Post-transcriptional Silencing Research Articles

Drosophila Modulo is Essential for Transposon Silencing and Developmental Robustness.

Transposable element (TE) silencing in the germline is crucial for preserving genome integrity; its absence results in sterility and diminished developmental robustness. The Piwi-interacting RNA (piRNA) pathway is the primary small non-coding RNA mechanism by which TEs are silenced in the germline. Three piRNA binding proteins promote the piRNA pathway function in the germline- P-element-induced wimpy testis (Piwi), Aubergine (Aub), and Argonaute 3 (Ago3). Piwi mediates transcriptional silencing of TEs by promoting the deposition of the heterochromatin mark Histone 3 lysine nine trimethylation (H3K9me3) at TE genomic sites. Aub and Ago3 facilitate post-transcriptional silencing of TEs. Proteins and mechanisms that promote piRNA function in TE silencing are still being discovered. This study demonstrates that the Drosophila Modulo protein, a homolog of mammalian Nucleolin and an epigenetic regulator, is crucial for the enrichment of H3K9me3 at TEs. We show that Modulo interacts with Piwi and operates downstream of the Piwi-piRNA complex's entry into the nucleus. Lack of Modulo function impairs Piwi-interacting protein Panoramix's ability to target transposon RNAs. Furthermore, the reduced function of Modulo in the mother undermines developmental robustness and exacerbates neomorphic Kr[If-1]-induced ectopic eye outgrowths in the offspring. Maternal Modulo enhances developmental robustness by inhibiting TE activation and transcriptome variability associated with intrinsic genetic variation. Thus, Modulo is an essential component of the mechanism that operates in the maternal germline to facilitate TE silencing and ensure developmental robustness in the ensuing generation.

A pipeline for validation of Serendipita indica effector-like sRNA suggests cross-kingdom communication in the symbiosis with Arabidopsis.

Bidirectional communication between pathogenic microbes and their plant hosts via small (s)RNA-mediated cross-kingdom RNA interference (ckRNAi) is a key element for successful host colonisation. Whether mutualistic fungi of the Serendipitaceae family, known for their extremely broad host range, use sRNAs to colonize plant roots is still under debate. To address this question, we developed a pipeline to validate the accumulation, translocation, and activity of fungal sRNAs in post-transcriptional silencing of Arabidopsis thaliana genes. Using stem-loop RT-qPCR, we detected the expression of a specific set of Serendipita indica (Si)sRNAs, targeting host genes involved in cell wall organization, hormonal signalling regulation, immunity, and gene regulation. To confirm the gene silencing activity of these sRNAs in plant cells, SisRNAs were transiently expressed in protoplasts. Stem-loop PCR confirmed sRNAs expression and accumulation, while qPCR validated post-transcriptional gene silencing of their predicted target genes. Furthermore, Arabidopsis ARGONAUTE 1 immunoprecipitation (AtAGO1-IP) revealed the loading of fungal SisRNAs into the plant RNAi machinery, suggesting the translocation of SisRNA from the fungus into root cells. In conclusion, this study provides a blueprint for rapid selection and analysis of sRNA effectors and further supports the model of cross-kingdom communication in the Sebacinoid symbiosis.

Cucurbit Crops Acquired Silencing: Virus-Induced Post-Transcriptional Silencing Is Transmitted Across the Graft Union

Plant grafting is one of the oldest agricultural technologies, capable of improving biotic and abiotic stress resistance, regulating plant growth, and increasing crop output and quality. Recent analytical technology has revealed that massive numbers of RNAs can move via the phloem to regulate the gene expression in the scion. However, there are currently no high-throughput methods for validating the role of these long-distance RNAs in grafting, which limits the development of rootstock resources. In this study, methodologies were developed for infection of TRSV vectors in germinating melon seeds. Efficient infection was achieved in Védrantais with completely white first-true leaves and above. Grafting was performed after the gene silencing phenotype appeared in the rootstock, resulting in gene silencing of cucurbit crops as the scion. Furthermore, different RNA viral vectors were utilized to demonstrate that the transfer of viral vectors across the graft union induced gene silencing of the scion. The approach established in this study can provide a valuable research tool for future transfer RNA function studies and rootstock breeding.

