Complementary Target RNAs Research Articles

Argonaute and the Nuclear RNAs: New Pathways for RNA-Mediated Control of Gene Expression

Small RNAs are a commonly used tool for gene silencing and a promising platform for nucleic acid drug development. They are almost exclusively used to silence gene expression post-transcriptionally through degradation of mRNA. Small RNAs, however, can have a broader range of function by binding to Argonaute proteins and associating with complementary RNA targets in the nucleus, including long noncoding RNAs (lncRNAs) and pre-mRNA. Argonaute-RNA complexes can regulate nuclear events like transcription, genome maintenance, and splicing. Thousands of lncRNAs and alternatively spliced pre-mRNA isoforms exist in humans, and these RNAs may serve as natural targets for regulation and therapeutic intervention. This review describes nuclear mechanisms for Argonaute proteins and small RNAs, new pathways for sequence-specific targeting, and the potential for therapeutic development of small RNAs with nuclear targets.

Argonaute protein identity and pairing geometry determine cooperativity in mammalian RNA silencing

Small RNAs loaded into Argonaute proteins direct silencing of complementary target mRNAs. It has been proposed that multiple, imperfectly complementary small interfering RNAs or microRNAs, when bound to the 3' untranslated region of a target mRNA, function cooperatively to silence target expression. We report that, in cultured human HeLa cells and mouse embryonic fibroblasts, Argonaute1 (Ago1), Ago3, and Ago4 act cooperatively to silence both perfectly and partially complementary target RNAs bearing multiple small RNA-binding sites. Our data suggest that for Ago1, Ago3, and Ago4, multiple, adjacent small RNA-binding sites facilitate cooperative interactions that stabilize Argonaute binding. In contrast, small RNAs bound to Ago2 and pairing perfectly to an mRNA target act independently to silence expression. Noncooperative silencing by Ago2 does not require the endoribonuclease activity of the protein: A mutant Ago2 that cannot cleave its mRNA target also silences noncooperatively. We propose that Ago2 binds its targets by a mechanism fundamentally distinct from that used by the three other mammalian Argonaute proteins.

Increased siRNA duplex stability correlates with reduced off-target and elevated on-target effects

Argonaute (Ago) proteins form the core of RNA-induced silencing complexes (RISCs) and mediate small RNA-guided gene silencing. In RNAi, short interfering RNAs (siRNAs) guide RISCs to complementary target RNAs, leading to cleavage by the endonuclease Ago2. Noncatalytic Ago proteins, however, contribute to RNAi as well but cannot cleave target RNA and often generate off-target effects. Here we show that synthetic siRNA duplexes interact with all Ago proteins, but a functional RISC rapidly assembles only around Ago2. By stabilizing the siRNA duplex, we show that the noncatalytic Ago proteins Ago1, -3, and -4 can be selectively blocked and do not form functional RISCs. In addition, stabilized siRNAs form an Ago2-RISC more efficiently, leading to increased silencing activity. Our data suggest novel parameters for the design of siRNAs with selective activation of the endonuclease Ago2.

Target-Mediated Protection of Endogenous MicroRNAs in C. elegans

MicroRNAs (miRNAs) are tightly regulated through transcriptional and posttranscriptional mechanisms, including degradation by nucleases. Here, we report that in C. elegans, target mRNAs can protect their cognate miRNAs from degradation invivo. We show that the let-7(n2853) mutation destabilizes the mature let-7 miRNA by impairing this protection. Moreover, presence of a cognate target or depletion of the xrn-1 (XRN1) or xrn-2 (XRN2/Rat1p) exoribonucleases enforces accumulation of certain miRNA passenger (miR(∗)) strands. Thus, following biased miRNA strand loading into Argonaute, elimination of nonfunctional RNAs can further refine miRNA strand selection. Conversely, by aligning the levels of miRNAs with those of their targets, the opposing activities of mature miRNA degradation and target-mediated miRNA protection (TMMP) may enable dynamic expression of either mature strand ofa pre-miRNA, and evolution of miRNAs. Thus, it seems that mRNAs are more than inert targets and function with miRNAs in a network of mutual regulation.

