RNA-induced Silencing Complex Complex Research Articles

Biogenesis and Function of miRNAs and Their Role in Cancer

MicroRNA (miRNA) is a small RNA molecule that regulates post-transcriptional gene expression. miRNA combines with the RNA-induced silencing complex (RISC) and carries out its function in controlling translation. The formation of miRNA involves enzymes and proteins that cut it in its processing as well as protein complexes in the cytoplasm that make the miRNA mature. Several changes, for example deletion or overexpression, can occur in miRNAs, causing cancer growth. The aim of this study was to investigate the process of miRNA biogenesis as well as analyze how changes in miRNA expression can affect the biological pathways involved in cancer. This type of research is a literature review. The data that has been collected is then analyzed in three stages, namely data reduction, data presentation and drawing conclusions. The results show that the role of miRNAs is in the post-transcriptional regulation of genes. MiRNAs associate with the RISC complex and interact with mRNA through complementary base pairing thereby halting translation and promoting mRNA degradation. MiRNAs are deregulated in cancer, including over-expression of oncogenic miRNAs or deletion of miRNA characteristics as tumor inhibitors. The results of this research also provide a foundation for further education and advanced research in the field of molecular biology, particularly in genetic regulation and disease mechanisms.

Gene silencing of Helicobacter pylori through newly designed siRNA convenes the treatment of gastric cancer.

Helicobacter pylori is a gastric pathogen that is responsible for causing chronic inflammation and increasing the risk of gastric cancer development. It is capable of persisting for decades in the harsh gastric environment because of the inability of the host to eradicate the infection. Several treatment strategies have been developed against this bacterium using different antibiotics. But the effectiveness of treating H. pylori has significantly decreased due to widespread antibiotic resistance, including an increased risk of gastric cancer. The small interfering RNAs (siRNA), which is capable of sequence-specific gene-silencing can be used as a new therapeutic approach for the treatment of a variety of such malignancies. In the current study, we rationally designed two siRNA molecules to silence the cytotoxin-associated gene A (CagA) and vacuolating cytotoxin A (VacA) genes of H. pylori for their significant involvement in developing cancer. We selected a common region of all the available transcripts from different countries of CagA and VacA to design the siRNA molecules. The final siRNA candidate was selected based on the results from machine learning algorithms, off-target similarity, and various thermodynamic properties. Further, we utilized molecular docking and all atom molecular dynamics (MD) simulations to assess the binding interactions of the designed siRNAs with the major components of the RNA-induced silencing complex (RISC) and results revealed the ability of the designed siRNAs to interact with the proteins of RISC complex in comparable to those of the experimentally reported siRNAs. These designed siRNAs should effectively silence the CagA and VacA genes of H. pylori during siRNA mediated treatment in gastric cancer.

Molecular modeling of PARP1 inhibition through miRNA gene silencing for the treatment of retinitis pigmentosa.

Retinitis Pigmentosa (RP) is a category of diseases that are characterized by the death of rod photoreceptors and subsequent death of cone receptors. Parthanatos, a form of regulated necrosis, has been reported as being the underlying mechanism of cell death in RP. Parthanatos in RP occurs with the downstream expression of poly-ADP-ribose-polymerase (PARP) and the subsequent nuclear translocation of the mitochondrial apoptosis-inducing factor (AIF), leading to condensing of chromatin and DNA fragmentation. Extracellular vesicles (EVs) are small-scale liposomes that carry miRNAs and the RNA-induced silencing complex (RISC) associated with gene regulation. miRNAs, loaded on the argonaute protein 2 (Ago-2) bind to the mature mRNA and initiate the recruitment of the RISC complex triggering the degradation of the mRNA, dramatically scaling down gene expression. In vitro validation of gene silencing is a time expensive process and computational resources provide faster and cheaper options. Here, we identified the miRNAs target sequences on the mature PARP1 mRNA construct from the TargetScan database and obtained their complementary miRNA sequences from the miRbase. Using the human Ago-2 crystal structure deposited to the PDB database (PDB ID: 4W5Q) as a template, mutational studies were conducted on the protein/mRNA/miRNA complex to reproduce PARP1 mRNA seed pairing to miRNAs. Upon initial geometry optimization, further conformational search studies were done for thorough exploration of the conformational space of the protein/RNA complex by implementing conformational flooding (CF) molecular dynamics (MD) simulations. CF MD introduces a flooding potential to the force field, thus lowering energy barriers and facilitating observations of macroscale conformational changes. Clustering of the CF trajectories reveal the stable conformations of the Ago-2/miRNA/mRNA complexes. Energetically favorable miRNAs discovered in silico will further be validated with in vitro silencing assays.