The N6-methyladenosine writer METTL3 promotes breast cancer progression through YTHDF2-dependent posttranscriptional silencing of GSDMD.

Cell pyroptosis is a form of programmed cell death, with Gasdermin-D (GSDMD) acting as its key executor. While activating pyroptosis represents a promising therapeutic strategy for cancer, the regulatory mechanisms governing GSDMD expression during cell death remain poorly understood. In this study, we identified METTL3 as a negative regulator of GSDMD-mediated pyroptosis, with high expression in breast cancer (BC) cells. YTHDF2 was found to recognize the m6A modification of GSDMD, thereby decreasing its stability. Finally, in vivo experiments further demonstrated the inhibitory effect of the METTL3 inhibitor STM2457 on tumors. Overall, these findings suggest that inhibition of METTL3 can enhance GSDMD-mediated pyroptosis and reveal a novel regulatory mechanism governing GSDMD expression, presenting a novel strategy for cancer treatment.

TMIC-05. AGING-SPECIFIC MICRORNA EXPRESSION SIGNATURE IN ELDERLY GLIOBLASTOMA

Abstract PURPOSE The rise in aging population worldwide is increasing death from cancer, including glioblastoma. MicroRNAs (miRNAs/miRs) are small non-coding RNAs that mediate the posttranscriptional silencing of specific target mRNAs and that are currently recognized as important regulators of tumorigenesis and development. Here, we explore the impact of brain aging and aging-specific miRNAs on elderly glioblastomas. METHODS We comprehensively analyzed miRNAs expression using the TCGA dataset and identified miRNAs that are specifically expressed in elderly glioblastomas. To create the Ehime Glioma Genome Atlas (EGGA), RNA sequencing was performed using tumor tissues from 8 glioblastoma patients (4 elderly and 4 non-elderly). First, we evaluated the impact of the identified miRNAs on prognosis using TCGA and CGGA datasets and searched for target genes of specific miRNAs using TargetScan. The expression patterns of the identified target genes were verified using EGGA, TCGA, and CGGA datasets. RESULTS We divided the TCGA dataset into elderly and non-elderly groups based on three ages (75 years-old, 70 years-old, and 65 years-old) and analyzed the expression of miRNAs in tumor tissues. There were 3 miRNAs that showed abnormalities (1 increased miRNA, 2 decreased miRNAs), and among these, miR-210 (increased expression) and miR-507 (increased expression) were identified as miRNAs related to prognosis. Next, we analyzed the expression of EGGA and identified 410 mRNAs (368 increased mRNAs, 42 decreased mRNAs) that showed significantly abnormal expression in the elderly group. The target genes searched using TargetScan were 4,048 genes for miR-210 and 4,704 genes for miR-507. When compared with the 410 genes identified using EGGA, PLA2G3, a target gene to miR-507, was identified. The expression of PLA2G3 was also specifically increased in elderly glioblastoma in the TCGA dataset, and it had a strong influence on prognosis. CONCLUSION Our results found an aging-specific miRNA “miR-507” and its target gene “PLA2G3” that suggested as potential new therapeutic target molecules for improving the prognosis of elderly glioblastomas.

