RNA Interference Components Research Articles

Identification of an active RNAi pathway in Candida albicans

RNA interference (RNAi) is a fundamental regulatory pathway with a wide range of functions, including regulation of gene expression and maintenance of genome stability. Although RNAi is widespread in the fungal kingdom, well-known species, such as the model yeast Saccharomyces cerevisiae, have lost the RNAi pathway. Until now evidence has been lacking for a fully functional RNAi pathway in Candida albicans, a human fungal pathogen considered critically important by the World Health Organization. Here, we demonstrated that the widely used C. albicans reference strain (SC5314) contains an inactivating missense mutation in the gene encoding for the central RNAi component Argonaute. In contrast, most other C. albicans isolates contain a canonical Argonaute protein predicted to be functional and RNAi-active. Indeed, using high-throughput small and long RNA sequencing combined with seamless CRISPR/Cas9-based gene editing, we demonstrate that an active C. albicans RNAi machinery represses expression of subtelomeric gene families. Thus, an intact and functional RNAi pathway exists in C. albicans, highlighting the importance of using multiple reference strains when studying this dangerous pathogen.

Therapeutic siRNAs: small molecules with bigger function

In the realm of molecular medicine, therapeutic small-interfering RNA (siRNA) is a promising class of molecules with exciting new opportunities in healthcare. An essential component of RNA interference (RNAi) is siRNA. In clinical setup, siRNA may be engineered to specifically target genes linked to certain diseases, providing a focused and accurate method of modulating gene expression. Many important factors characterize the use of therapeutic siRNA. Target gene expression is selectively silenced by therapeutic siRNA through the use of the RNAi pathway. Guided by corresponding messenger RNA (mRNA) sequences, the double-stranded siRNA molecule is integrated into the RNA-induced silencing complex (RISC). This mechanism causes translational repression or mRNA cleavage, which in turn causes the relevant protein to be downregulated. Therapeutic siRNA is a promising avenue in several areas. siRNA can be used in oncology to target oncogenes or other important regulators, inhibiting cancer cell proliferation. Furthermore, by modifying the expression of genes linked to disease, siRNA may be used to treat viral infections, neurological conditions, and other rare and hereditary diseases. The selectivity of therapeutic siRNA is one of its main advantages. siRNA sequences can be engineered and customized to target specific genes linked to different diseases like cancer or genetic disorders. This focused strategy improves the accuracy of the therapeutic intervention and reduces off-target effects. In a recent article by Nissen et al., siRNA targeting LP(a) in a trial with 48 participants without cardiovascular disease and with lipoprotein(a) concentrations of 75 nmol/L or greater (or ≥30 mg/dL), was used to evaluate its tolerability and efficacy to reduce LP(a). Results showed that Lepodisiran was found to be well tolerated and resulted in dose-dependent, long-lasting reduction in serum lipoprotein(a) concentrations in phase 1 trial with elevated lipoprotein(a) levels. Therapeutic siRNA-based diagnosis is rapidly advancing, and several siRNA-based medications are now entering clinical trials. These trials aim to assess the pharmacokinetics, safety, and effectiveness of siRNA therapeutics in human subjects, offering crucial new information about their potential as a treatment option. Despite the therapeutic potential of siRNA, clinical translation of siRNA faces a challenge in its effective delivery. Naked siRNA molecules are more likely to degrade with a poor half-life and poor cellular uptake. Therefore, to increase the stability and delivery of therapeutic siRNA to target cells, a variety of delivery systems, such as lipid nanoparticles, viral vectors, and polymer-based carriers, are being investigated.
 

Tissue-specific overexpression of systemic RNA interference components limits lifespan in C. elegans