Fluorescent oligonucleotides containing a novel perylene 2′-amino-α-L-LNA monomer: Synthesis and analytical potential

Herein, a novel fluorescent nucleotide analogue, perylene-2′-amino-α-L-LNA, has been prepared and studied within synthetic oligonucleotides of different sequences. The phosphoramidite reagent was synthesized in 85% overall yield starting from 2′-amino-α-L-LNA nucleoside. Incorporation efficiency of the resulting perylene-2′-amino-α-L-LNA monomer (T*) into synthetic oligonucleotides was significantly improved by replacement of the typically used 1H-tetrazole activator with pyridine hydrochloride. Generally, oligonucleotides containing monomerT* showed high binding affinity towards complementary DNA and RNA targets, batochromically shifted excitation/emission wavelengths with respect to the often applied polyaromatic hydrocarbon pyrene, high fluorescent quantum yields and very low target detection limits (5–10 nM). Fluorescence of single stranded LNA/DNA mixmer oligonucleotide having two incorporations of monomersT* was quenched (quantum yield ΦF= 0.21) relative to duplexes of this probe with complementary DNA and RNA (ΦF= 0.42 and 0.35, respectively). On the contrary, a strong fluorescence quenching upon target binding was demonstrated by two short oligonucleotides of analogues sequences containing monomersT* at 5′- and 3′-terminal positions. We explain the hybridization-induced light-up effect observed for double-labeled probe by a reduction of fluorescence quenching due to precise positioning of the fluorophores within the double-stranded complexes. Furthermore, we propose that a covalent link between twoT* monomers in the double-labeled probe provides a remarkable degree of rigidity in the double helix which enforces positioning of the bulky perylene moieties in the nonpolar groove resulting in reduced fluorescence quenching.

Antisense Treatment in Human Prostate Cancer and Melanoma

Antisense reagents and technology have developed as extraordinarily useful tools for analysis of gene function. The capacity of antisense to reduce expression of RNA (including protein-encoding mRNA and non-coding RNA) important in a multitude of diseases (including cancer) has led to the concept of using antisense molecules as drugs to treat those diseases. Both antisense RNA (RNAi) and antisense oligonucleotides (ASOs) are being developed for this purpose, with ASOs currently the most advanced in clinical testing. ASOs inhibit translation or induce degradation of complementary target RNA, and both Phase I and Phase II trials are either completed or in progress for a number of diseases. In this review, we focus on antisense approaches to treatment of two cancers (melanoma and hormone-resistant prostate cancer) where the early application of ASOs has provided important information revealing both potential for success and lessons for future preclinical and clinical investigation of ASOs as anti-cancer drugs. The progress of clinical application of two ASOs showing promise in treatment of human cancers--Oblimersen (G3139), targeting BCL2 for the treatment of metastatic melanoma, and Custirsen (OGX-11), targeting clusterin for the treatment of hormone refractory prostate cancer (HRPC)--is examined.

In Vitro Assembly of Plant RNA-Induced Silencing Complexes Facilitated by Molecular Chaperone HSP90

RNA-induced silencing complexes (RISCs) play central roles in posttranscriptional gene silencing. In plants, the mechanism of RISC assembly has remained elusive due to the lack of cell-free systems that recapitulate the process. In this report, we demonstrate that plant AGO1 protein synthesized by in vitro translation using an extract of evacuolated tobacco protoplasts incorporates synthetic small interfering RNA (siRNA) and microRNA (miRNA) duplexes to form RISCs that sequester the single-stranded siRNA guide strand and miRNA strand, respectively. The formed RISCs were able to recognize and cleave the complementary target RNAs. In this system, the siRNA duplex was incorporated into HSP90-bound AGO1, and subsequent removal of the passenger strand was triggered by ATP hydrolysis by HSP90. Removal of the siRNA passenger strand required the ribonuclease activity of AGO1, while that of the miRNA star strand did not. Based on these results, the mechanism of plant RISC formation is discussed.

RETRACTED: Small RNA duplexes function as mobile silencing signals between plant cells.