Endometriotic lesions express microRNAs that potentially regulate the transcription of hydroxysteroid 17-beta dehydrogenase 2 (HSD17B2): a pilot study

ObjectiveEndometriosis is an estrogen-dependent disease. A theory of deficient HSD17B2 activity in endometriotic lesions leading to high local levels of estradiol has been described as a potential molecular mechanism for the disease progression. However, HSD17B2 mRNA is normally expressed in endometriotic tissue, leading us to question whether an excess of microRNAS (miRNAs) could be involved in post-transcriptional silencing of HSD17B2. MethodsThis was a prospective, cross-sectional, pilot study. The study included seven samples of superficial peritoneal endometriosis and 22 samples of eutopic endometrium from 22 reproductive-aged patients submitted to laparoscopy for symptomatic endometriosis. We searched in an experimentally validated microRNA-target interaction online database (miRTarBase) for miRNAs that target the HSD17B2 gene and performed RT-PCR to evaluate their expression in endometriotic lesions and eutopic endometrium. ResultsWe found two miRNAs on the miRTarBase search that interact with HSD17B2: miR-124-3p and miR-26b-5p. RT-PCR analysis showed that both miRNAs were expressed in ectopic tissues as well as in eutopic endometrium. Quantitatively, no significant difference existed between the two tissue groups in either miR-26b-5p (p = 0.08) or miR-124-3p (p = 0.09) expression levels, although the observed trend was towards lower levels of both miRNAs in the endometriotic lesions. ConclusionsThe expression of miRNAs miR-26b-5p and miR-124-3p is not increased in peritoneal endometriosis compared to eutopic endometrium. The regulation of HSD17B2 expression may be more complex than assumed, and it must be further investigated in different clinical scenarios.

MicroRNA mediated suppression of airway lactoperoxidase by TGF-β1 and cigarette smoke promotes airway inflammation

Transforming Growth Factor Beta1 (TGF-β1) signaling is upregulated in Chronic Obstructive Pulmonary disease (COPD), smokers, and people living with HIV. Cigarette smoking and HIV are also independent risk factors for COPD. Chronic inflammation is a hallmark of COPD. However, the underlying mechanisms remain unknown. Previous research has suggested that TGF-β1 alters the airway epithelial microRNAome and transcriptome, potentially contributing to lung inflammation. The Lactoperoxidase (LPO) system is an integral component of innate immunity within the airway. LPO plays a crucial role in host defense by catalyzing the oxidation of thiocyanate to hypothiocyanite in the presence of hydrogen peroxide (H2O2), generating a potent antibacterial and antiviral agent. Additionally, the LPO system potentially aids in maintaining cellular redox balance by reducing the levels of H2O2, thus mitigating oxidative stress within the airway epithelium. LPO dysfunction can impair immune responses and exacerbate inflammatory processes in respiratory diseases.In this study, primary bronchial epithelial cells and bronchial cell lines were treated with TGF-β1 and exposed to cigarette smoke to characterize the effect of these factors on LPO and their downstream effects. RT-qPCR and Western Blot were applied to quantify mRNA and proteins’ expression. The levels of H2O2 were detected using the Amplex Red Assay. Magnetofection and transfection were applied to probe the effect of miR-449b-5p. Staining procedures using the MitoTracker Green and C12FDG dyes were used to establish mitochondria mass and senescence. The levels of pro-inflammatory cytokines were measured via Luminex assays.We found that TGF-β1 and cigarette smoke suppressed airway LPO expression, increasing H2O2 levels. This increase in H2O2 had downstream effects on mitochondrial homeostasis, epithelial cellular senescence, and the pro-inflammatory cytokine response. We demonstrate for the first time that airway LPO is regulated by TGF-β1-induced miRNA-mediated post-transcriptional silencing through miR-449b-5p in the lungs. Further, we identify and validate miR-449-5p as the candidate miRNA upregulated by TGF-β1, which is involved in LPO suppression. This paper demonstrates a new mechanism by which TGF-β1 can lead to altered redox status in the airway.

