microRNA-induced Silencing Complex Research Articles

Interaction between a J-domain co-chaperone and a specific Argonaute protein contributes to microRNA function in animals.

MicroRNAs (miRNAs) are essential regulators of several biological processes. They are loaded onto Argonaute (AGO) proteins to achieve their repressive function, forming the microRNA-Induced Silencing Complex known asmiRISC. While several AGO proteins are expressed in plants and animals, it is still unclear why specific AGOs are strictly binding miRNAs. Here, we identified the co-chaperone DNJ-12 as a new interactor of ALG-1, one of the two major miRNA-specific AGOs in Caenorhabditis elegans. DNJ-12 does not interact with ALG-2, the other major miRNA-specific AGO, and PRG-1 and RDE-1, two AGOs involved in other small RNA pathways, making it a specific actor in ALG-1-dependent miRNA-mediated gene silencing. The loss of DNJ-12 causes developmental defects associated with defective miRNA function. Using the Auxin Inducible Degronsystem, a powerful tool to acutely degrade proteins in specific tissues, we show that DNJ-12 depletion hampers ALG-1 interaction with HSP70, a chaperone required for miRISC loading in vitro. Moreover, DNJ-12 depletion leads to the decrease of several miRNAs and prevents their loading onto ALG-1. This study uncovers the importance of a co-chaperone for the miRNA function in vivo and provides insights to explain how different small RNAs associate with specific AGO in animals.

Phosphorylation of AGO2 by TBK1 Promotes the Formation of Oncogenic miRISC in NSCLC.

Non-small-cell lung cancer (NSCLC) is a highly lethal tumor that often develops resistance to targeted therapy. It is shown that Tank-binding kinase 1 (TBK1) phosphorylates AGO2 at S417 (pS417-AGO2), which promotes NSCLC progression by increasing the formation of microRNA-induced silencing complex (miRISC). High levels of pS417-AGO2 in clinical NSCLC specimens are positively associated with poor prognosis. Interestingly, the treatment with EGFR inhibitor Gefitinib can significantly induce pS417-AGO2, thereby increasing the formation and activity of oncogenic miRISC, which may contribute to NSCLC resistance to Gefitinib. Based on these, two therapeutic strategies is developed. One is jointly to antagonize multiple oncogenic miRNAs highly expressed in NSCLC and use TBK1 inhibitor Amlexanox reducing the formation of oncogenic miRISC. Another approach is to combine Gefitinib with Amlexanox to inhibit the progression of Gefitinib-resistant NSCLC. This findings reveal a novel mechanism of oncogenic miRISC regulation by TBK1-mediated pS417-AGO2 and suggest potential therapeutic approaches for NSCLC.

An RNAi screen for conserved kinases that enhance microRNA activity after dauer in Caenorhabditis elegans.

Gene regulation in changing environments is critical for maintaining homeostasis. Some animals undergo a stress-resistant diapause stage to withstand harsh environmental conditions encountered during development. MicroRNAs are one mechanism for regulating gene expression during and after diapause. MicroRNAs downregulate target genes posttranscriptionally through the activity of the microRNA-induced silencing complex. Argonaute is the core microRNA-induced silencing complex protein that binds to both the microRNA and to other microRNA-induced silencing complex proteins. The 2 major microRNA Argonautes in the Caenorhabditis elegans soma are ALG-1 and ALG-2, which function partially redundantly. Loss of alg-1 [alg-1(0)] causes penetrant developmental phenotypes including vulval defects and the reiteration of larval cell programs in hypodermal cells. However, these phenotypes are essentially absent if alg-1(0) animals undergo a diapause stage called dauer. Levels of the relevant microRNAs are not higher during or after dauer, suggesting that activity of the microRNA-induced silencing complex may be enhanced in this context. To identify genes that are required for alg-1(0) mutants to develop without vulval defects after dauer, we performed an RNAi screen of genes encoding conserved kinases. We focused on kinases because of their known role in modulating microRNA-induced silencing complex activity. We found RNAi knockdown of 4 kinase-encoding genes, air-2, bub-1, chk-1, and nekl-3, caused vulval defects and reiterative phenotypes in alg-1(0) mutants after dauer, and that these defects were more penetrant in an alg-1(0) background than in wild type. Our results implicate these kinases as potential regulators of microRNA-induced silencing complex activity during postdauer development in C. elegans.

