Deadenylation kinetics of mixed poly(A) tails at single-nucleotide resolution.

Shortening of messenger RNA poly(A) tails, or deadenylation, is a rate-limiting step in mRNA decay and is highly regulated during gene expression. The incorporation of non-adenosines in poly(A) tails, or 'mixed tailing', has been observed in vertebrates and viruses. Here, to quantitate the effect of mixed tails, we mathematically modeled deadenylation reactions at single-nucleotide resolution using an in vitro deadenylation system reconstituted with the complete human CCR4-NOT complex. Applying this model, we assessed the disrupting impact of single guanosine, uridine or cytosine to be equivalent to approximately 6, 8 or 11 adenosines, respectively. CCR4-NOT stalls at the 0, -1 and -2 positions relative to the non-adenosine residue. CAF1 and CCR4 enzyme subunits commonly prefer adenosine but exhibit distinct sequence selectivities and stalling positions. Our study provides an analytical framework to monitor deadenylation and reveals the molecular basis of tail sequence-dependent regulation of mRNA stability.

Alternative polyadenylation determines the functional landscape of inverted Alu repeats.

Inverted Alu repeats (IRAlus) are abundantly found in the transcriptome, especially in introns and 3' untranslated regions (UTRs). Yet, the biological significance of IRAlus embedded in 3' UTRs remains largely unknown. Here, we find that 3' UTR IRAlus silences genes involved in essential signaling pathways. We utilize J2 antibody to directly capture and map the double-stranded RNA structure of 3' UTR IRAlus in the transcriptome. Bioinformatic analysis reveals alternative polyadenylation as a major axis of IRAlus-mediated gene regulation. Notably, the expression of mouse double minute 2 (MDM2), an inhibitor of p53, is upregulated by the exclusion of IRAlus during UTR shortening, which is exploited to silence p53 during tumorigenesis. Moreover, the transcriptome-wide UTR lengthening in neural progenitor cells results in the global downregulation of genes associated with neurodegenerative diseases, including amyotrophic lateral sclerosis, via IRAlus inclusion. Our study establishes the functional landscape of 3' UTR IRAlus and its role in human pathophysiology.

The Synergistic Effects of Environmental and Genetic Factors on the Regulation of Anthocyanin Accumulation in Plant Tissues.

Anthocyanin accumulation is responsible for the coloration of apple fruit, and their accumulation depends on the expression of anthocyanin biosynthesis-related genes. Light is an environmental stimulus that induces fruit color by regulating genes involved in the anthocyanin biosynthesis pathway. In this study, the roles of light and genetic factors on fruit coloration and anthocyanin accumulation in apple fruit were investigated. Three genes in the anthocyanin biosynthesis pathway, MdCHS, MdANS, and MdUFGT1, were synthesized and cloned into a viral-based expression vector system for transient expression in 'Ruby S' apple fruits. Apple fruits were agroinfiltrated with expression vectors harboring MdCHS, MdANS, and MdUFGT1. Agroinfiltrated apple fruits were then either kept in the dark (bagged fruits) or exposed to light (exposed fruits). The agroinfiltrated fruits showed significantly different coloration patterns, transcript expression levels, and anthocyanin accumulation compared to the control fruits. Moreover, these parameters were higher in exposed fruits than in bagged fruits. For stable expression, MdCHS was introduced into a binary vector under the control of the rice α-amylase 3D (RAmy3D) promoter. The ectopic overexpression of MdCHS in transgenic rice calli showed a high accumulation of anthocyanin content. Taken together, our findings suggest that light, together with the overexpression of anthocyanin biosynthesis genes, induced the coloration and accumulation of anthocyanin content in apple fruits by upregulating the expression of the genes involved in the anthocyanin biosynthesis pathway.

Identification of genes associated with the regulation of cold tolerance and the RNA movement in the grafted apple.