Circular RNAs regulate neuron size and migration of midbrain dopamine neurons during development

Midbrain dopamine (mDA) neurons play an essential role in cognitive and motor behaviours and are linked to different brain disorders. However, the molecular mechanisms underlying their development, and in particular the role of non-coding RNAs (ncRNAs), remain incompletely understood. Here, we establish the transcriptomic landscape and alternative splicing patterns of circular RNAs (circRNAs) at key developmental timepoints in mouse mDA neurons in vivo using fluorescence-activated cell sorting followed by short- and long-read RNA sequencing. In situ hybridisation shows expression of several circRNAs during early mDA neuron development and post-transcriptional silencing unveils roles for different circRNAs in regulating mDA neuron morphology. Finally, in utero electroporation and time-lapse imaging implicate circRmst, a circRNA with widespread morphological effects, in the migration of developing mDA neurons in vivo. Together, these data for the first time suggest a functional role for circRNAs in developing mDA neurons and characterise poorly defined aspects of mDA neuron development.

Remodeling of perturbed chromatin can initiate de novo transcriptional and post-transcriptional silencing

In eukaryotes, repetitive DNA can become silenced de novo, either transcriptionally or post-transcriptionally, by processes independent of strong sequence-specific cues. The mechanistic nature of such processes remains poorly understood. We found that in the fungus Neurospora crassa, de novo initiation of both transcriptional and post-transcriptional silencing was linked to perturbed chromatin, which was produced experimentally by the aberrant activity of transcription factors at the tetO operator array. Transcriptional silencing was mediated by canonical constitutive heterochromatin. On the other hand, post-transcriptional silencing resembled repeat-induced quelling but occurred normally when homologous recombination was inactivated. All silencing of the tetO array was dependent on SAD-6, fungal ortholog of the SWI/SNF chromatin remodeler ATRX (Alpha Thalassemia/Mental Retardation Syndrome X-Linked), which was required to maintain nucleosome occupancy at the perturbed locus. In addition, we found that two other types of sequences (the lacO array and native AT-rich DNA) could also undergo recombination-independent quelling associated with perturbed chromatin. These results suggested a model in which the de novo initiation of transcriptional and post-transcriptional silencing is coupled to the remodeling of perturbed chromatin.

Extracellular matrix proteins Pericardin and Lonely heart mediate periostial hemocyte aggregation in the mosquito Anopheles gambiae

An infection induces the migration of immune cells called hemocytes to the insect heart, where they aggregate around heart valves called ostia and phagocytose pathogens in areas of high hemolymph flow. Here, we investigated whether the cardiac extracellular matrix proteins, Pericardin (Prc) and Lonely heart (Loh), regulate the infection-induced aggregation of periostial hemocytes in the mosquito, An. gambiae. We discovered that RNAi-based post-transcriptional silencing of Prc or Loh did not affect the resident population of periostial hemocytes in uninfected mosquitoes, but that knocking down these genes decreases the infection-induced migration of hemocytes to the heart. Knocking down Prc or Loh did not affect the proportional distribution of periostial hemocytes along the periostial regions. Moreover, knocking down Prc or Loh did not affect the number of sessile hemocytes outside the periostial regions, suggesting that the role of these proteins is cardiac-specific. Finally, knocking down Prc or Loh did not affect the amount of melanin at the periostial regions, or the intensity of an infection at 24 h after challenge. Overall, we demonstrate that Prc and Loh are positive regulators of the infection-induced migration of hemocytes to the heart of mosquitoes.

An RNA-seq study in Friedreich ataxia patients identified hsa-miR-148a-3p as a putative prognostic biomarker of the disease