MicroRNA biogenesis. Part 2. Formation of mature miRNAs. Maturation of non-canonical miRNAs

The scientific review presents the biogenesis of miRNAs. To write the article, information was searched using databases Scopus, Web of Science, MedLine, PubMed, Google Scholar, EMBASE, Global Health, The Cochrane Library, CyberLeninka. The article shows the stages of formation of mature miRNAs. It is noted that duplex RNAs resulting from DICER-mediated cleavage interact with Argonaute (AGO) proteins to form an effector RNA-induced silencing complex (RISC). It is shown that the deficiency of AGO proteins leads to a significant decrease in the amount of miRs, and overexpression of AGO proteins is accompanied by an increase in the level of miRs. The main stages of assembling a fully functional RISC are presented. The first stage is the loading of duplex miRs on AGO proteins. The second stage is the promotion of duplex miRs. Human diseases associated with processing disorders in the cytoplasm of the cell are presented. Numerous alternative mechanisms involved in the formation of functionally active miRs are is characterized. There are three classes of mirtrons: typical mirtrons, 5’-tailed mirtrons and 3’-tailed mirtrons. Endogenous csRNAs resemble Drosha-independent synthetic csRNAs used to experimentally induce gene knockout. Chimeric hairpins of non-canonical miR genes are transcribed in tandem or as a part of another type of small RNA gene. Thus, the formation of mature miRs occurs due to the formation of the RISC complex. The core of the RISC complex consists of microRNA, AGO and protein with a trinucleotide repeat 6. Loading dsRNA on AGO proteins and subsequent promotion of duplex RNA are the main stages of assembly of a fully functional RISC. Disorders of processing in the cytoplasm of the cell are associated with the development of some human diseases. There are alternative mechanisms involved in the formation of functionally active miRs: mirtrons, endogenous short RNAs containing hairpins, chimeric hairpins.

Abstract B39: Characterization of the interaction between KRAS and Argonaute 2

Abstract KRAS is one of three members of the RAS family of small GTPases, and nearly one third of all human cancers harbor mutations in the KRAS gene. These mutations lead to a constitutively active GTP-bound form of KRAS that results in aberrant activation of MAPK and PI3K pathways that control cell differentiation, growth, and survival. While much has been learned about the effectors of KRAS and its complex signaling network, little progress has been made in successfully targeting it in the treatment of cancer. Recently, we identified a functional protein-protein interaction between KRAS and Argonaute 2 (AGO2), a core component of the RNA-induced silencing complex (RISC). Investigation of this interaction revealed that the switch II domain of KRAS (both wild-type [WT] and mutant) binds to the N-terminal (Nt) domain of AGO2. Mutant KRAS-AGO2 binding results in attenuation of the RNAi function of AGO2 by preventing microRNA unwinding, a necessary step in the formation of the mature RISC complex (model 1). Analysis of this interaction revealed that KRAS-AGO2 binding is direct through Tyr64 of KRAS and Lys112/Glu114 of AGO2. Recently, using genetically engineered mouse models (GEMM), we showed that mutant KRAS is dependent on AGO2 for the development of pancreatic ductal adenocarcinoma (PDAC) from early precursor lesions in the pancreas, suggesting a critical role for the KRAS-AGO2 interaction in vivo. To further characterize KRAS-AGO2 binding, we purified recombinant proteins individually, isolated complex via size exclusion chromatography (SEC), performed differential scanning fluorimetry (DSF), isothermal titration calorimetry (ITC), and small angle x-ray scattering (SAXS). Additionally, in order to evaluate endogenous regulators of the KRAS-AGO2 interaction we investigated the role of the epidermal growth factor receptor (EGFR), which is known to regulate the RNAi function of AGO2 via phosphorylation at Tyr393. Interestingly, we observed that phosphorylation at this residue disrupts the interaction of AGO2 with WT but not mutant KRAS, providing a basis for differential regulation of the KRAS-AGO2 interaction through growth factor activation. Finally, to examine structural changes induced by EGFR phosphorylation of AGO2 at Tyr393 that may affect KRAS binding, we performed parallel molecular dynamics simulations (MDS) of nonphosphorylated and phospho-Tyr393 AGO2. These studies revealed immediate structural reorganization of the RAS binding site and an outward rotation of the entire Nt domain of AGO2. Taken together, these data shed light on a potential mechanism underlying the differential regulation of mutant vs. WT KRAS. Citation Format: Jessica Waninger, Sunita Shankar, Ronald Siebenaler, Malay Mody, Jean Tien, Vijaya Dommeti, Tyler Beyett, Richard Smith, Chandan Kumar-Sinha, Heather Carlson, John Tesmer, Arul Chinnaiyan. Characterization of the interaction between KRAS and Argonaute 2 [abstract]. In: Proceedings of the AACR Special Conference on Targeting RAS-Driven Cancers; 2018 Dec 9-12; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(5_Suppl):Abstract nr B39.