In grafted apple, rootstock-derived signals influence scion cold tolerance by initiating physiological changes to survive over the winter. To understand the underlying molecular interactions between scion and rootstock responsive to cold, we developed transcriptomics and metabolomics data in the stems of two scion/rootstock combinations, 'Gala'/'G202' (cold resistant rootstock) and 'Gala'/'M9' (cold susceptible rootstock). Outer layers of scion and rootstock stem, including vascular tissues, were collected from the field-grown grafted apple during the winter. The clustering of differentially expressed genes (DEGs) and gene ontology enrichment indicated distinct expression dynamics in the two graft combinations, which supports the dependency of scion cold tolerance on the rootstock genotypes. We identified 544 potentially mobile mRNAs of DEGs showing highly-correlated seasonal dynamics between scion and rootstock. The mobility of a subset of 544 mRNAs was validated by translocated genome-wide variants and the measurements of selected RNA mobility in tobacco and Arabidopsis. We detected orthologous genes of potentially mobile mRNAs in Arabidopsis thaliana, which belong to cold regulatory networks with RNA mobility. Together, our study provides a comprehensive insight into gene interactions and signal exchange between scion and rootstock responsive to cold. This will serve for future research to enhance cold tolerance of grafted tree crops.

Introducing MdTFL1 Promotes Heading Date and Produces Semi-Draft Phenotype in Rice.

Flowering time (in rice, termed the heading date), plant height, and grain number are crucial agronomic traits for rice productivity. The heading date is controlled via environmental factors (day length and temperature) and genetic factors (floral genes). TERMINAL FLOWER 1 (TFL1) encodes a protein that controls meristem identity and participates in regulating flowering. In this study, a transgenic approach was used to promote the heading date in rice. We isolated and cloned apple MdTFL1 for early flowering in rice. Transgenic rice plants with antisense MdTFL1 showed an early heading date compared with wild-type plants. A gene expression analysis suggested that introducing MdTFL1 upregulated multiple endogenous floral meristem identity genes, including the (early) heading date gene family FLOWERING LOCUS T and MADS-box transcription factors, thereby shortening vegetable development. Antisense MdTFL1 also produced a wide range of phenotypic changes, including a change in overall plant organelles that affected an array of traits, especially grain productivity. The transgenic rice exhibited a semi-draft phenotype, increased leaf inclination angle, restricted flag leaf length, reduced spikelet fertility, and fewer grains per panicle. MdTFL1 plays a central role in regulating flowering and in various physiological aspects. These findings emphasize the role of TFL1 in regulating flowering in shortened breeding and expanding its function to produce plants with semi-draft phenotypes.

Heterologous vaccination utilizing viral vector and protein platforms confers complete protection against SFTSV.

Severe fever with thrombocytopenia syndrome virus was first discovered in 2009 as the causative agent of severe fever with thrombocytopenia syndrome. Despite its potential threat to public health, no prophylactic vaccine is yet available. This study developed a heterologous prime-boost strategy comprising priming with recombinant replication-deficient human adenovirus type 5 (rAd5) expressing the surface glycoprotein, Gn, and boosting with Gn protein. This vaccination regimen induced balanced Th1/Th2 immune responses and resulted in potent humoral and T cell-mediated responses in mice. It elicited high neutralizing antibody titers in both mice and non-human primates. Transcriptome analysis revealed that rAd5 and Gn proteins induced adaptive and innate immune pathways, respectively. This study provides immunological and mechanistic insight into this heterologous regimen and paves the way for future strategies against emerging infectious diseases.

Prediction of anti-vascular endothelial growth factor agent-specific treatment outcomes in neovascular age-related macular degeneration using a generative adversarial network.

To develop an artificial intelligence (AI) model that predicts anti-vascular endothelial growth factor (VEGF) agent-specific anatomical treatment outcomes in neovascular age-related macular degeneration (AMD), thereby assisting clinicians in selecting the most suitable anti-VEGF agent for each patient. This retrospective study included patients diagnosed with neovascular AMD who received three loading injections of either ranibizumab or aflibercept. Training was performed using optical coherence tomography (OCT) images with an attention generative adversarial network (GAN) model. To test the performance of the AI model, the sensitivity and specificity to predict the presence of retinal fluid after treatment were calculated for the AI model, an experienced (Examiner 1), and a less experienced (Examiner 2) human examiners. A total of 1684 OCT images from 842 patients (419 treated with ranibizumab and 423 treated with aflibercept) were used as the training set. Testing was performed using images from 98 patients. In patients treated with ranibizumab, the sensitivity and specificity, respectively, were 0.615 and 0.667 for the AI model, 0.385 and 0.861 for Examiner 1, and 0.231 and 0.806 for Examiner 2. In patients treated with aflibercept, the sensitivity and specificity, respectively, were 0.857 and 0.881 for the AI model, 0.429 and 0.976 for Examiner 1, and 0.429 and 0.857 for Examiner 2. In 18.5% of cases, the fluid status of synthetic posttreatment images differed between ranibizumab and aflibercept. The AI model using GAN might predict anti-VEGF agent-specific short-term treatment outcomes with relatively higher sensitivity than human examiners. Additionally, there was a difference in the efficacy in fluid resolution between the anti-VEGF agents. These results suggest the potential of AI in personalized medicine for patients with neovascular AMD.