Intertissue RNA transport recently emerged as a novel signaling mechanism. In mammals, mounting evidence suggests that small RNA transfer between cells is widespread and used in various physiological contexts. In the nematode C. elegans, a similar mechanism is conferred by the systemic RNAi pathway. Members of the Systemic RNA Interference Defective (SID) family act at different steps of cellular RNA uptake and export. The limiting step in systemic RNA interference (RNAi) is the import of extracellular RNAs via the conserved double-stranded (dsRNA)-gated dsRNA channel SID-1. To better understand the role of RNAs as intertissue signaling molecules, we modified the function of SID-1 in specific tissues of C. elegans. We observed that sid-1 loss-of-function mutants are as healthy as wild-type worms. Conversely, overexpression of sid-1 in C. elegans intestine, muscle, or neurons rendered worms short-lived. The effects of intestinal sid-1 overexpression were attenuated by silencing the components of systemic RNAi sid-1, sid-2 and sid-5, implicating systemic RNA signaling in the lifespan reduction. Accordingly, tissue-specific overexpression of sid-2 and sid-5 also reduced worm lifespan. Additionally, an RNAi screen for components of several non-coding RNA pathways revealed that silencing the miRNA biogenesis proteins PASH-1 and DCR-1 rendered the lifespan of worms with intestinal sid-1 overexpression similar to controls. Collectively, our data support the notion that systemic RNA signaling must be tightly regulated, and unbalancing that process provokes a reduction in lifespan. We termed this phenomenon Intercellular/Extracellular Systemic RNA imbalance (InExS).

RNA interference of Aspergillus flavus in response to Aspergillus flavus partitivirus 1 infection.

RNA interference (RNAi) is one of the important defense responses against viral infection, but its mechanism and impact remain unclear in mycovirus infections. In our study, reverse genetics and virus-derived small RNA sequencing were used to show the antiviral responses of RNAi components in Aspergillus flavus infected with Aspergillus flavus partitivirus 1 (AfPV1). qRT-PCR revealed that AfPV1 infection induced the expression of the RNAi components in A. flavus compared with noninfected A. flavus. Knock mutants of each RNAi component were generated, but the mutants did not exhibit any obvious phenotypic changes compared with the A. flavus parental strain. However, after AfPV1 inoculation, production of AfPV1 was significantly less than in the parental strain. Furthermore, sporulation was greater in each AfPV1-infected mutant compared with the AfPV1-infected parental A. flavus. We also investigated the sensitivity of virus-free and AfPV1-infected RNAi mutants and the parental strain to cell wall stress, osmotic stress, genotoxic stress, and oxidative stress. The mutants of DCLs and AGOs infected by AfPV1 displayed more changes than RDRP mutants in response to the first three stresses. Small RNA sequencing analysis suggested that AfPV1 infection reduced the number of unique reads of sRNA in A. flavus, although there were many vsiRNA derived from the AfPV1 genome. GO term and KEGG pathway analyses revealed that the functions of sRNA affected by AfPV1 infection were closely related to vacuole production. These results provide a better understanding of the functional role of RNAi in the impact of AfPV1 on the hypovirulence of A. flavus.

Structural mechanism of R2D2 and Loqs-PD synergistic modulation on DmDcr-2 oligomers

Small interference RNAs are the key components of RNA interference, a conserved RNA silencing or viral defense mechanism in many eukaryotes. In Drosophila melanogaster, Dicer-2 (DmDcr-2)-mediated RNAi pathway plays important roles in defending against viral infections and protecting genome integrity. During the maturation of siRNAs, two cofactors can regulate DmDcr-2’s functions: Loqs-PD that is required for dsRNA processing, and R2D2 that is essential for the subsequent loading of siRNAs into effector Ago2 to form RISC complexes. However, due to the lack of structural information, it is still unclear whether R2D2 and Loqs-PD affect the functions of DmDcr-2 simultaneously. Here we present several cryo-EM structures of DmDcr-2/R2D2/Loqs-PD complex bound to dsRNAs with various lengths by the Helicase domain. These structures revealed that R2D2 and Loqs-PD can bind to different regions of DmDcr-2 without interfering with each other. Furthermore, the cryo-EM results demonstrate that these complexes can form large oligomers and assemble into fibers. The formation and depolymerization of these oligomers are associated with ATP hydrolysis. These findings provide insights into the structural mechanism of DmDcr-2 and its cofactors during siRNA processing.

Near-infrared light and PIF4 promote plant antiviral defense by enhancing RNA interference

The molecular mechanism underlying phototherapy and light treatment, which utilize various wavelength spectra of light, including near-infrared (NIR), to cure human and plant diseases, is obscure. Here we revealed that NIR light confers antiviral immunity by positively regulating PHYTOCHROME-INTERACTING FACTOR 4 (PIF4)-activated RNA interference (RNAi) in plants. PIF4, a central transcription factor involved in light signaling, accumulates to high levels under NIR light in plants. PIF4 directly induces the transcription of two essential components of RNAi, RNA-DEPENDENT RNA POLYMERASE 6 (RDR6) and ARGONAUTE 1 (AGO1), which play important roles in resistance to both DNA and RNA viruses. Moreover, the pathogenic determinant βC1 protein, which is evolutionarily conserved and encoded by betasatellites, interacts with PIF4 and inhibits its positive regulation of RNAi by disrupting PIF4 dimerization. These findings shed light on the molecular mechanism of PIF4-mediated plant defense and provide a new perspective for the exploration of NIR antiviral treatment.