In the plant RNA interference (RNAi) pathway, 21-nucleotide duplexes of small interfering RNA (siRNA) are processed from longer double-stranded RNA precursors by the RNaseIII Dicer-like 4 (DCL4). Single-stranded siRNAs then guide Argonaute 1 (AGO1) to execute posttranscriptional silencing of complementary target RNAs. RNAi is not cell-autonomous in higher plants, but the nature of the mobile nucleic acid(s) signal remains unknown. Using cell-specific rescue of DCL4 function and cell-specific inhibition of RNAi movement, we genetically establish that exogenous and endogenous siRNAs, as opposed to their precursor molecules, act as mobile silencing signals between plant cells. We further demonstrate physical movement of mechanically delivered, labeled siRNA duplexes that functionally recapitulate transgenic RNAi spread. Cell-to-cell movement is unlikely to involve AGO1-bound siRNA single strands, but instead likely involves siRNA duplexes.

Scrutiny on Significant Factors of Hemin Catalyzed Ag Nanoparticle as Biosensor for RNA

A responsive electrical detection technique of nucleic acids has been demonstrated on submicrogapped biosensor. This method contain immobilizing of Peptide Nucleic Acid (PNA) probes in the gap areas of a pair of interdigited microelectrodes and subsequently hybridizing with their complementary target RNA. After hybridization, hemin molecules were introduced into the RNA strand via zirconium-phosphate and zirconium-carbonate chemistries. The newly attached hemin molecules act as a catalyst to accelerate reducing ammoniacal Ag ion to form Ag nanoparticles, which span the gap of the interdigitated microelectrode. The conductance of the Ag nanoparticles directly correlated with the number of the hybridized RNA molecules. Nearly 1 fM sensitivity was achieved under optimal conditions.

Scrutiny on Significant Factors of Hemin Catalyzed Ag Nanoparticle as Biosensor for RNA

A responsive electrical detection technique of nucleic acids has been demonstrated on submicrogapped biosensor. This method contain immobilizing of Peptide Nucleic Acid (PNA) probes in the gap areas of a pair of interdigited microelectrodes and subsequently hybridizing with their complementary target RNA. After hybridization, hemin molecules were introduced into the RNA strand via zirconium-phosphate and zirconium-carbonate chemistries. The newly attached hemin molecules act as a catalyst to accelerate reducing ammoniacal Ag ion to form Ag nanoparticles, which span the gap of the interdigitated microelectrode. The conductance of the Ag nanoparticles directly correlated with the number of the hybridized RNA molecules. Nearly 1 fM sensitivity was achieved under optimal conditions.

RNAi: Prokaryotes Get in on the Act

The small CRISPR-derived RNAs of bacteria and archaea provide adaptive immunity by targeting the DNA of invading viruses and plasmids. Hale et al. (2009) now report on a new variant CRISPR/Cas complex in the archaeon Pyrococcus furiosus that uses guide RNAs to specifically target and cleave RNA not DNA.

RNA-Guided RNA Cleavage by a CRISPR RNA-Cas Protein Complex

Compelling evidence indicates that the CRISPR-Cas system protects prokaryotes from viruses and other potential genome invaders. This adaptive prokaryotic immune system arises from the clustered regularly interspaced short palindromic repeats (CRISPRs) found in prokaryotic genomes, which harbor short invader-derived sequences, and the CRISPR-associated (Cas) protein-coding genes. Here, we have identified a CRISPR-Cas effector complex that is comprised of small invader-targeting RNAs from the CRISPR loci (termed prokaryotic silencing (psi)RNAs) and the RAMP module (or Cmr) Cas proteins. The psiRNA-Cmr protein complexes cleave complementary target RNAs at a fixed distance from the 3' end of the integral psiRNAs. In Pyrococcus furiosus, psiRNAs occur in two size forms that share a common 5' sequence tag but have distinct 3' ends that direct cleavage of a given target RNA at two distinct sites. Our results indicate that prokaryotes possess a unique RNA silencing system that functions by homology-dependent cleavage of invader RNAs.

Nucleation, propagation and cleavage of target RNAs in Ago silencing complexes.

The slicer activity of the RNA-induced silencing complex resides within its Argonaute (Ago) component, in which the PIWI domain provides the catalytic residues governing guide-strand mediated site-specific cleavage of target RNA. Here we report on structures of ternary complexes of Thermus thermophilus Ago catalytic mutants with 5'-phosphorylated 21-nucleotide guide DNA and complementary target RNAs of 12, 15 and 19 nucleotides in length, which define the molecular basis for Mg(2+)-facilitated site-specific cleavage of the target. We observe pivot-like domain movements within the Ago scaffold on proceeding from nucleation to propagation steps of guide-target duplex formation, with duplex zippering beyond one turn of the helix requiring the release of the 3'-end of the guide from the PAZ pocket. Cleavage assays on targets of various lengths supported this model, and sugar-phosphate-backbone-modified target strands showed the importance of structural and catalytic divalent metal ions observed in the crystal structures.