Short poly(A) tails are protected from deadenylation by the LARP1-PABP complex.

Deadenylation generally constitutes the first and pivotal step in eukaryotic messenger RNA decay. Despite its importance in posttranscriptional regulations, the kinetics of deadenylation and its regulation remain largely unexplored. Here we identify La ribonucleoprotein 1, translational regulator (LARP1) as a general decelerator of deadenylation, which acts mainly in the 30-60-nucleotide (nt) poly(A) length window. We measured the steady-state and pulse-chased distribution of poly(A)-tail length, and found that deadenylation slows down in the 30-60-nt range. LARP1 associates preferentially with short tails and its depletion results in accelerated deadenylation specifically in the 30-60-nt range. Consistently, LARP1 knockdown leads to a global reduction of messenger RNA abundance. LARP1 interferes with the CCR4-NOT-mediated deadenylation in vitro by forming a ternary complex with poly(A)-binding protein (PABP) and poly(A). Together, our work reveals a dynamic nature of deadenylation kinetics and a role of LARP1 as a poly(A) length-specific barricade that creates a threshold for deadenylation.

Functional and molecular dissection of HCMV long non-coding RNAs.

Small, compact genomes confer a selective advantage to viruses, yet human cytomegalovirus (HCMV) expresses the long non-coding RNAs (lncRNAs); RNA1.2, RNA2.7, RNA4.9, and RNA5.0. Little is known about the function of these lncRNAs in the virus life cycle. Here, we dissected the functional and molecular landscape of HCMV lncRNAs. We found that HCMV lncRNAs occupy ~ 30% and 50-60% of total and poly(A)+viral transcriptome, respectively, throughout virus life cycle. RNA1.2, RNA2.7, and RNA4.9, the three abundantly expressed lncRNAs, appear to be essential in all infection states. Among these three lncRNAs, depletion of RNA2.7 and RNA4.9 results in the greatest defect in maintaining latent reservoir and promoting lytic replication, respectively. Moreover, we delineated the global post-transcriptional nature of HCMV lncRNAs by nanopore direct RNA sequencing and interactome analysis. We revealed that the lncRNAs are modified with N6-methyladenosine (m6A) and interact with m6A readers in all infection states. In-depth analysis demonstrated that m6A machineries stabilize HCMV lncRNAs, which could account for the overwhelming abundance of viral lncRNAs. Our study lays the groundwork for understanding the viral lncRNA-mediated regulation of host-virus interaction throughout the HCMV life cycle.

Antisense Expression of Apple TFL1-like Gene (MdTFL1) Promotes Early Flowering and Causes Phenotypic Changes in Tobacco.

Apples (Malus × domestica Borkh.) require up to several years for flowering and bearing fruits. The transition from vegetative to reproductive phase is controlled by floral regulators such as TERMINAL FLOWER 1 (TFL1) and FLOWERING LOCUS T (FT). TFL1 mediates the maintenance of vegetative phase, unlike the antagonistic function of FT to promote the transition into reproductive phase. In this study, we isolated apple TFL1-like gene (MdTFL1) to elucidate various phenotypic traits triggered by the antisense expression of MdTFL1 in tobacco apart from its floral induction function. Early flowering was observed in the tobacco line with MdTFL1 knockout, indicating the reduced time for transition to vegetative phases. Quantitative reverse-transcription PCR showed upregulation of genes involved in the regulation of floral induction, including NtAP1, NtSOC1, NFL1, and NtFTs, and downregulation of carotenoid cleavage dioxygenases (CCDs) and CEN-like genes in transgenic lines. Interestingly, transgenic tobacco expressing antisense MdTFL1 exhibited distinct morphological changes in lateral shoot outgrowth, internode length, and the development of leaves, flowers, and fruits. The results suggested that using the antisense expression of MdTFL1 gene is one of the approaches to shorten the vegetable phase and proposed improvement of plant architecture in horticultural crops.