Friedreich ataxia (FRDA) is a life-threatening hereditary ataxia; its incidence is 1:50,000 individuals in the Caucasian population. A unique therapeutic drug for FRDA, the antioxidant Omaveloxolone, has been recently approved by the US Food and Drug Administration (FDA). FRDA is a multi-systemic neurodegenerative disease; in addition to a progressive neurodegeneration, FRDA is characterized by hypertrophic cardiomyopathy, diabetes mellitus and musculoskeletal deformities. Cardiomyopathy is the predominant cause of premature death. The onset of FRDA typically occurs between the ages of 5 and 15. Given the complexity and heterogeneity of clinical features and the variability of their onset, the identification of biomarkers capable of assessing disease progression and monitoring the efficacy of treatments is essential to facilitate decision making in clinical practice. We conducted an RNA-seq analysis in peripheral blood mononuclear cells from FRDA patients and healthy donors, identifying a signature of small non-coding RNAs (sncRNAs) capable of distinguishing healthy individuals from the majority of FRDA patients. Among the differentially expressed sncRNAs, microRNAs are a class of small non-coding endogenous RNAs that regulate posttranscriptional silencing of target genes. In FRDA plasma samples, hsa-miR-148a-3p resulted significantly upregulated. The analysis of the Receiver Operating Characteristic (ROC) curve, combining the circulating expression levels of hsa-miR-148a-3p and hsa-miR-223-3p (previously identified by our group), revealed an Area Under the Curve (AUC) of 0.86 (95%, Confidence Interval 0.77–0.95; p-value < 0.0001). An in silico prediction analysis indicated that the IL6ST gene, an interesting marker of neuroinflammation in FRDA, is a common target gene of both miRNAs. Our findings support the evaluation of combined expression levels of different circulating miRNAs as potent epi-biomarkers in FRDA. Moreover, we found hsa-miR-148a-3p significantly over-expressed in Intermediate and Late-Onset Friedreich Ataxia patients’ group (IOG and LOG, respectively) compared to healthy individuals, indicating it as a putative prognostic biomarker in this pathology.

The multifaceted role of RNA-based regulation in plant stress memory.

Plants have evolved interconnected regulatory pathways which enable them to respond and adapt to their environments. In plants, stress memory enhances stress tolerance through the molecular retention of prior stressful experiences, fostering rapid and robust responses to subsequent challenges. Mounting evidence suggests a close link between the formation of stress memories and effective future stress responses. However, the mechanism by which environmental stressors trigger stress memory formation is poorly understood. Here, we review the current state of knowledge regarding the RNA-based regulation on stress memory formation in plants and discuss research challenges and future directions. Specifically, we focus on the involvement of microRNAs (miRNAs), small interfering RNAs (siRNAs), long non-coding RNAs (lncRNAs), and alternative splicing (AS) in stress memory formation. miRNAs regulate target genes via post-transcriptional silencing, while siRNAs trigger stress memory formation through RNA-directed DNA methylation (RdDM). lncRNAs guide protein complexes for epigenetic regulation, and AS of pre-mRNAs is crucial to plant stress memory. Unraveling the mechanisms underpinning RNA-mediated stress memory formation not only advances our knowledge of plant biology but also aids in the development of improved stress tolerance in crops, enhancing crop performance and global food security.

Efficient deletion of microRNAs using CRISPR/Cas9 with dual guide RNAs.

MicroRNAs (miRNAs) are short non-coding RNAs that play crucial roles in gene regulation, exerting post-transcriptional silencing, thereby influencing cellular function, development, and disease. Traditional loss-of-function methods for studying miRNA functions, such as miRNA inhibitors and sponges, present limitations in terms of specificity, transient effects, and off-target effects. Similarly, CRISPR/Cas9-based editing of miRNAs using single guide RNAs (sgRNAs) also has limitations in terms of design space for generating effective gRNAs. In this study, we introduce a novel approach that utilizes CRISPR/Cas9 with dual guide RNAs (dgRNAs) for the rapid and efficient generation of short deletions within miRNA genomic regions. Through the expression of dgRNAs through single-copy lentiviral integration, this approach achieves over a 90% downregulation of targeted miRNAs within a week. We conducted a comprehensive analysis of various parameters influencing efficient deletion formation. In addition, we employed doxycycline (Dox)-inducible expression of Cas9 from the AAVS1 locus, enabling homogeneous, temporal, and stage-specific editing during cellular differentiation. Compared to miRNA inhibitory methods, the dgRNA-based approach offers higher specificity, allowing for the deletion of individual miRNAs with similar seed sequences, without affecting other miRNAs. Due to the increased design space, the dgRNA-based approach provides greater flexibility in gRNA design compared to the sgRNA-based approach. We successfully applied this approach in two human cell lines, demonstrating its applicability for studying the mechanisms of human erythropoiesis and pluripotent stem cell (iPSC) biology and differentiation. Efficient deletion of miR-451 and miR-144 resulted in blockage of erythroid differentiation, and the deletion of miR-23a and miR-27a significantly affected iPSC survival. We have validated the highly efficient deletion of genomic regions by editing protein-coding genes, resulting in a significant impact on protein expression. This protocol has the potential to be extended to delete multiple miRNAs within miRNA clusters, allowing for future investigations into the cooperative effects of the cluster members on cellular functions. The protocol utilizing dgRNAs for miRNA deletion can be employed to generate efficient pooled libraries for high-throughput comprehensive analysis of miRNAs involved in different biological processes.