A systematic study on the influence of thermodynamic asymmetry of 5\u2032-ends of siRNA duplexes in relation to their silencing potency

siRNA molecules possess high potential as molecular tools and can be used as effective therapeutics in humans. One of the key steps in the action of these molecules is the choice of antisense strand by the RNA-induced silencing complex (RISC). To explain this process, we verified the theory which states that antisense strand selection is based on the thermodynamically less stable 5′ end of siRNA. Based on the studies presented herein, we observed that for the tested siRNA duplexes, the difference in the thermodynamic stability of the terminal, penultimate and pre-penultimate pairs in the duplex siRNA is not the dominant factor in antisense strand selection. We found that both strands in each tested siRNA molecule are used as an antisense strand. The introduction of modified nucleotides, whose impact on the thermodynamic stability of siRNA duplexes was studied, results in changes in antisense strand selection by the RISC complex. The presence of a modified residue often caused predominant selection of only one antisense strand which is at variance with the theory of siRNA strand bias.

Argonaute 2 drives miR-145-5p-dependent gene expression program in breast cancer cells

To perform their regulatory functions, microRNAs (miRNAs) must assemble with any of the four mammalian Argonaute (Ago) family of proteins, Ago1–4, into an effector complex known as the RNA-induced silencing complex (RISC). While the mature miRNA guides the RISC complex to its target mRNA, the Ago protein represses mRNA translation. The specific roles of the various Ago members in mediating miRNAs activity, however, haven’t been clearly established. In this study, we investigated the contribution of Ago2, the only human Ago protein endowed with nuclease activity, to the function of tumor-suppressor miR-145-5p in breast cancer (BC). We show that miR-145-5p and Ago2 protein are concomitantly downregulated in BC tissues and that restoration of miR-145-5p expression in BC cells leads to Ago2 protein induction through the loosening of Ago2 mRNA translational repression. Functionally, miR-145-5p exerts its inhibitory activity on cell migration only in presence of Ago2, while, upon Ago2 depletion, we observed increased miR-145/Ago1 complex and enhanced cell motility. Profiling by microarray of miR-145-5p target mRNAs, in BC cells depleted or not of Ago2, revealed that miR-145-5p drives Ago2-dependent and -independent activities. Our results highlight that the Ago2 protein in cancer cells strictly dictates miR-145-5p tumor suppressor activity.

Cosilencing Intestinal Transglutaminase-2 and Interleukin-15 Using Gelatin-Based Nanoparticles in an in Vitro Model of Celiac Disease.