Light Induces Carotenoid Biosynthesis-Related Gene Expression, Accumulation of Pigment Content, and Expression of the Small Heat Shock Protein in Apple Fruit.

The coloration of the apple fruit (Malus × domestica Borkh.) depends on pigment content. Light stimulus activates a broad range of photosynthesis-related genes, including carotenoids. The effect of light on two red commercial apple cultivars, 'Summer Prince' and 'Arisoo' at the juvenile stage were examined. Apple fruits were either bagged to reduce light irradiation or were exposed to direct, enhanced sunlight (reflected). The pigment content and the expression of carotenoid metabolism genes in the peel and flesh of apple fruits were significantly different between the shaded and the reflected parts. These parameters were also different in the two cultivars, highlighting the contribution of the genetic background. Further, a combination of light and transient overexpression of carotenogenic genes increased fruit coloration and pigment content in the variety 'RubyS'. Western blot analysis showed the expression of small heat shock proteins (smHSP) in lysates extracted from the reflected part of the fruits but not in the bagged fruits, indicating the activation of smHSP in response to heat generated by the reflected light. Therefore, the synergy between the genes and the environment dictates the color of apple fruits.

The m6A(m)-independent role of FTO in regulating WNT signaling pathways.

FTO and ALKBH5 are the two enzymes responsible for mRNA demethylation. Hence, the functional study of FTO has been focused on its mechanistic role in dynamic mRNA modification, and how this post-transcriptional regulation modulates signaling pathways. Here, we report that the functional landscape of FTO is largely associated with WNT signaling pathways but in a manner that is independent of its enzymatic activity. Re-analyses of public datasets identified the bifurcation of canonical and noncanonical WNT pathways as the major role of FTO. In FTO-depleted cells, we find that the canonical WNT/ß-Catenin signaling is attenuated in a non-cell autonomous manner via the up-regulation of DKK1. Simultaneously, this up-regulation of DKK1 promotes cell migration via activating the noncanonical WNT/PCP pathway. Unexpectedly, this regulation of DKK1 is independent of its RNA methylation status but operates at the transcriptional level, revealing a noncanonical function of FTO in gene regulation. In conclusion, this study places the functional context of FTO at the branch point of multiple WNT signaling pathways and extends its mechanistic role in gene regulation.

Identification of Genes Associated with Nitrogen Stress Responses in Apple Leaves.

Nitrogen (N) is an essential macronutrient that regulates diverse physiological processes for plant survival and development. In apple orchards, inappropriate N conditions can cause imbalanced growth and subsequent physiological disorders in trees. In order to investigate the molecular basis underlying the physiological signals for N stress responses, we examined the metabolic signals responsive to contrasting N stress conditions (deficient/excessive) in apple leaves using transcriptome approaches. The clustering of differentially expressed genes (DEGs) showed the expression dynamics of genes associated with each N stress group. Functional analyses of gene ontology and pathway enrichments revealed the potential candidates of metabolic signals responsible for N-deficient/excessive stress responses. The functional interactions of DEGs in each cluster were further explored by protein-protein interaction network analysis. Our results provided a comprehensive insight into molecular signals responsive to N stress conditions, and will be useful in future research to enhance the nutrition tolerance of tree crops.

The SARS-CoV-2 RNA interactome.