An integrated ion-exchange membrane-based microfluidic device for irreversible dissociation and quantification of miRNA from ribonucleoproteins.

Ribonucleoproteins (RNPs), particularly microRNA-induced silencing complex (miRISC), have been associated with cancer-related gene regulation. Specific RNA-protein associations in miRISC complexes or those found in let-7 lin28A complexes can downregulate tumor-suppressing genes and can be directly linked to cancer. The high protein-RNA electrostatic binding affinity is a particular challenge for the quantification of the associated microRNAs (miRNAs). We report here the first microfluidic point-of-care assay that allows direct quantification of RNP-associated RNAs, which has the potential to greatly advance RNP profiling for liquid biopsy. Key to the technology is an integrated cation-anion exchange membrane (CEM/AEM) platform for rapid and irreversible dissociation (k = 0.0025 s-1) of the RNP (Cas9-miR-21) complex and quantification of its associated miR-21 in 40 minutes. The CEM-induced depletion front is used to concentrate the RNP at the depletion front such that the high electric field (>100 V cm-1) within the concentration boundary layer induces irreversible dissociation of the low KD (∼0.5 nM) complex, with ∼100% dissociation even though the association rate (kon = 6.1 s-1) is 1000 times higher. The high field also electrophoretically drives the dissociated RNA out of the concentrated zone without reassociation. A detection limit of 1.1 nM is achieved for Cy3 labelled miR-21.

Roles of the Core Components of the Mammalian miRISC in Chromatin Biology.

The Argonaute (AGO) and the Trinucleotide Repeat Containing 6 (TNRC6) family proteins are the core components of the mammalian microRNA-induced silencing complex (miRISC), the machinery that mediates microRNA function in the cytoplasm. The cytoplasmic miRISC-mediated post-transcriptional gene repression has been established as the canonical mechanism through which AGO and TNRC6 proteins operate. However, growing evidence points towards an additional mechanism through which AGO and TNRC6 regulate gene expression in the nucleus. While several mechanisms through which miRISC components function in the nucleus have been described, in this review we aim to summarize the major findings that have shed light on the role of AGO and TNRC6 in mammalian chromatin biology and on the implications these novel mechanisms may have in our understanding of regulating gene expression.

Modulation of miRISC-Mediated Gene Silencing in Eukaryotes.

Gene expression is regulated at multiple levels in eukaryotic cells. Regulation at the post-transcriptional level is modulated by various trans-acting factors that bind to specific sequences in the messenger RNA (mRNA). The binding of different trans factors influences various aspects of the mRNA such as degradation rate, translation efficiency, splicing, localization, etc. MicroRNAs (miRNAs) are short endogenous ncRNAs that combine with the Argonaute to form the microRNA-induced silencing complex (miRISC), which uses base-pair complementation to silence the target transcript. RNA-binding proteins (RBPs) contribute to post-transcriptional control by influencing the mRNA stability and translation upon binding to cis-elements within the mRNA transcript. RBPs have been shown to impact gene expression through influencing the miRISC biogenesis, composition, or miRISC-mRNA target interaction. While there is clear evidence that those interactions between RBPs, miRNAs, miRISC and target mRNAs influence the efficiency of miRISC-mediated gene silencing, the exact mechanism for most of them remains unclear. This review summarizes our current knowledge on gene expression regulation through interactions of miRNAs and RBPs.

De novo variants in AGO1 recapitulate a heterogeneous neurodevelopmental disorder phenotype

AGO1, as one of the rare genes in neurodevelopmental disorders, is involved in the microRNA-induced silencing complex. Here, we describe the clinical and genetic features of 18 individuals with de novo AGO1 variants: four new and 14 previously reported. Three variants are identified: two in-frame deletion variants and one missense variant. The spectrum of AGO1-related disorders included global development delay (GDD), intellectual disability (ID) with or without epilepsy, autism spectrum disorder, hypotonia and dysmorphisms. Focal seizures are the most common type of seizure, occasionally with atypical absence. Mild deafness may be a new phenotype of AGO1-releated disease. Gly199Ser may be a hot-spot variant of AGO1 with the same phenotype: GDD/ID, intractable epilepsy, remarkably with Rolandic discharges, and even reaching electrical status epilepticus during sleep.