Disruption of the Aspergillus fumigatus RNA interference machinery alters the conidial transcriptome.

The RNA interference (RNAi) pathway has evolved numerous functionalities in eukaryotes, with many on display in Kingdom Fungi. RNAi can regulate gene expression, facilitate drug resistance, or even be altogether lost to improve growth potential in some fungal pathogens. In the WHO fungal priority pathogen, Aspergillus fumigatus, the RNAi system is known to be intact and functional. To extend our limited understanding of A. fumigatus RNAi, we first investigated the genetic variation in RNAi-associated genes in a collection of 217 environmental and 83 clinical genomes, where we found that RNAi components are conserved even in clinical strains. Using endogenously expressed inverted-repeat transgenes complementary to a conditionally essential gene (pabA) or a nonessential gene (pksP), we determined that a subset of the RNAi componentry is active in inverted-repeat transgene silencing in conidia and mycelium. Analysis of mRNA-seq data from RNAi double-knockout strains linked the A. fumigatus dicer-like enzymes (DclA/B) and RNA-dependent RNA polymerases (RrpA/B) to regulation of conidial ribosome biogenesis genes; however, surprisingly few endogenous small RNAs were identified in conidia that could explain this broad change. Although RNAi was not clearly linked to growth or stress response defects in the RNAi knockouts, serial passaging of RNAi knockout strains for six generations resulted in lineages with diminished spore production over time, indicating that loss of RNAi can exert a fitness cost on the fungus. Cumulatively, A. fumigatus RNAi appears to play an active role in defense against double-stranded RNA species alongside a previously unappreciated housekeeping function in regulation of conidial ribosomal biogenesis genes.

PSMC3 promotes RNAi by maintaining AGO2 stability through USP14

BackgroundArgonaute 2 (AGO2), the only protein with catalytic activity in the human Argonaute family, is considered as a key component of RNA interference (RNAi) pathway. Here we performed a yeast two-hybrid screen using the human Argonaute 2 PIWI domain as bait to screen for new AGO2-interacting proteins and explored the specific mechanism through a series of molecular biology and biochemistry experiments.MethodsThe yeast two-hybrid system was used to screen for AGO2-interacting proteins. Co-immunoprecipitation and immunofluorescence assays were used to further determine interactions and co-localization. Truncated plasmids were constructed to clarify the interaction domain. EGFP fluorescence assay was performed to determine the effect of PSMC3 on RNAi. Regulation of AGO2 protein expression and ubiquitination by PSMC3 and USP14 was examined by western blotting. RT-qPCR assays were applied to assess the level of AGO2 mRNA. Rescue assays were also performed.ResultsWe identified PSMC3 (proteasome 26S subunit, ATPase, 3) as a novel AGO2 binding partner. Biochemical and bioinformatic analysis demonstrates that this interaction is performed in an RNA-independent manner and the N-terminal coiled-coil motif of PSMC3 is required. Depletion of PSMC3 impairs the activity of the targeted cleavage mediated by small RNAs. Further studies showed that depletion of PSMC3 decreased AGO2 protein amount, whereas PSMC3 overexpression increased the expression of AGO2 at a post-translational level. Cycloheximide treatment indicated that PSMC3 depletion resulted in a decrease in cytoplasmic AGO2 amount due to an increase in AGO2 protein turnover. The absence of PSMC3 promoted ubiquitination of AGO2, resulting in its degradation by the 26S proteasome. Mechanistically, PSMC3 assists in the interaction of AGO2 with the deubiquitylase USP14(ubiquitin specific peptidase 14) and facilitates USP14-mediated deubiquitination of AGO2. As a result, AGO2 is stabilized, which then promotes RNAi.ConclusionOur findings demonstrate that PSMC3 plays an essential role in regulating the stability of AGO2 and thus in maintaining effective RNAi.

Comparative profiling of canonical and non-canonical small RNAs in the rice blast fungus, Magnaporthe oryzae.