Hemorrhage and Subsequent Allogenic Red Blood Cell Transfusion are Associated With Characteristic Monocyte Messenger RNA Expression Patterns in Patients After Multiple Injury—A Genome Wide View

As outcome to severe trauma is frequently affected by massive blood loss and consecutive hemorrhagic shock, replacement of red blood cell (RBC) units remains indispensable. Administration of RBC units is an independent risk factor for adverse outcome in patients with trauma. The impact of massive blood transfusion or uncrossmatched blood transfusion on the patients' immune response in the early posttraumatic period remains unclear. Thirteen patients presenting with blunt multiple injuries (Injury Severity Score >16) were studied. Monocytes were obtained on admission and at 6, 12, 24, 48, and 72 hours after trauma. Biotinylated complementary RNA targets were hybridized to Affymetrix HG U 133A microarrays. The data were analyzed by a supervised analysis based on whether the patients received massive blood transfusions, and then subsequently, by hierarchical clustering, and by Ingenuity pathway analysis. Supervised analysis identified 224 probe sets to be differentially expressed (p < 0.001) in patients who received massive blood transfusion, when compared with those who did not. In addition, 331 probe sets were found differentially expressed (p < 0.001) in patients who received uncrossmatched RBC units in comparison with those who exclusively gained crossmatched ones. Functional pathway analysis of the respectively identified gene expression profiles suggests a contributory role by the AKT/PI3Kinase pathway, the mitogen-activated protein-kinase pathway, the Ubiquitin pathway, and the diverse inflammatory networks. We exhibited for the first time a serial, sequential screening analysis of monocyte messenger RNA expression patterns in patients with multiple trauma indicating a strongly significant association between the patients' genomic response in blood monocytes and massive or uncrossmatched RBC substitution.

Loss of MicroRNA Leads to Abnormal Function and Development of the Adult Mouse Uterus.

MicroRNAs (miRNA) are 19-22 nucleotide long, non-coding RNA molecules that post-transcriptionally regulate gene expression by base-pairing with the 3′untranslated region of complementary messenger RNA targets. To date, over 700 miRNAs have been identified in animal cells and have been reported to play a role in numerous vital processes, including: embryonic development, cellular differentiation and proliferation, and apoptosis. Our laboratory recently demonstrated that conditional deletion of Dicer in ovarian granulosa cells and derivatives of the Mullerian duct (i.e. oviduct, uterus, and cervix) by Cre/loxP recombination (Dicerfl/fl;Amhr2-Cre, Dicer-cKO) led to female infertility. This was shown to be the result of a severe oviductal developmental defect that prevented fertilized embryos from entering the uterus. While the uterus of Dicer-cKO females was also disrupted, as evidenced by shortened uterine horns and reduced numbers of uterine glands, its functional ability to establish and maintain pregnancy is not yet known. We first evaluated whether the uterus of pseudopregnant mice could undergo a decidual reaction. To test uterine decidualization, sesame oil was injected into one uterine horn of 3.5 day pseudopregnant mice, while the contralateral horn served as the non-injected control. We next completed an embryo transfer experiment, where 5 blastocysts collected from naturally mated CD-1 mice on day 3.5 (day 0.5 = plug) were transferred into each uterine horn of day 2.5 pseudopregnant adult female Dicer-cKO (n=13) and wild-type mice (Dicerfl/fl or Dicerfl/wt littermates lacking the Amhr2-Cre allele, n=7). Recipient females were then sacrificed on day 7 of pregnancy and the number of implantation sites per mouse recorded. Results of the decidualization experiment indicated that uteri of Dicer-cKO mice (n=11) failed to exhibit a normal decidualization response when compared to wild-type mice (n=7), which exhibited a 3.6-fold increase (p<0.05) in uterine wet weight (mg) after body weight (g) correction. The uteri were 11.8 ± 4.3 mg and 19.7 ± 3.8 mg for the non-injected and 15.0 ± 6.1 mg and 70.8 ± 28.0 mg for sesame oil injected, for the Dicer cKO and wild-type mice, respectively. In the embryo transfer experiment, implantation occurred in 4 of 13 Dicer-cKO recipient females. Interestingly, all implantation sites in the pregnant Dicer-cKO mice were located at the cervical end of the uterine horns. Implantation sites in Dicer-cKO mice appeared less vascular, as evidenced by a blanched color in comparison to the dark red implantation sites observed in wild-type mice. Embryos failed to space appropriately in the two Dicer-cKO recipients with multiple embryos. Histological analysis, however, indicated that the uteri of the pregnant Dicer-cKO mice were able to mount what appeared to be a normal decidualization response with normal embryonic development. We next examined adult female Dicer-cKO uterine tissue histology at 60, 180, 230, and 330 days of age. Uteri from Dicer-cKO mice exhibited adenomyosis, a condition of abnormal endometrial growth within the myometrial layer, which progressively worsened as these animals aged. In conclusion, uterine function in Dicer-cKO mice was compromised and miRNA mediated post-transcriptional gene regulation appears to play a significant role in normal uterine development and function. (poster)