MicroRNA-mediated regulation of microRNA machinery controls cell fate decisions.

microRNAs associate with Argonaute proteins, forming the microRNA-induced silencing complex (miRISC), to repress target gene expression post-transcriptionally. Although microRNAs are critical regulators in mammalian cell differentiation, our understanding of how microRNA machinery, such as the miRISC, are regulated during development is still limited. We previously showed that repressing the production of one Argonaute protein, Ago2, by Trim71 is important for mouse embryonic stem cells (mESCs) self-renewal (Liu et al., 2021). Here, we show that among the four Argonaute proteins in mammals, Ago2 is the major developmentally regulated Argonaute protein in mESCs. Moreover, in pluripotency, besides the Trim71-mediated regulation of Ago2 (Liu et al., 2021), Mir182/Mir183 also repress Ago2. Specific inhibition of this microRNA-mediated repression results in stemness defects and accelerated differentiation through the let-7 microRNA pathway. These results reveal a microRNA-mediated regulatory circuit on microRNA machinery that is critical to maintaining pluripotency.

Evaluation of expression profile of miRNAs with regulatory functions in metabolic pathways and signaling of parasitic diseases

Background: MicroRNAs (miRNAs) are a subset of small regulatory RNAs that are expressed by almost all metazoans and protozoans. They express genes either by direct cleavage or by suppressing the translation of target mRNAs by partial complementary base pairing. The active and functional unit of miRNA is a complex of Argonaute proteins known as microRNA-induced silencing complex (miRISC). They are known to regulate various growth and physiological processes. Irregular expression of miRNA in human cells is associated with a variety of disorders such as cancer, cardiovascular dysfunction, liver damage, immune dysfunction, metabolic syndromes, and pathogenic infections. A growing number of studies have shown that miRNAs are in fact a major component of host interactions and pathogens and play an important role in host immune responses to microorganisms. Emerging miRNAs are recognized as important tools for the genetic study, therapeutic development, and diagnosis of human pathogenic infections caused by various pathogenic organisms such as viruses, bacteria, parasites, and fungi. Many pathogens use the host miRNA system for their own benefits, including pathogenesis, survival within the host cell, and crossing some host immune barriers. Other pathogens express their miRNA within the host and contribute to their replication, survival, or delay. This article aims to review the role and importance of miRNA in relation to some important parasitic diseases.

Poly(ADP-ribosyl)ation enhances HuR oligomerization and contributes to pro-inflammatory gene mRNA stabilization

Poly(ADP-ribosyl)ation (PARylation) is an important post-translational modification mainly catalyzed by poly-ADP-ribose polymerase 1 (PARP1). In addition to having important roles in DNA damage detection and repair, it functions in gene expression regulation, especially at the posttranscriptional level. Embryonic lethal abnormal vision-like 1/human antigen R (ELAVL/HuR), a canonical 3′ untranslated region AU-rich element-binding protein, is a crucial mRNA-stabilizing protein that protects target mRNAs from RNA-destabilizing protein- or microRNA-induced silencing complex (miRISC)-mediated degradation. Additionally, in some cases, HuR itself either promotes or suppresses translation. Here, we demonstrated that in response to inflammatory stimuli, the PARylation of HuR, mostly at the conserved D226 site, by PARP1 increased the formation of the HuR oligomer/multimer, and HuR oligomerization promoted the disassociation of miRISC and stabilized the pro-inflammatory gene mRNAs. The prevention of PARP1 activation or HuR oligomerization attenuated lipopolysaccharide-induced inflammatory gene expression and the airway recruitment of neutrophils in mouse lungs. The present study verified a novel mechanism of PARP1 and HuR PARylation in the RNA stability regulation, increasing our understanding of how PARP1 regulates gene expression.

Suppression of Endothelial AGO1 Promotes Adipose Tissue Browning and Improves Metabolic Dysfunction.