SARS-CoV-2 is an RNA virus whose success as a pathogen relies on its abilities to repurpose host RNA-binding proteins (RBPs) and to evade antiviral RBPs. To uncover the SARS-CoV-2 RNA interactome, we here develop a robust ribonucleoprotein (RNP) capture protocol and identify 109 host factors that directly bind to SARS-CoV-2 RNAs. Applying RNP capture on another coronavirus, HCoV-OC43, revealed evolutionarily conserved interactions between coronaviral RNAs and host proteins. Transcriptome analyses and knockdown experiments delineated 17 antiviral RBPs, including ZC3HAV1, TRIM25, PARP12, and SHFL, and 8 proviral RBPs, such as EIF3D and CSDE1, which are responsible for co-opting multiple steps of the mRNA life cycle. This also led to the identification of LARP1, a downstream target of the mTOR signaling pathway, as an antiviral host factor that interacts with the SARS-CoV-2 RNAs. Overall, this study provides a comprehensive list of RBPs regulating coronaviral replication and opens new avenues for therapeutic interventions.

Collagen type VI­α1 and 2 repress the proliferation, migration and invasion of bladder cancer cells.

The bladder cancer (BCa) microenvironment comprises heterogeneous tumor cell populations, the surrounding stroma and the extracellular matrix (ECM). Collagen, the scaffold of the tumor microenvironment, regulates ECM remodeling to promote tumor infiltration, angiogenesis, invasion and migration. The present study examined how collagen type VI­α (COL6A) 1 and 2 function during BCa pathogenesis and progression, with the aim of facilitating the development of precision therapeutics, risk stratification and molecular diagnosis. COL6A1 and COL6A2 mRNA expression in non­muscle invasive BCa (NMIBC) and MIBC tissue samples was measured using reverse transcription­quantitative PCR. In addition, the tumor­suppressive effects of COL6A1 and COL6A2 in human BCa EJ cells (MGH­U1) were assessed. Compared with normal controls, COL6A1 and COL6A2 mRNA expression was downregulated in both NMIBC and MIBC tissue samples (P<0.05, respectively). COL6A1 and COL6A2 effectively inhibited the proliferation of human BCa EJ cells (MGH­U1) and induced cell cycle arrest at the G1 phase. Additionally, COL6A1 and COL6A2 served roles in MAPK and AKT signaling by increasing p38 MAPK phosphorylation and decreasing AKT phosphorylation. Finally, COL6A1 and COL6A2 inhibited wound healing and invasion by suppressing the activity of matrix metalloproteinase (MMP)­2 and MMP­9. In conclusion, COL6A1 and COL6A2 may act as classical collagens by forming a physical barrier to inhibit BCa tumor growth and invasion.

Cold stress triggers premature fruit abscission through ABA-dependent signal transduction in early developing apple.

Fruit abscission is a complex physiological process that is regulated by internal and environmental factors. During early development, apple fruit are exposed to extreme temperature fluctuations that are associated with premature fruit drop; however, their effect on fruit abscission is largely unknown. We hypothesized that fruit abscission is triggered by cold stress and investigated the molecular basis of premature fruit drop using RNA-Seq and metabolomics data from apple fruit undergoing abscission following cold stress in the field. Genes responsive to abscisic acid signaling and cell wall degradation were upregulated during abscission, consistent with the increased abscisic acid concentrations detected by liquid chromatography-mass spectrometry. We performed ex vivo cold shock experiments with excised tree subunits consisting of a branch, pedicel, and fruit. Abscission induction occurred in the cold-stressed subunits with concurrent upregulation of abscisic acid biosynthesis (MdNCED1) and metabolism (MdCYP707A) genes, and ethylene biosynthesis (MdACS1) and receptor (MdETR2) genes in the pedicel. Another key finding was the activation of cytoplasmic streaming in abscission-zone cells detected by electron microscopy. Our results provide a novel insight into the molecular basis of fruit abscission physiology in response to cold stress in apple.

Noncanonical immune response to the inhibition of DNA methylation by Staufen1 via stabilization of endogenous retrovirus RNAs.