RNA interference (RNAi) is divided into canonical, Dicer-dependent and non-canonical, Dicer-independent pathways according to Dicer protein dependency. However, sRNAs processed in a Dicer-independent manner have not been reported in plant pathogenic fungi, including Magnaporthe oryzae. We comparatively profiled the Dicer-dependent and -independent sRNAs of M. oryzae. Dicer-dependent sRNAs were 19–24-nt in length, had low strand-specificity, and showed a preference for uracil at the 5′-end. By contrast, Dicer-independent sRNAs presented irregular patterns in length distribution, high strand-specificity, and a preference for cytosine at the penultimate position. Dicer-dependent sRNA loci were mainly associated with LTR-transposons, while Dicer-independent sRNAs were associated with protein-coding genes and transposons. We identified MoERI-1, a non-canonical RNAi component, and profiled the sRNA and mRNA transcriptomes of ΔMoeri-1 at the mycelia and conidiation stages, as the mutant showed increased conidiation. We found that genes involved in conidiation and cell cycle were upregulated by MoERI-1 deletion. Furthermore, a comparison between sRNA and mRNA transcriptome revealed that MoERI-1-dependent sRNAs mediate the regulation of gene expression. Overall, these results showed that M. oryzae has non-canonical RNAi pathways distinct to the Dicer-dependent manner and exploits MoERI-1-dependent sRNAs to regulate the conidiation process.

Comprehensive In Silico Analysis of RNA Silencing-Related Genes and Their Regulatory Elements in Wheat (Triticum aestivum L.).

Dicer-like (DCL), Argonaute (AGO), and RNA-dependent RNA polymerase (RDR) are known as the three major gene families that act as the critical components of RNA interference or silencing mechanisms through the noncoding small RNA molecules (miRNA and siRNA) to regulate the expressions of protein-coding genes in eukaryotic organisms. However, most of their characteristics including structures, chromosomal location, subcellular locations, regulatory elements, and gene networking were not rigorously studied. Our analysis identified 7 TaDCL, 39 TaAGO, and 16 TaRDR genes as RNA interference (RNAi) genes from the wheat genome. Phylogenetic analysis of predicted RNAi proteins with the RNAi proteins of Arabidopsis and rice showed that the predicted proteins of TaDCL, TaAGO, and TaRDR groups are clustered into four, eight, and four subgroups, respectively. Domain, 3D protein structure, motif, and exon-intron structure analyses showed that these proteins conserve identical characteristics within groups and maintain differences between groups. The nonsynonymous/synonymous mutation ratio (Ka/Ks) < 1 suggested that these protein sequences conserve some purifying functions. RNAi genes networking with TFs revealed that ERF, MIKC-MADS, C2H2, BBR-BPC, MYB, and Dof are the key transcriptional regulators of the predicted RNAi-related genes. The cis-regulatory element (CREs) analysis detected some important CREs of RNAi genes that are significantly associated with light, stress, and hormone responses. Expression analysis based on an online database exhibited that almost all of the predicted RNAi genes are expressed in different tissues and organs. A case-control study from the gene expression level showed that some RNAi genes significantly responded to the drought and heat stresses. Overall results would therefore provide an excellent basis for in-depth molecular investigation of these genes and their regulatory elements for wheat crop improvement against different stressors.

Uncontrolled transposition following RNAi loss causes hypermutation and antifungal drug resistance in clinical isolates of Cryptococcus neoformans.

Cryptococcus neoformans infections cause approximately 15% of AIDS-related deaths owing to a combination of limited antifungal therapies and drug resistance. A collection of clinical and environmental C. neoformans isolates were assayed for increased mutation rates via fluctuation analysis, and we identified two hypermutator C. neoformans clinical isolates with increased mutation rates when exposed to the combination of rapamycin and FK506. Sequencing of drug target genes found that Cnl1 transposon insertions conferred the majority of resistance to rapamycin and FK506 and could also independently cause resistance to 5-fluoroorotic acid and the clinically relevant antifungal 5-flucytosine. Whole-genome sequencing revealed both hypermutator genomes harbour a nonsense mutation in the RNA-interference component ZNF3 and hundreds of Cnl1 elements organized into massive subtelomeric arrays on each of the fourteen chromosomes. Quantitative trait locus mapping in 28 progeny derived from a cross between a hypermutator and wild-type identified a locus associated with hypermutation that included znf3. CRISPR editing of the znf3 nonsense mutation abolished hypermutation and restored small-interfering-RNA production. We conclude that hypermutation and drug resistance in these clinical isolates result from RNA-interference loss and accumulation of Cnl1 elements.