Metabolic disorders such as obesity and diabetes mellitus can cause dysfunction of endothelial cells (ECs) and vascular rarefaction in adipose tissues. However, the modulatory role of ECs in adipose tissue function is not fully understood. Other than vascular endothelial growth factor-vascular endothelial growth factor receptor-mediated angiogenic signaling, little is known about the EC-derived signals in adipose tissue regulation. We previously identified Argonaute 1 (AGO1; a key component of microRNA-induced silencing complex) as a crucial regulator in hypoxia-induced angiogenesis. In this study, we intend to determine the AGO1-mediated EC transcriptome, the functional importance of AGO1-regulated endothelial function in vivo, and the relevance to adipose tissue function and obesity. We generated and subjected mice with EC-AGO1 deletion (EC-AGO1-knockout [KO]) and their wild-type littermates to a fast food-mimicking, high-fat high-sucrose diet and profiled the metabolic phenotypes. We used crosslinking immunoprecipitation- and RNA-sequencing to identify the AGO1-mediated mechanisms underlying the observed metabolic phenotype of EC-AGO1-KO. We further leveraged cell cultures and mouse models to validate the functional importance of the identified molecular pathway, for which the translational relevance was explored using human endothelium isolated from healthy donors and donors with obesity/type 2 diabetes mellitus. We identified an antiobesity phenotype of EC-AGO1-KO, evident by lower body weight and body fat, improved insulin sensitivity, and enhanced energy expenditure. At the organ level, we observed the most significant phenotype in the subcutaneous and brown adipose tissues of KO mice, with greater vascularity and enhanced browning and thermogenesis. Mechanistically, EC-AGO1 suppression results in inhibition of thrombospondin-1 (THBS1/TSP1), an antiangiogenic and proinflammatory cytokine that promotes insulin resistance. In EC-AGO1-KO mice, overexpression of TSP1 substantially attenuated the beneficial phenotype. In human endothelium isolated from donors with obesity or type 2 diabetes mellitus, AGO1 and THBS1 are expressed at higher levels than the healthy controls, supporting a pathological role of this pathway. Our study suggests a novel mechanism by which ECs, through the AGO1-TSP1 pathway, control vascularization and function of adipose tissues, insulin sensitivity, and whole-body metabolic state.

MicroRNA-668 represses MTP18 to preserve mitochondrial dynamics in ischemic acute kidney injury

The pathogenesis of ischemic diseases remains unclear. Here we demonstrate the induction of microRNA-668 (miR-668) in ischemic acute kidney injury (AKI) in human patients, mice, and renal tubular cells. The induction was HIF-1 dependent, as HIF-1 deficiency in cells and kidney proximal tubules attenuated miR-668 expression. We further identified a functional HIF-1 binding site in the miR-668 gene promoter. Anti-miR-668 increased apoptosis in renal tubular cells and enhanced ischemic AKI in mice, whereas miR-668 mimic was protective. Mechanistically, anti-miR-668 induced mitochondrial fragmentation, whereas miR-668 blocked mitochondrial fragmentation during hypoxia. We analyzed miR-668 target genes through immunoprecipitation of microRNA-induced silencing complexes followed by RNA deep sequencing and identified 124 protein-coding genes as likely targets of miR-668. Among these genes, only mitochondrial protein 18 kDa (MTP18) has been implicated in mitochondrial dynamics. In renal cells and mouse kidneys, miR-668 mimic suppressed MTP18, whereas anti-miR-668 increased MTP18 expression. Luciferase microRNA target reporter assay further verified MTP18 as a direct target of miR-668. In renal tubular cells, knockdown of MTP18 suppressed mitochondrial fragmentation and apoptosis. Together, the results suggest that miR-668 is induced via HIF-1 in ischemic AKI and that, upon induction, miR-668 represses MTP18 to preserve mitochondrial dynamics for renal tubular cell survival and kidney protection.

P38 activation induces the dissociation of tristetraprolin from Argonaute 2 to increase ARE-mRNA stabilization.

ARE-mRNAs are actively degraded with tristetraprolin (TTP) in resting cells while they turn into stable messengers in activated cells. P38 plays a crucial role in stabilizing ARE-mRNA. Here we reveal that P38 activation represses the interaction between TTP and Ago2, thus restraining TTP from being targeted into processing bodies and stabilizing ARE-mRNA.