Utilization of Bacteriophage phi6 for the Production of High-Quality Double-Stranded RNA Molecules.

Double-stranded RNA (dsRNA) molecules are mediators of RNA interference (RNAi) in eukaryotic cells. RNAi is a conserved mechanism of post-transcriptional silencing of genes cognate to the sequences of the applied dsRNA. RNAi-based therapeutics for the treatment of rare hereditary diseases have recently emerged, and the first sprayable dsRNA biopesticide has been proposed for registration. The range of applications of dsRNA molecules will likely expand in the future. Therefore, cost-effective methods for the efficient large-scale production of high-quality dsRNA are in demand. Conventional approaches to dsRNA production rely on the chemical or enzymatic synthesis of single-stranded (ss)RNA molecules with a subsequent hybridization of complementary strands. However, the yield of properly annealed biologically active dsRNA molecules is low. As an alternative approach, we have developed methods based on components derived from bacteriophage phi6, a dsRNA virus encoding RNA-dependent RNA polymerase (RdRp). Phi6 RdRp can be harnessed for the enzymatic production of high-quality dsRNA molecules. The isolated RdRp efficiently synthesizes dsRNA in vitro on a heterologous ssRNA template of any length and sequence. To scale up dsRNA production, we have developed an in vivo system where phi6 polymerase complexes produce target dsRNA molecules inside Pseudomonas cells.

Transcriptome and small RNAome profiling uncovers how a recombinant begomovirus evades RDRγ-mediated silencing of viral genes and outcompetes its parental virus in mixed infection.

Tomato yellow leaf curl virus (TYLCV, genus Begomovirus, family Geminiviridae) causes severe disease of cultivated tomatoes. Geminiviruses replicate circular single-stranded genomic DNA via rolling-circle and recombination-dependent mechanisms, frequently generating recombinants in mixed infections. Circular double-stranded intermediates of replication also serve as templates for Pol II bidirectional transcription. IS76, a recombinant derivative of TYLCV with a short sequence in the bidirectional promoter/origin-of-replication region acquired from a related begomovirus, outcompetes TYLCV in mixed infection and breaks disease resistance in tomato Ty-1 cultivars. Ty-1 encodes a γ-clade RNA-dependent RNA polymerase (RDRγ) implicated in Dicer-like (DCL)-mediated biogenesis of small interfering (si)RNAs directing gene silencing. Here, we profiled transcriptome and small RNAome of Ty-1 resistant and control susceptible plants infected with TYLCV, IS76 or their combination at early and late infection stages. We found that RDRγ boosts production rates of 21, 22 and 24 nt siRNAs from entire genomes of both viruses and modulates DCL activities in favour of 22 and 24 nt siRNAs. Compared to parental TYLCV, IS76 undergoes faster transition to the infection stage favouring rightward transcription of silencing suppressor and coat protein genes, thereby evading RDRγ activity and facilitating its DNA accumulation in both single and mixed infections. In coinfected Ty-1 plants, IS76 efficiently competes for host replication and transcription machineries, thereby impairing TYLCV replication and transcription and forcing its elimination associated with further increased siRNA production. RDRγ is constitutively overexpressed in Ty-1 plants, which correlates with begomovirus resistance, while siRNA-generating DCLs (DCL2b/d, DCL3, DCL4) and genes implicated in siRNA amplification (α-clade RDR1) and function (Argonaute2) are upregulated to similar levels in TYLCV- and IS76-infected susceptible plants. Collectively, IS76 recombination facilitates replication and promotes expression of silencing suppressor and coat proteins, which allows the recombinant virus to evade the negative impact of RDRγ-boosted production of viral siRNAs directing transcriptional and posttranscriptional silencing.