Structural insights into dsRNA processing by Drosophila Dicer-2–Loqs-PD

Small interfering RNAs (siRNAs) are the key components for RNA interference (RNAi), a conserved RNA-silencing mechanism in many eukaryotes1,2. In Drosophila, an RNase III enzyme Dicer-2 (Dcr-2), aided by its cofactor Loquacious-PD (Loqs-PD), has an important role in generating 21 bp siRNA duplexes from long double-stranded RNAs (dsRNAs)3,4. ATP hydrolysis by the helicase domain of Dcr-2 is critical to the successful processing of a long dsRNA into consecutive siRNA duplexes5,6. Here we report the cryo-electron microscopy structures of Dcr-2–Loqs-PD in the apo state and in multiple states in which it is processing a 50 bp dsRNA substrate. The structures elucidated interactions between Dcr-2 and Loqs-PD, and substantial conformational changes of Dcr-2 during a dsRNA-processing cycle. The N-terminal helicase and domain of unknown function 283 (DUF283) domains undergo conformational changes after initial dsRNA binding, forming an ATP-binding pocket and a 5′-phosphate-binding pocket. The overall conformation of Dcr-2–Loqs-PD is relatively rigid during translocating along the dsRNA in the presence of ATP, whereas the interactions between the DUF283 and RIIIDb domains prevent non-specific cleavage during translocation by blocking the access of dsRNA to the RNase active centre. Additional ATP-dependent conformational changes are required to form an active dicing state and precisely cleave the dsRNA into a 21 bp siRNA duplex as confirmed by the structure in the post-dicing state. Collectively, this study revealed the molecular mechanism for the full cycle of ATP-dependent dsRNA processing by Dcr-2–Loqs-PD.

In silico analysis of RNA interference components and miRNAs-like RNAs in the seed-borne necrotrophic fungus Alternariabrassicicola

RNA interference is a mechanism of suppressing gene expression in plants, animals and fungi. This regulation mechanism involves three main enzymes, Dicers (Dcr), Argonautes (Ago) and RNA Dependent RNA Polymerases (Rdrp) allowing to produce smallRNAs. RNA interference and smallRNAs have a role in the plant–microorganisms interaction, either in a pathogenic or in a symbiotic relationships. Alternaria brassicicola is a pathogenic fungus of the Brassicaceae plants. During plant infection, it is able to transmit itself vertically and horizontally, giving advantages for new infection and dissemination. To investigate RNA interference and the presence of smallRNAs in A. brassicicola, an in silico analysis was achieved. Two DCR, 4 AGO and 3 RDRP genes were identified comforting the presence of smallRNAs in A. brassicicola. SmallRNA sequencing from wild-type strain and DCR deleted mutants allowed the identifcation of 17 miRNAs in A. brassicicola. The synthesis of these miRNAs is only weakly influenced by the inactivation of DCR genes suggesting the possible existence of an alternative Dicer-independent miRNA synthesis pathway. Target's prediction of A. brassicicola miRNAs identified genes in the fungus and in the plant model Arabidopsis thaliana. Some miRNAs were predicted to target A. thaliana genes involved in the methylation of histone and in the disease resistance.

Functional Characterization of the Cnidarian Antiviral Immune Response Reveals Ancestral Complexity.

Animals evolved a broad repertoire of innate immune sensors and downstream effector cascades for defense against RNA viruses. Yet, this system varies greatly among different bilaterian animals, masking its ancestral state. In this study, we aimed to characterize the antiviral immune response of the cnidarian Nematostella vectensis and decipher the function of the retinoic acid-inducible gene I (RIG-I)-like receptors (RLRs) known to detect viral double-stranded RNA (dsRNA) in bilaterians but activate different antiviral pathways in vertebrates and nematodes. We show that polyinosinic:polycytidylic acid (poly(I:C)), a mimic of long viral dsRNA and a primary ligand for the vertebrate RLR melanoma differentiation-associated protein 5 (MDA5), triggers a complex antiviral immune response bearing features distinctive for both vertebrate and invertebrate systems. Importantly, a well-characterized agonist of the vertebrate RIG-I receptor does not induce a significant transcriptomic response that bears signature of the antiviral immune response, which experimentally supports the results of a phylogenetic analysis indicating clustering of the two N. vectensis RLR paralogs (NveRLRa and NveRLRb) with MDA5. Furthermore, the results of affinity assays reveal that NveRLRb binds poly(I:C) and long dsRNA and its knockdown impairs the expression of putative downstream effector genes including RNA interference components. Our study provides for the first time the functional evidence for the conserved role of RLRs in initiating immune response to dsRNA that originated before the cnidarian–bilaterian split and lay a strong foundation for future research on the evolution of the immune responses to RNA viruses.