FUS Regulates Activity of MicroRNA-Mediated Gene Silencing

MicroRNA-mediated gene silencing is a fundamental mechanism in the regulation of gene expression. It remains unclear how the efficiency of RNA silencing could be influenced by RNA-binding proteins associated with the microRNA-induced silencing complex (miRISC). Here we report that fused in sarcoma (FUS), an RNA-binding protein linked to neurodegenerative diseases including amyotrophic lateral sclerosis (ALS), interacts with the core miRISC component AGO2 and is required for optimal microRNA-mediated gene silencing. FUS promotes gene silencing by binding to microRNA and mRNA targets, as illustrated by its action on miR-200c and its target ZEB1. A truncated mutant form of FUS that leads its carriers to an aggressive form of ALS, R495X, impairs microRNA-mediated gene silencing. The C.elegans homolog fust-1 also shares a conserved role in regulating the microRNA pathway. Collectively, our results suggest a role for FUS in regulating the activity of microRNA-mediated silencing.

Argonaute Utilization for miRNA Silencing Is Determined by Phosphorylation-Dependent Recruitment of LIM-Domain-Containing Proteins

SummaryAs core components of the microRNA-induced silencing complex (miRISC), Argonaute (AGO) proteins interact with TNRC6 proteins, recruiting other effectors of translational repression/mRNA destabilization. Here, we show that LIMD1 coordinates the assembly of an AGO-TNRC6 containing miRISC complex by binding both proteins simultaneously at distinct interfaces. Phosphorylation of AGO2 at Ser 387 by Akt3 induces LIMD1 binding, which in turn enables AGO2 to interact with TNRC6A and downstream effector DDX6. Conservation of this serine in AGO1 and 4 indicates this mechanism may be a fundamental requirement for AGO function and miRISC assembly. Upon CRISPR-Cas9-mediated knockout of LIMD1, AGO2 miRNA-silencing function is lost and miRNA silencing becomes dependent on a complex formed by AGO3 and the LIMD1 family member WTIP. The switch to AGO3 utilization occurs due to the presence of a glutamic acid residue (E390) on the interaction interface, which allows AGO3 to bind to LIMD1, AJUBA, and WTIP irrespective of Akt signaling.

Regulation of cardiac CACNB2 by microRNA-499: Potential role in atrial fibrillation

The L-type calcium channel (LTCC) is one of the major ion channels that are known to be associated with the electrical remodeling of atrial fibrillation (AF). In AF, there is significant downregulation of the LTCC, but the underlying mechanism for such downregulation is not clear. We have previously reported that microRNA-499 (miR-499) is significantly upregulated in patients with permanent AF and that KCNN3, the gene that encodes the small-conductance calcium-activated potassium channel 3 (SK3), is a target of miR-499. We found that CACNB2, an important subunit of the LTCC, is also a target of miR-499. We hypothesize that miR-499 plays an important role in AF electrical remodeling by regulating the expression of CACNB2 and the LTCC. In atrial tissue from patients with permanent AF, CACNB2 was significantly downregulated by 67% (n=4, p<0.05) compared to those from patients with no history of AF. Transfection of miR-499 mimic into HL-1 cells, a mouse hyperplastic atrial cardiac myocyte cell-line, resulted in the downregulation of CACNB2 protein expression, while that of miR-499 inhibitor upregulated CACNB2 protein expression. Binding of miR-499 to the 3′ untranslated region of CACNB2 was confirmed by luciferase reporter assay and by the increased presence of CACNB2 mRNA in Argonaute pulled-down microRNA-induced silencing complexes after transfection with the miR-499 mimic. In addition, downregulation of CACNB2 resulted in the downregulation of protein levels of the pore-forming α-subunit (CACNA1C). In conclusion, upregulation of atrial miR-499 induces the downregulation of CACNB2 expression and may contribute to the electrical remodeling in AF.

GW182-Free microRNA Silencing Complex Controls Post-transcriptional Gene Expression during Caenorhabditis elegans Embryogenesis.

MicroRNAs and Argonaute form the microRNA induced silencing complex or miRISC that recruits GW182, causing mRNA degradation and/or translational repression. Despite the clear conservation and molecular significance, it is unknown if miRISC-GW182 interaction is essential for gene silencing during animal development. Using Caenorhabditis elegans to explore this question, we examined the relationship and effect on gene silencing between the GW182 orthologs, AIN-1 and AIN-2, and the microRNA-specific Argonaute, ALG-1. Homology modeling based on human Argonaute structures indicated that ALG-1 possesses conserved Tryptophan-binding Pockets required for GW182 binding. We show in vitro and in vivo that their mutations severely altered the association with AIN-1 and AIN-2. ALG-1 tryptophan-binding pockets mutant animals retained microRNA-binding and processing ability, but were deficient in reporter silencing activity. Interestingly, the ALG-1 tryptophan-binding pockets mutant phenocopied the loss of alg-1 in worms during larval stages, yet was sufficient to rescue embryonic lethality, indicating the dispensability of AINs association with the miRISC at this developmental stage. The dispensability of AINs in miRNA regulation is further demonstrated by the capacity of ALG-1 tryptophan-binding pockets mutant to regulate a target of the embryonic mir-35 microRNA family. Thus, our results demonstrate that the microRNA pathway can act independently of GW182 proteins during C. elegans embryogenesis.