In this study, we have developed a type B gelatin nanoparticle based siRNA delivery system for silencing of intestinal transglutaminase-2 (TG2) and interleukin-15 (IL-15) genes in cultured human intestinal epithelial cells (Caco-2) and murine alveolar macrophage cells (J774A.1). Small interfering RNA (siRNA) targeting the TG2 or IL-15 gene was encapsulated within gelatin nanoparticles using ethanol-water solvent displacement method. Size, charge, and morphology of gelatin nanoparticles were evaluated using a Zetasizer instrument and transmission electron microscopy. siRNA encapsulation efficiency was determined using an siRNA specific stem-loop quantitative polymerase chain reaction (qPCR) assay. Cellular uptake of siRNA-containing gelatin nanoparticles was determined using fluorescent microscopy and stem-loop qPCR assay. siRNA loading in the RISC (RNA-induced silencing complex) was determined by immunoprecipitation of argonaute 2 (AGO2) protein followed by stem-loop qPCR for siRNA quantification. Gene expression analysis of TG2, IL-15, and the proinflammatory cytokines, tumor necrosis factor alpha (TNF-α) and interferon gamma (IFN-γ), was performed using qPCR assays. Efficacy of silencing TG2 and IL-15 knockdown was evaluated in an in vitro model of celiac disease by utilizing immunogenic α-gliadin peptide p31-43 in cultured J774A.1 cells. siRNA-containing gelatin nanoparticles were spherical in shape with mean particle size and charge of 217 ± 8.39 nm and -6.2 ± 0.95 mV, respectively. siRNA loading efficiency within gelatin nanoparticles was found to be 89.3 ± 3.05%. Evaluations of cellular uptake using fluorescent microscopy showed rapid internalization of gelatin nanoparticles within 2 h of dosing, with cytosolic localization of delivered siRNA in Caco-2 cells. Gelatin nanoparticles showed greater intracellular siRNA exposure with a longer half-life, when compared to Lipofectamine-mediated siRNA delivery. Approximately 0.1% of total intracellular siRNA was associated in the RISC complex. A maximum knockdown of 60% was observed at 72 h post siRNA treatment for both TG2 and IL-15 genes, which corresponded to ∼200 copies of RISC associated siRNA. Further, efficacy of gelatin nanoparticle mediated knockdown of TG2 and IL-15 mRNA was tested in an in vitro model of celiac disease. Significant suppression in the levels of proinflammatory cytokines (TNF-α and IFN-γ) was observed in p31-43 stimulated J774A.1 cells upon either IL-15 or IL-15 + TG2 siRNA treatment. The results from this study indicate that gelatin nanoparticle mediated TG2 and IL-15 siRNA gene silencing is a very promising approach for the treatment of celiac disease.

Dysregulated RNA-Induced Silencing Complex (RISC) Assembly within CNS Corresponds with Abnormal miRNA Expression during Autoimmune Demyelination.

MicroRNAs (miRNAs) associate with Argonaute (Ago), GW182, and FXR1 proteins to form RNA-induced silencing complexes (RISCs). RISCs represent a critical checkpoint in the regulation and bioavailability of miRNAs. Recent studies have revealed dysregulation of miRNAs in multiple sclerosis (MS) and its animal model, experimental autoimmune encephalomyelitis (EAE); however, the function of RISCs in EAE and MS is largely unknown. Here, we examined the expression of Ago, GW182, and FXR1 in CNS tissue, oligodendrocytes (OLs), brain-infiltrating T lymphocytes, and CD3(+)splenocytes (SCs) of EAE mic, and found that global RISC protein levels were significantly dysregulated. Specifically, Ago2 and FXR1 levels were decreased in OLs and brain-infiltrating T cells in EAE mice. Accordingly, assembly of Ago2/GW182/FXR1 complexes in EAE brain tissues was disrupted, as confirmed by immunoprecipitation experiments. In parallel with alterations in RISC complex content in OLs, we found downregulation of miRNAs essential for differentiation and survival of OLs and myelin synthesis. In brain-infiltrating T lymphocytes, aberrant RISC formation contributed to miRNA-dependent proinflammatory helper T-cell polarization. In CD3(+) SCs, we found increased expression of both Ago2 and FXR1 in EAE compared with nonimmunized mice. Therefore, our results demonstrate a gradient in expression of miRNA between primary activated T cells in the periphery and polarized CNS-infiltrating T cells. These results suggest that, in polarized autoreactive effector T cells, miRNA synthesis is inhibited in response to dysregulated RISC assembly, allowing these cells to maintain a highly specific proinflammatory program. Therefore, our findings may provide a mechanism that leads to miRNA dysregulation in EAE/MS.

The microRNA Machinery.