RNA Interference-Screening of Potentially Lethal Gene Targets in the White-Backed Planthopper Sogatella furcifera via a Spray-Induced and Nanocarrier-Delivered Gene Silencing System.

RNA interference (RNAi) is a widespread post-transcriptional silencing mechanism that targets homologous mRNA sequences for specific degradation. An RNAi-based pest management strategy is target-specific and considered a sustainable biopesticide. However, the specific genes targeted and the efficiency of the delivery methods can vary widely across species. In this study, a spray-induced and nanocarrier-delivered gene silencing (SI-NDGS) system that incorporated gene-specific dsRNAs targeting conserved genes was used to evaluate phenotypic effects in white-backed planthopper (WBPH). At 2 days postspraying, transcript levels for all target genes were significantly reduced and knockdown of two gene orthologs, hsc70-3 and PP-α, resulted in an elevated mortality (>60%) and impaired ecdysis. These results highlight the utility of the SI-NDGS system for identifying genes involved in WBPH growth and development that could be potentially exploitable as high mortality target genes to develop an alternative method for WBPH control.

Non-Coding RNAs in Human Cancer and Other Diseases: Overview of the Diagnostic Potential.

Non-coding RNAs (ncRNAs) are abundant single-stranded RNA molecules in human cells, involved in various cellular processes ranging from DNA replication and mRNA translation regulation to genome stability defense. MicroRNAs are multifunctional ncRNA molecules of 18-24 nt in length, involved in gene silencing through base-pair complementary binding to target mRNA transcripts. piwi-interacting RNAs are an animal-specific class of small ncRNAs sized 26-31 nt, responsible for the defense of genome stability via the epigenetic and post-transcriptional silencing of transposable elements. Long non-coding RNAs are ncRNA molecules defined as transcripts of more than 200 nucleotides, their function depending on localization, and varying from the regulation of cell differentiation and development to the regulation of telomere-specific heterochromatin modifications. The current review provides recent data on the several forms of small and long non-coding RNA's potential to act as diagnostic, prognostic or therapeutic target for various human diseases.

Role of miRNAs in sucrose stress response, reactive oxygen species, and anthocyanin biosynthesis in Arabidopsis thaliana.

In plants, sucrose is the main transported disaccharide that is the primary product of photosynthesis and controls a multitude of aspects of the plant life cycle including structure, growth, development, and stress response. Sucrose is a signaling molecule facilitating various stress adaptations by crosstalk with other hormones, but the molecular mechanisms are not well understood. Accumulation of high sucrose concentrations is a hallmark of many abiotic and biotic stresses, resulting in the accumulation of reactive oxygen species and secondary metabolite anthocyanins that have antioxidant properties. Previous studies have shown that several MYeloBlastosis family/MYB transcription factors are positive and negative regulators of sucrose-induced anthocyanin accumulation and subject to microRNA (miRNA)-mediated post-transcriptional silencing, consistent with the notion that miRNAs may be "nodes" in crosstalk signaling by virtue of their sequence-guided targeting of different homologous family members. In this study, we endeavored to uncover by deep sequencing small RNA and mRNA transcriptomes the effects of exogenous high sucrose stress on miRNA abundances and their validated target transcripts in Arabidopsis. We focused on genotype-by-treatment effects of high sucrose stress in Production of Anthocyanin Pigment 1-Dominant/pap1-D, an activation-tagged dominant allele of MYB75 transcription factor, a positive effector of secondary metabolite anthocyanin pathway. In the process, we discovered links to reactive oxygen species signaling through miR158/161/173-targeted Pentatrico Peptide Repeat genes and two novel non-canonical targets of high sucrose-induced miR408 and miR398b*(star), relevant to carbon metabolic fluxes: Flavonoid 3'-Hydroxlase (F3'H), an important enzyme in determining the B-ring hydroxylation pattern of flavonoids, and ORANGE a post-translational regulator of Phytoene Synthase expression, respectively. Taken together, our results contribute to understanding the molecular mechanisms of carbon flux shifts from primary to secondary metabolites in response to high sugar stress.