On the road to reading the RNA-interference code

The finding that sequence-specific gene silencing occurs in response to the presence of double-stranded RNAs has had an enormous impact on biology, uncovering an unsuspected level of regulation of gene expression. This process, known as RNA interference (RNAi) or RNA silencing, involves small non-coding RNAs, which associate with nuclease-containing regulatory complexes and then pair with complementary messenger RNA targets, thereby preventing the expression of these mRNAs. Remarkable progress has been made towards understanding the underlying mechanisms of RNAi, raising the prospect of deciphering the 'RNAi code' that, like transcription factors, allows the fine-tuning and networking of complex suites of gene activity, thereby specifying cellular physiology and development.

Target site effects in the RNA interference and microRNA pathways

In RNAi (RNA interference), siRNAs (small interfering RNAs) are loaded into the RISC (RNA-induced silencing complex), which then mediates endonucleolytic cleavage of complementary target RNAs. Although RNAi has become one of the most powerful tools in molecular biology to assess gene function, there remains a great number of ineffective siRNAs. It is already known that the assembly and activation of RISC is a crucial determinant of RNAi activity, but downstream effects such as target accessibility have not been analysed extensively. Therefore we assessed the effect of target site accessibility and found that it significantly improves the potency of siRNAs. Similarly, miRNAs (microRNAs) act by repressing protein synthesis through imperfect base-pairing to the 3'-UTR (untranslated region) of target mRNAs. We found that predicted target sites reside in regions of high accessibility and tested whether this criterion could be used in the search of functional miRNA targets. In addition, we performed reporter gene assays to test whether accessibility correlates with measured mRNA suppression levels. The results of our initial study suggest that secondary structures might add a so far underrepresented layer of complexity in the recognition of RNA targets by miRNAs.

Hairpin ribozyme-antisense RNA constructs can act as molecular lassos

We have developed a novel class of antisense agents, RNA Lassos, which are capable of binding to and circularizing around complementary target RNAs. The RNA Lasso consists of a fixed sequence derived from the hairpin ribozyme and an antisense segment whose size and sequence can be varied to base pair with accessible sites in the target RNA. The ribozyme catalyzes self-processing of the 5′- and 3′-ends of a transcribed Lasso precursor and ligates the processed ends to produce a circular RNA. The circular and linear forms of the self-processed Lasso coexist in an equilibrium that is dependent on both the Lasso sequence and the solution conditions. Lassos form strong, noncovalent complexes with linear target RNAs and form true topological linkages with circular targets. Lasso complexes with linear RNA targets were detected by denaturing gel electrophoresis and were found to be more stable than ordinary RNA duplexes. We show that expression of a fusion mRNA consisting of a sequence from the murine tumor necrosis factor-α (TNF-α) gene linked to luciferase reporter can be specifically and efficiently blocked by an anti-TNF Lasso. We also show in cell culture experiments that Lassos directed against Fas pre-mRNA were able to induce a change in alternative splicing patterns.

Dicer Function Is Essential for Female Fertility and Normal Development of the Female Reproductive Organ System.