MicroRNAs (miRNAs) are short (~22 nucleotides) single-stranded RNA molecules that primarily function to negatively regulate gene expression at the post-transcriptional level. miRNAs have thus been implicated in the regulation of a wide variety of normal cell functions and pathophysiological conditions. The miRNA machinery consists of a series of protein complexes which act to: (1) cleave the precursor-miRNA hairpin from its primary transcript (i.e. DROSHA and DGCR8); (2) traffic the miRNA hairpin between nucleus and cytoplasm (i.e. XPO5); (3) remove the loop sequence of the hairpin by a second nucleolytic cleavage reaction (i.e. DICER1); (4) facilitate loading of the mature miRNA sequence into an Argonaute protein (typically AGO2) as part of the RNA-Induced Silencing Complex (RISC); (5) guide the loaded RISC complex to complementary, or semi-complementary, target transcripts and (6) facilitate gene silencing via one of several possible mechanisms.

Abstract 3537: Suppression of miR-145 by long noncoding RNA RoR in colon cancer

Abstract Background: The human genome makes a large number of non-coding RNAs including long non-coding RNAs (lncRNAs) and microRNAs. Compared to well-characterized microRNAs, much less is known about lncRNAs. However, emerging evidence has implicated lncRNAs as master gene regulators; and they are often aberrantly expressed in a variety of human diseases including cancer. In particular, several studies suggest that lncRNAs can modulate the concentration and biological functions of microRNAs through a competing endogenous RNA (ceRNA) mechanism. We have previously demonstrated that lncRNA-RoR is a strong negative regulator of p53 in response to DNA damage and miR-145 is a direct transcription target for p53. Therefore, it is important to further characterize this p53 regulatory network involving lncRNA-RoR and miR-145, and to determine their role in tumorigenesis. Methods: To determine cell growth, we performed MTT assays. Protein levels from treated cells were analyzed by western blot. Real-time RT-PCR was used to determine relative expression of genes. Interaction between lncRNA-RoR and the key component (AGO2) of the RNA-induced silencing complex (RISC) was confirmed by RNA co-immunoprecipitation and RNA precipitation. Dual fluorescent in situ hybridization (FISH) was used to determine the relative levels of lncRNA-RoR and miR-145, and their co-localizations in colon cancer specimens. The effect of lncRNA-RoR on tumor growth was determined by a xenograft mouse model. Results: LncRNA-RoR suppressed miR-145 and upregulated c-Myc, one of major miR-145 target genes. In contrast, RoR-siRNA increased miR-145 expression and inhibited c-Myc. Of interest, miR-145 can also repress lncRNA-RoR expression. This reciprocal repression between lncRNA-RoR and miR-145 appeared to work through the CeRNA mechanism. Mechanistically, we showed that lncRNA-RoR directly interacted with AGO2, a key component of RISC complex, by RNA precipitation assays. This interaction was further confirmed by RNA immunoprecipitation with AGO2 antibody. In consistent with these finding, lncRNA-RoR stimulated proliferation in different cancer cells. Moreover, ectopic expression of lncRNA-RoR in HCT-116 cells significantly promoted tumor growth in a xenograft mouse model. Finally, experiments with dual FISH revealed an inverse correlation between lncRNA-RoR and miR-145 in colon cancer specimens. LncRNA-RoR was upregulated in colon cancer whereas miR-145 was downregulated in the same specimens. Conclusion: Taken together, these results suggest that lncRNA-RoR plays an oncogene role in colon cancer in part through suppression of the tumor suppressor miR-145. Thus, a better understanding of the p53-RoR-miR-145 regulatory system will provide new insight of colon cancer therapy. Citation Format: Jianguo Huang, Yin-Yuan Mo. Suppression of miR-145 by long noncoding RNA RoR in colon cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3537. doi:10.1158/1538-7445.AM2014-3537

A facile method for interfering with off-target silencing mediated by the sense strand.

Although siRNA duplexes are widely used for gene silencing, several unwanted effects such as activation of innate immunity and off-target gene silencing can limit their therapeutic use. Off-targeting can be identified for both the sense and antisense siRNA strands. Some avenues of obstructing the incorporation of the sense strand into the RNA-induced silencing complex (RISC) are currently being pursued. Herein, a biotin group at the 5'-end of the sense strand was used to inhibit its incorporation into the RISC complex. In contrast to chemical modifications, biotin is a naturally occurring compound and its presence in siRNA sequences will not induce side effects.