DNA-methyltransferase inhibitors (DNMTis), such as azacitidine and decitabine, are used clinically to treat myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). Decitabine activates the transcription of endogenous retroviruses (ERVs), which can induce immune response by acting as cellular double-stranded RNAs (dsRNAs). Yet, the posttranscriptional regulation of ERV dsRNAs remains uninvestigated. Here, we find that the viral mimicry and subsequent cell death in response to decitabine require the dsRNA-binding protein Staufen1 (Stau1). We show that Stau1 directly binds to ERV RNAs and stabilizes them in a genome-wide manner. Furthermore, Stau1-mediated stabilization requires a long noncoding RNA TINCR, which enhances the interaction between Stau1 and ERV RNAs. Analysis of a clinical patient cohort reveals that MDS and AML patients with lower Stau1 and TINCR expressions exhibit inferior treatment outcomes to DNMTi therapy. Overall, our study reveals the posttranscriptional regulatory mechanism of ERVs and identifies the Stau1-TINCR complex as a potential target for predicting the efficacy of DNMTis and other drugs that rely on dsRNAs.

RNA demethylation by FTO stabilizes the FOXJ1 mRNA for proper motile ciliogenesis.

Adenosine N6-methylation (m6A) is one of the most pervasive mRNA modifications, and yet the physiological significance of m6A removal (demethylation) remains elusive. Here, we report that the m6A demethylase FTO functions as a conserved regulator of motile ciliogenesis. Mechanistically, FTO demethylates and thereby stabilizes the mRNA that encodes the master ciliary transcription factor FOXJ1. Depletion of Fto in Xenopus laevis embryos caused widespread motile cilia defects, and Foxj1 was identified as one of the major phenocritical targets. In primary human airway epithelium, FTO depletion also led to FOXJ1 mRNA destabilization and a severe loss of ciliated cells with an increase of neighboring goblet cells. Consistently, Fto knockout mice showed strong asthma-like phenotypes upon allergen challenge, a result owing to defective ciliated cells in the airway epithelium. Altogether, our study reveals a conserved role of the FTO-FOXJ1 axis in embryonic and homeostatic motile ciliogenesis.

Preharvest Application of Chitosan Improves the Postharvest Life of 'Garmrok' Kiwifruit through the Modulation of Genes Related to Ethylene Biosynthesis, Cell Wall Modification, and Lignin Metabolism.

The influence of the preharvest application of chitosan on physicochemical properties and changes in gene expression of 'Garmrok' kiwifruit during postharvest cold storage (0 °C; RH 90-95%; 90 days) was investigated. Preharvest treatment of chitosan increased the fruit weight but had no significant effect on fruit size. The chitosan treatment suppressed the ethylene production and respiration rate of kiwifruit during the cold storage. The reduction of ethylene production of chitosan-treated kiwifruit was accompanied with the suppressed expression of ethylene biosynthesis genes. Moreover, preharvest application of chitosan diminished weight loss and delayed the changes in physicochemical properties that include firmness, soluble solids content, titratable acidity, total sugars, total acids, total phenols, and total lignin. As a result, the preharvest application of chitosan delayed the maturation and ripening of fruit. Expression of genes related to cell wall modification was down-regulated during the early maturation (ripening) period, while those related to gene expression for lignin metabolism were up-regulated at the later stages of ripening. These results demonstrate that the preharvest application of chitosan maintained the fruit quality and extends the postharvest life of 'Garmrok' kiwifruit, possibly through the modulation of genes related to ethylene biosynthesis, cell wall modification, and lignin metabolism.

Viral hijacking of the TENT4-ZCCHC14 complex protects viral RNAs via mixed tailing.

TENT4 enzymes generate 'mixed tails' of diverse nucleotides at 3' ends of RNAs via nontemplated nucleotide addition to protect messenger RNAs from deadenylation. Here we discover extensive mixed tailing in transcripts of hepatitis B virus (HBV) and human cytomegalovirus (HCMV), generated via a similar mechanism exploiting the TENT4-ZCCHC14 complex. TAIL-seq on HBV and HCMV RNAs revealed that TENT4A and TENT4B are responsible for mixed tailing and protection of viral poly(A) tails. We find that the HBV post-transcriptional regulatory element (PRE), specifically the CNGGN-type pentaloop, is critical for TENT4-dependent regulation. HCMV uses a similar pentaloop, an interesting example of convergent evolution. This pentaloop is recognized by the sterile alpha motif domain-containing ZCCHC14 protein, which in turn recruits TENT4. Overall, our study reveals the mechanism of action of PRE, which has been widely used to enhance gene expression, and identifies the TENT4-ZCCHC14 complex as a potential target for antiviral therapeutics.