Designing an effective therapeutic siRNA to silence RdRp gene of SARS-CoV-2.

The devastating outbreak of COVID-19 has spread all over the world and has become a global health concern. There is no specific therapeutics to encounter the COVID-19. Small interfering RNA (siRNA)-based therapy is an efficient strategy to control human viral infections employing post-transcriptional gene silencing (PTGS) through neutralizing target complementary mRNA. RNA-dependent RNA polymerase (RdRp) encoded by the viral RdRp gene as a part of the replication-transcription complex can be adopted as an acceptable target for controlling SARS-CoV-2 mediated infection. Therefore, in the current study, accessible siRNA designing tools, including significant algorithms and parameters, were rationally used to design the candidate siRNAs against SARS-COV-2 encoded RdRp. The designed siRNA molecules possessed adequate nucleotide-based and other features for potent gene silencing. The targets of the designed siRNAs revealed no significant matches within the whole human genome, ruling out any possibilities for off-target silencing by the siRNAs. Characterization with different potential parameters of efficacy allowed selecting the finest siRNA among all the designed siRNA molecules. Further, validation assessment and target site accessibility prediction also rationalized the suitability of this siRNA molecule. Molecular docking study between the selected siRNA molecule and component of RNA interference (RNAi) pathway gave an excellent outcome. Molecular dynamics of two complexes: siRNA and argonaute complex, guide RNA, and target protein complex, have shown structural stability of these proteins. Therefore, the designed siRNA molecule might act as an effective therapeutic agent against the SARS-CoV-2 at the genome level and can prevent further outbreaks of COVID-19 in humans.

Testing the CRISPR-Cas9 and glmS ribozyme systems in Leishmania tarentolae

Leishmania parasites include important pathogens and model organisms and are even used for the production of recombinant proteins. However, functional genomics and the characterization of essential genes are often limited in Leishmania because of low-throughput technologies for gene disruption or tagging and the absence of components for RNA interference. Here, we tested the T7 RNA polymerase-dependent CRISPR-Cas9 system by Beneke et al. and the glmS ribozyme-based knock-down system in the model parasite Leishmania tarentolae. We successfully deleted two reference genes encoding the flagellar motility factor Pf16 and the salvage-pathway enzyme adenine phosphoribosyltransferase, resulting in immotile and drug-resistant parasites, respectively. In contrast, we were unable to disrupt the gene encoding the mitochondrial flavoprotein Erv. Cultivation of L. tarentolae in standard BHI medium resulted in a constitutive down-regulation of an episomal mCherry-glmS reporter by 40 to 60%. For inducible knock-downs, we evaluated the growth of L. tarentolae in alternative media and identified supplemented MEM, IMDM and McCoy’s 5A medium as candidates. Cultivation in supplemented MEM allowed an inducible, glucosamine concentration-dependent down-regulation of the episomal mCherry-glmS reporter by more than 70%. However, chromosomal glmS-tagging of the genes encoding Pf16, adenine phosphoribosyltransferase or Erv did not reveal a knock-down phenotype. Our data demonstrate the suitability of the CRISPR-Cas9 system for the disruption and tagging of genes in L. tarentolae as well as the limitations of the glmS system, which was restricted to moderate efficiencies for episomal knock-downs and caused no detectable phenotype for chromosomal knock-downs.