Long-lived microRNA–Argonaute complexes in quiescent cells can be activated to regulate mitogenic responses

Cellular proliferation depends on the integration of mitogenic stimuli with environmental conditions. Increasing evidence suggests that microRNAs play a regulatory role in this integration. Here we show that during periods of cellular quiescence, mature microRNAs are stabilized and stored in Argonaute protein complexes that can be activated by mitogenic stimulation to repress mitogen-stimulated targets, thus influencing subsequent cellular responses. In quiescent cells, the majority of microRNAs exist in low molecular weight, Argonaute protein-containing complexes devoid of essential components of the RNA-induced silencing complex (RISC). For at least 3 wk, this pool of Argonaute-associated microRNAs is stable and can be recruited into RISC complexes subsequent to mitogenic stimulation. Using several model systems, we demonstrate that stable Argonaute protein-associated small RNAs are capable of repressing mitogen-induced transcripts. Therefore, mature microRNAs may represent a previously unappreciated form of cellular memory that allows cells to retain posttranscriptional regulatory information over extended periods of cellular quiescence.

Modification of the siRNA Passenger Strand by 5‐Nitroindole Dramatically Reduces its Off‐Target Effects

During the formation of RNA-induced silencing complex (RISC), the passenger and guide strand of an siRNA duplex separate from each other to generate an active RISC complex. Accumulating evidence shows that an siRNA passenger strand can also assemble into a RISC complex and mediate RNA interference, thereby causing undesired off-target effects. To reduce this effect, the so-called "universal base" 5-nitroindole nucleotides were incorporated into an siRNA passenger strand. Melting temperature and circular dichroism spectrum measurements showed no significant changes compared to the unmodified duplex, thus indicating the formation of normal A-form conformation. Using a dual luciferase reporter assay, we have further shown that 5-nitroindole modification at position 15 of the siRNA passenger strand drastically decreased the RNAi (RNA interfering) potency of this strand, whereas the potency of the RNA guide strand was not much affected. These results could provide a practical approach for reducing off-target effects mediated by the siRNA passenger strand.

Slicing-Independent RISC Activation Requires the Argonaute PAZ Domain

Small RNAs regulate genetic networks through a ribonucleoprotein complex called the RNA-induced silencing complex (RISC), which, in mammals, contains at its center one of four Argonaute proteins (Ago1-Ago4). A key regulatory event in the RNA interference (RNAi) and microRNA (miRNA) pathways is Ago loading, wherein double-stranded small-RNA duplexes are incorporated into RISC (pre-RISC) and then become single-stranded (mature RISC), a process that is not well understood. The Agos contain an evolutionarily conserved PAZ (Piwi/Argonaute/Zwille) domain whose primary function is to bind the 3' end of small RNAs. We created multiple PAZ-domain-disrupted mutant Ago proteins and studied their biochemical properties and biological functionality in cells. We found that the PAZ domain is dispensable for Ago loading of slicing-competent RISC. In contrast, in the absence of slicer activity or slicer-substrate duplex RNAs, PAZ-disrupted Agos bound duplex small interfering RNAs, but were unable to unwind or eject the passenger strand and form functional RISC complexes. We have discovered that the highly conserved PAZ domain plays an important role in RISC activation, providing new mechanistic insights into how miRNAs regulate genes, as well as new insights for future design of miRNA- and RNAi-based therapeutics.

Modified siRNA Structure With a Single Nucleotide Bulge Overcomes Conventional siRNA-mediated Off-target Silencing

Off-target gene silencing is a major concern when using RNA interference. Imperfect pairing of the antisense strand with unintended mRNA targets is one of the main causes of small interfering RNA (siRNA) off-target silencing. To overcome this, we have developed "bulge-siRNA," a modified siRNA backbone structure with a single nucleotide (nt) bulge placed in the antisense strand. We found that siRNAs with a bulge at position 2 of the antisense strand were able to discriminate better between perfectly matched and mismatched targets, with no loss in silencing of the intended target. Genome-wide analysis also revealed that the bulge-siRNAs significantly reduced off-target silencing of transcripts with complementarity to the seed region of the siRNA antisense strand. When compared to 2'-methoxy ribosyl (2'-OMe) modified siRNAs previously developed to alleviate antisense off-target silencing; the bulge modification could better discriminate between on- versus off-targets. Our results suggest that the bulge-siRNA structure is a simple, yet superior alternative to chemical modifications for minimizing off-target silencing triggered by conventional siRNA structures.