Synonymous sites for accessibility around microRNA binding sites in bacterial spot and speck disease resistance genes of tomato.

The major causes of mass tomato infections in both covered and open ground are agents of bacterial spot and bacterial speck diseases. MicroRNAs (miRNAs) are 16-21 nucleotides in length, non-coding RNAs that inhibit translation and trigger mRNA degradation. MiRNAs play a significant part in plant resistance to abiotic and biotic stresses by mediating gene regulation via post-transcriptional RNA silencing. In this study, we analyzed a collection of bacterial resistance genes of tomato and their binding sites for tomato miRNAs and Pseudomonas syringe pv. tomato miRNAs. Our study found that two genes, bacterial spot disease resistance gene (Bs4) and bacterial speck disease resistance gene (Prf), have a 7mer-m8 perfect seed match with miRNAs. Bs4 was targeted by one tomato miRNA (sly-miR9470-3p) and three Pseudomonas syringe pv. tomato miRNAs (PSTJ4_3p_27246, PSTJ4_3p_27246, and PSTJ4_3p_27246). Again, Prf gene was found to be targeted by two tomato miRNAs namely, sly-miR9469-5p and sly-miR9474-3p. The accessibility of the miRNA-target site and its flanking regions and the relationship between relative synonymous codon usage and tRNAs were compared. Strong access to miRNA targeting regions and decreased rate of translations suggested that miRNAs might be efficient in binding to their particular targets. We also found the existence of rare codons, which suggests that it could enhance miRNA targeting even more. The codon usage pattern analysis of the two genes revealed that both were AT-rich (Bs4 = 63.2%; Prf = 60.8%). We found a low codon usage bias in both genes, suggesting that selective restriction might regulate them. The silencing property of miRNAs would allow researchers to discover the involvement of plant miRNAs in pathogen invasion. However, the efficient validation of direct targets of miRNAs is an urgent need that might be highly beneficial in enhancing plant resistance to multiple pathogenic diseases.

Pinostrobin, a fingerroot compound, regulates miR-181b-5p and induces acute leukemic cell apoptosis

Pinostrobin (PN) is the most abundant flavonoid found in fingerroot. Although the anti-leukemic properties of PN have been reported, its mechanisms are still unclear. MicroRNAs (miRNAs) are small RNA molecules that function in posttranscriptional silencing and are increasingly being used in cancer therapy. The aims of this study were to investigate the effects of PN on proliferation inhibition and induction of apoptosis, as well as the involvement of miRNAs in PN-mediated apoptosis in acute leukemia. The results showed that PN reduced cell viability and induced apoptosis in acute leukemia cells via both intrinsic and extrinsic pathways. A bioinformatics approach and Protein–Protein Interaction (PPI) network analysis revealed that ataxia-telangiectasia mutated kinase (ATM), one of the p53 activators that responds to DNA damage-induced apoptosis, is a crucial target of PN. Four prediction tools were used to predict ATM-regulated miRNAs; miR-181b-5p was the most likely candidate. The reduction in miR-181b-5 after PN treatment was found to trigger ATM, resulting in cellular apoptosis. Therefore, PN could be developed as a drug for acute leukemia; in addition, miR-181b-5p and ATM may be promising therapeutic targets.