Mature microRNAs (miRNA) are non-coding RNAs approximately 22 nucleotides long that regulate gene expression post-transcriptionally by base-pairing with the 3' untranslated-region of complementary messenger RNA targets. MicroRNAs are synthesized through a multi-step process that includes the formation of an ~70 to 110 nucleotide long precursor stemloop RNA transcript referred to as a pre-miRNA. Dicer mediated cleavage of these pre-miRNA transcripts generate the mature miRNAs that ultimately regulate gene expression. Our laboratories and others have established that miRNAs are expressed in the ovary and reproductive tract, and we have recently shown that miRNA expression is dramatically regulated in both ovarian and uterine tissues in response to hormonal treatments (i.e., LH and estradiol, respectively). To date, however, the functional roles that miRNAs play in ovarian and uterine function have remained unknown. To determine whether ovarian granulosa cell and uterine/oviductal miRNAs play an important role in female fertility and development of the reproductive tract, a line of mice with Dicer knocked-down specifically in the ovary and reproductive tract was established using Cre-lox/P recombination. Mice with loxP insertions in the Dicer gene (Dicerfl/fl) were crossed with mice expressing Cre recombinase driven by the granulosa cell/reproductive tract-specific Amhr2 promoter (Amhr2Cre). Female Dicer fl/fl;Amhr2Cre mice (n=5) 42 days of age were mated to known fertile wild-type males to assess their fertility and over a three month period these mice failed to generate offspring. To determine whether Dicerfl/fl;Amhr2Cre mice exhibit estrus and are capable of ovulation, females (n=4) were exposed to known fertile males and mating was confirmed. These mice were ovariectomized on the day following mating and the ovaries and oviducts processed for general histology. To determine if the failure to reproduce was at the ovarian or reproductive tract level, ovaries, oviducts and uterine tissue were isolated from the Dicerfl/fl;Amhr2Cre and wild-type littermates, fixed in Bouin's solution, and subjected to hematoxylin and eosin staining, and characterized for general histology. Ovaries from Dicerfl/fl;Amhr2Cre mice appeared to have follicles at all stages of follicular development and corpora lutea (CL). Trapped oocytes were observed in some CL of the Dicerfl/fl;AmhR2Cre mice but was not observed in the control wild-type mice. Marked gross morphologic changes in the uterus and oviduct of the Dicerfl/fl;Amhr2Cre were evident. The uterine horns of the Dicerfl/fl;AmhR2Cre were thicker and shorter in length compared to wild type littermates (approxiately1/2 the length). The oviduct exhibited the most dramatic phenotypic alteration resulting from loss of Dicer expression as evidenced by the presence of multiple fluid-filled vesicular sacs. Histological evaluation of these tissues, indicated that the musculature was much less developed in both the oviduct and myometrium. Further study using this conditional Dicer knock-down mouse line should provide important insight into the role of miRNAs have in the female reproductive tract development and function.

Lipoplex and peptide-based strategies for the delivery of steric-block oligonucleotides

Synthetic oligonucleotides offer interesting prospects for the control of gene expression but clinical applications have been severely limited by their poor bioavailability. Cationic lipids have been widely used for the delivery of charged oligonucleotide (ON) analogues but most of the commercial formulations are toxic and poorly stable in the presence of serum proteins. We have developed a DOGS/DOPE liposome formulation named DLS (for delivery liposomal system), that allows for the efficient nuclear delivery of negatively charged antisense ON analogues as monitored by fluorescence microscopy and by their ability to correct deficient pre-mRNA splicing, even in serum-supplemented cell culture. Uncharged DNA mimics such as peptide nucleic acids (PNA), or phosphorodiamidate morpholino (PMO) ON are particularly interesting for their high metabolic stability and affinity for complementary RNA targets but they cannot be delivered with cationic lipids. Cell penetrating peptides (CPP), such as Tat or penetratin, have been used widely as conjugates for the delivery of various biomolecules and might be appropriate for neutral ON analogues. However, entrapment within endocytic vesicles severely limits the efficiency of PNA delivery by CPPs in the absence of endosomolytic drugs, such as chloroquine. The conjugation of new arginine-rich CPPs to PNA allows efficient nuclear delivery in the absence of chloroquine as monitored in a splicing correction assay. Both strategies have their advantages but DLS-mediated delivery remains more efficient than CPP delivery for the nuclear targeting of splice correcting ON analogues in vitro.