Integrative analysis of the RNA interference toolbox in two Salicaceae willow species, and their roles in stress response in poplar (Populus trichocarpa Torr. & Gray)

Regulation of gene expression related to chromatin modification at the transcriptional silencing and RNA interference (RNAi) at the post-transcriptional level. RNA-dependent RNA polymerase (RDR) and Argonaute (AGO), along with Dicer-like (DCL) from the core components of RNAi, play integral roles in these processes. Here, 14 PtAGOs, 5 PtDCLs, and 9 PtRDRs were identified in P. trichocarpa and compared them with those of another Salicaceae willow (Salix suchowensis Cheng). Maximum-likelihood trees revealed that each AGO, DCL, and RDR family members were divided into four subfamilies. Forty-three orthologous pairs were identified between the P. trichocarpa and S. suchowensis RNAi-toolbox genes. Sixteen collinear gene pairs were detected in highly microsynteny regions with containing more than ten pairs of conserved flanking-genes, indicated that they were considered to have evolved from the large-scale duplication events. Many of the RNAi-toolbox genes were up-regulated, suggesting P. trichocarpa should have evolved specialized regulatory mechanisms in response to cold, salt, drought and heat stresses. Some RNAi-toolbox genes were most highly expressed in stem, suggesting these genes may function in the regulation of small RNAs during P. trichocarpa stem development. Our results provided the integrative analysis and highlighted the function and duplication of the RNAi-toolbox genes in P. trichocarpa.

Predominant Distribution of the RNAi Machinery at Apical Adherens Junctions in Colonic Epithelia Is Disrupted in Cancer.

The RNA interference (RNAi) machinery is an essential component of the cell, regulating miRNA biogenesis and function. RNAi complexes were thought to localize either in the nucleus, such as the microprocessor, or in the cytoplasm, such as the RNA-induced silencing complex (RISC). We recently revealed that the core microprocessor components DROSHA and DGCR8, as well as the main components of RISC, including Ago2, also associate with the apical adherens junctions of well-differentiated cultured epithelial cells. Here, we demonstrate that the localization of the core RNAi components is specific and predominant at apical areas of cell-cell contact of human normal colon epithelial tissues and normal primary colon epithelial cells. Importantly, the apical junctional localization of RNAi proteins is disrupted or lost in human colon tumors and in poorly differentiated colon cancer cell lines, correlating with the dysregulation of the adherens junction component PLEKHA7. We show that the restoration of PLEKHA7 expression at adherens junctions of aggressively tumorigenic colon cancer cells restores the junctional localization of RNAi components and suppresses cancer cell growth in vitro and in vivo. In summary, this work identifies the apical junctional localization of the RNAi machinery as a key feature of the differentiated colonic epithelium, with a putative tumor suppressing function.

Conserved chromosomal functions of RNA interference.

RNA interference (RNAi), a cellular process through which small RNAs target and regulate complementary RNA transcripts, has well-characterized roles in post-transcriptional gene regulation and transposon repression. Recent studies have revealed additional conserved roles for RNAi proteins, such as Argonaute and Dicer, in chromosome function. By guiding chromatin modification, RNAi components promote chromosome segregation during both mitosis and meiosis and regulate chromosomal and genomic dosage response. Small RNAs and the RNAi machinery also participate in the resolution of DNA damage. Interestingly, many of these lesser-studied functions seem to be more strongly conserved across eukaryotes than are well-characterized functions such as the processing of microRNAs. These findings have implications for the evolution of RNAi since the last eukaryotic common ancestor, and they provide a more complete view of the functions of RNAi.

Expression of TNRC6 (GW182) Proteins Is Not Necessary for Gene Silencing by Fully Complementary RNA Duplexes.

The trinucleotide repeat containing 6 (TNRC6) family of proteins are core components of RNA interference (RNAi) and consist of three paralogs (TNRC6A, TNRC6B, and TNRC6C). The TNRC6 paralogs associate with argonaute (AGO) protein, the core RNAi factor, and bridge its interactions with other proteins. We obtained TNRC6A and TNRC6B single and double knockout cell lines to investigate how the TNRC6 paralogs contribute to RNAi. We found that TNRC6 proteins are not required for gene silencing when duplex RNAs are fully complementary. TNRC6 expression was necessary for regulation by a microRNA. TNRC6A, but not TNRC6B, expression was necessary for transcriptional activation by a duplex RNA targeting a gene promoter. By contrast, AGO2 is required for all three gene expression pathways. TNRC6A can affect the Dicer localization in cytoplasm versus the nucleus, but none of the three TNRC6 paralogs was necessary for nuclear localization of AGO2. Our data suggest that the roles of the TNRC6 paralogs differ in some details and that TNRC6 is not required for clinical therapeutic silencing mechanisms that involve fully complementary duplex RNAs.