Single-Molecule Fluorescence Energy Transfer Assays for the Characterization of Reaction Pathways of miRNA-Argonaute Complex.

Argonaute proteins are key components of the microRNA-induced silencing complexes (miRISCs) that mediate the posttranscriptional gene silencing of microRNAs and small interfering RNA (siRNAs). The complex reaction mechanism of miRISC is expected to be characterized by tracing the reaction pathways of miRISC at the single-molecule level in real time. In this chapter, we describe single-molecule fluorescence resonance energy transfer (FRET) assays to observe the target binding and reaction pathways of miRISC composed of a recombinant Argonaute and a small RNA.

The Smaug RNA-Binding Protein Is Essential for microRNA Synthesis During the Drosophila Maternal-to-Zygotic Transition

Metazoan embryos undergo a maternal-to-zygotic transition (MZT) during which maternal gene products are eliminated and the zygotic genome becomes transcriptionally active. During this process, RNA-binding proteins (RBPs) and the microRNA-induced silencing complex (miRISC) target maternal mRNAs for degradation. In Drosophila, the Smaug (SMG), Brain tumor (BRAT), and Pumilio (PUM) RBPs bind to and direct the degradation of largely distinct subsets of maternal mRNAs. SMG has also been shown to be required for zygotic synthesis of mRNAs and several members of the miR-309 family of microRNAs (miRNAs) during the MZT. Here, we have carried out global analysis of small RNAs both in wild-type and in smg mutants. Our results show that 85% of all miRNA species encoded by the genome are present during the MZT. Whereas loss of SMG has no detectable effect on Piwi-interacting RNAs (piRNAs) or small interfering RNAs (siRNAs), zygotic production of more than 70 species of miRNAs fails or is delayed in smg mutants. SMG is also required for the synthesis and stability of a key miRISC component, Argonaute 1 (AGO1), but plays no role in accumulation of the Argonaute family proteins associated with piRNAs or siRNAs. In smg mutants, maternal mRNAs that are predicted targets of the SMG-dependent zygotic miRNAs fail to be cleared. BRAT and PUM share target mRNAs with these miRNAs but not with SMG itself. We hypothesize that SMG controls the MZT, not only through direct targeting of a subset of maternal mRNAs for degradation but, indirectly, through production and function of miRNAs and miRISC, which act together with BRAT and/or PUM to control clearance of a distinct subset of maternal mRNAs.

Mammalian microRNA: an important modulator of host-pathogen interactions in human viral infections.

MicroRNAs (miRNAs), which are small non-coding RNAs expressed by almost all metazoans, have key roles in the regulation of cell differentiation, organism development and gene expression. Thousands of miRNAs regulating approximately 60 % of the total human genome have been identified. They regulate genetic expression either by direct cleavage or by translational repression of the target mRNAs recognized through partial complementary base pairing. The active and functional unit of miRNA is its complex with Argonaute proteins known as the microRNA-induced silencing complex (miRISC). De-regulated miRNA expression in the human cell may contribute to a diverse group of disorders including cancer, cardiovascular dysfunctions, liver damage, immunological dysfunction, metabolic syndromes and pathogenic infections. Current day studies have revealed that miRNAs are indeed a pivotal component of host-pathogen interactions and host immune responses toward microorganisms. miRNA is emerging as a tool for genetic study, therapeutic development and diagnosis for human pathogenic infections caused by viruses, bacteria, parasites and fungi. Many pathogens can exploit the host miRNA system for their own benefit such as surviving inside the host cell, replication, pathogenesis and bypassing some host immune barriers, while some express pathogen-encoded miRNA inside the host contributing to their replication, survival and/or latency. In this review, we discuss the role and significance of miRNA in relation to some pathogenic viruses.