partially reversed the steatosis amelioration induced by FLRL2 overexpression. Downstream Sirt1 was also inhibited in NAFLD and influenced by both FLRL2 and Arntl. In NAFLD mice, FLRL2 enhancement alleviated steatosis, activated the Arntl-Sirt1 axis, and inhibited lipogenesis, ER stress, and inflammation, providing preliminary evidence of the benefits of FLRL2-mediated gene therapy in NAFLD.-Chen, Y., Chen, X., Gao, J., Xu, C., Xu, P., Li, Y., Zhu, Y., Yu, C. Long noncoding RNA FLRL2 alleviated nonalcoholic fatty liver disease through Arntl-Sirt1 pathway.

Argonaute 4 as an Effector Protein in RNA-Directed DNA Methylation in Human Cells.

DNA methylation of specific genome locations contributes to the distinct functions of multicellular organisms. DNA methylation can be governed by RNA-dependent DNA methylation (RdDM). RdDM is carried out by endogenous small-RNA-guided epigenomic editing complexes that add a methyl group to a precise DNA location. In plants, the Argonaute 4 (AGO4) protein is one of the main catalytic components involved in RdDM. Although small interfering RNA or short hairpin RNA has been shown to be able to guide DNA methylation in human cells, AGO protein-regulated RdDM in humans has not yet been evaluated. This study aimed to identify a key regulatory AGO protein involved in human RdDM by bioinformatics and to explore its function in RdDM by a combination of AGO4 knockdown, Alu small interfering RNA transfection, AGO4-expressing plasmid transfection, chromatin immunoprecipitation, cell-penetrating peptide-tagged AGO4 combined Alu single-guide RNA transfection, and methylation analyses. We found that first, human AGO4 showed stronger genome-wide association with DNA methylation than AGO1–AGO3. Second, endogenous AGO4 depletion demethylated DNA of known AGO4 bound loci. Finally, exogenous AGO4 de novo methylated the bound DNA sequences. Therefore, we discovered that AGO4 plays a role in human RdDM.