Transcriptome-wide Analysis of Regulatory Interactions of the RNA-Binding Protein HuR

Posttranscriptional gene regulation relies on hundreds of RNA binding proteins (RBPs) but the function of most RBPs is unknown. The human RBP HuR/ELAVL1 is a conserved mRNA stability regulator. We used PAR-CLIP, a recently developed method based on RNA-protein crosslinking, to identify transcriptome-wide ∼26,000 HuR binding sites. These sites were on average highly conserved, enriched for HuR binding motifs and mainly located in 3' untranslated regions. Surprisingly, many sites were intronic, implicating HuR in mRNA processing. Upon HuR knockdown, mRNA levels and protein synthesis of thousands of target genes were downregulated, validating functionality. HuR and miRNA binding sites tended to reside nearby but generally did not overlap. Additionally, HuR knockdown triggered strong and specific upregulation of miR-7. In summary, we identified thousands of direct and functional HuR targets, found a human miRNA controlled by HuR, and propose a role for HuR in splicing.

Reversible Inhibition of PSD-95 mRNA Translation by miR-125a, FMRP Phosphorylation, and mGluR Signaling

The molecular mechanism for how RISC and microRNAs selectively and reversibly regulate mRNA translation in response to receptor signaling is unknown but could provide a means for temporal and spatial control of translation. Here we show that miR-125a targeting PSD-95 mRNA allows reversible inhibition of translation and regulation by gp1 mGluR signaling. Inhibition of miR-125a increased PSD-95 levels in dendrites and altered dendritic spine morphology. Bidirectional control of PSD-95 expression depends on miR-125a and FMRP phosphorylation status. miR-125a levels at synapses and its association with AGO2 are reduced in Fmr1 KO. FMRP phosphorylation promotes the formation of an AGO2-miR-125a inhibitory complex on PSD-95 mRNA, whereas mGluR signaling of translation requires FMRP dephosphorylation and release of AGO2 from the mRNA. These findings reveal a mechanism whereby FMRP phosphorylation provides a reversible switch for AGO2 and microRNA to selectively regulate mRNA translation at synapses in response to receptor activation.

A proteomic study of TAR-RNA binding protein (TRBP)-associated factors

BackgroundThe human TAR RNA-binding protein, TRBP, was first identified and cloned based on its high affinity binding to the small hairpin trans-activation responsive (TAR) RNA of HIV-1. TRBP has more recently been found to be a constituent of the RNA-induced silencing complex (RISC) serving as a Dicer co-factor in the processing of the ~70 nucleotide pre-microRNAs(miRNAs) to 21-25 nucleotide mature miRNAs.FindingsUsing co-immunoprecipitation and protein-identification by mass spectrometry, we characterized intracellular proteins that complex with TRBP. These interacting proteins include those that have been described to act in protein synthesis, RNA modifications and processing, DNA transcription, and cell proliferation.ConclusionsOur findings provide a proteome of factors that may cooperate with TRBP in activities such as miRNA processing and in RNA interference by the RISC complex.

Genome-wide identification of targets and function of individual MicroRNAs in mouse embryonic stem cells.

Mouse Embryonic Stem (ES) cells express a unique set of microRNAs (miRNAs), the miR-290-295 cluster. To elucidate the role of these miRNAs and how they integrate into the ES cell regulatory network requires identification of their direct regulatory targets. The difficulty, however, arises from the limited complementarity of metazoan miRNAs to their targets, with the interaction requiring as few as six nucleotides of the miRNA seed sequence. To identify miR-294 targets, we used Dicer1-null ES cells, which lack all endogenous mature miRNAs, and introduced just miR-294 into these ES cells. We then employed two approaches to discover miR-294 targets in mouse ES cells: transcriptome profiling using microarrays and a biochemical approach to isolate mRNA targets associated with the Argonaute2 (Ago2) protein of the RISC (RNA Induced Silencing Complex) effector, followed by RNA–sequencing. In the absence of Dicer1, the RISC complexes are largely devoid of mature miRNAs and should therefore contain only transfected miR-294 and its base-paired targets. Our data suggest that miR-294 may promote pluripotency by regulating a subset of c-Myc target genes and upregulating pluripotency-associated genes such as Lin28.