Target mRNA Sequences Research Articles

IntmiR: a complete catalogue of intronic miRNAs of human and mouse

IntmiR is a manually curated database of published intronic miRNAs of Human and Mouse genome. Each entry in the database, aims at providing a complete resource of intronic miRNA with their target gene and deregulation in various diseases with related tissues and pathways. The current release contains 426 intronic miRNA loci from human and 76 from mouse, expressing distinct target mRNA sequences. Database gives information on an intronic miRNA-disease relationship, including miRNA ID, pathaway connected and related tissues. All entries can be retrieved by miRNA ID or target gene. IntmiR is freely available at rgcb.res.in/intmir. The database is available for free at rgcb.res.in/intmir.

6 CLONING AND EXPRESSION OF BOVINE FACTOR IN THE GERMLINE ALPHA (FIGLA) IN OOCYTES AND EARLY EMBRYOS: A POTENTIAL TARGET OF microRNA-212

Factor In the GermLine Alpha (FIGLA), a basic helix-loop-helix transcription factor, was first identified in regulating coordinate expression of zona pellucida genes in mice. It plays a crucial role in the formation of primordial follicles and lack of FIGLA in mice alters the expression of many oocyte specific genes that are required for fertilization and early embryonic survival. The objective of this study was to characterise the expression and regulation of bovine FIGLA during early embryogenesis. The cloned bovine FIGLA cDNA is 660 bp in length, which encodes a protein of 165 amino acids. Expression of bovine FIGLA mRNA is restricted to ovarian tissue and can be detected in fetal ovaries harvested as early as 90 days of gestation when primordial follicles start to form. Expression analysis demonstrated that FIGLA mRNA is abundant in germinal vesicle and metaphase II stage oocytes, as well as in embryos from pronuclear to 8-cell stage, but barely detectable in embryos collected at morula and blastocyst stages, suggesting that FIGLA might be a maternal effect gene. Recent studies in zebrafish have highlighted the importance of non-coding small RNAs (microRNAs) as key regulatory molecules targeting maternal mRNAs for degradation during embryonic development. We hypothesised that FIGLA, as a maternal transcript, is regulated by microRNAs during early embryogenesis. Using microInspector, an algorithm for detection of possible interactions between microRNAs and target mRNA sequences, a microRNA binding site (miR-212) was identified in the 3′-UTR of the bovine FIGLA mRNA. Alignment of the 3′-UTR of FIGLA mRNAs from bovine, human and mouse shows complete conservation of the ‘seed’ region indicating that miR-212 might be a post-transcriptional regulator of FIGLA and the microRNA: mRNA interaction is evolutionary conserved. Expression analysis indicates that bovine miR-212 is expressed in oocytes and tends to increase at the 4-cell and 8-cell stage embryos followed by a decline at morula and blastocyst stages, indicating that miR-212 is presumably of maternal origin and potentially involved in maternal transcript degradation during the maternal-to-embryonic transition. To validate the role of miR-212 in silencing FIGLA, a luciferase reporter assay was performed using HeLa cells. The luciferase activity in cells expressing a luciferase construct containing the entire 3′ UTR of bovine FIGLA was suppressed by ∼40% in the presence of miR-212. We also investigated the stability of FIGLA mRNA in cells transfected with bovine FIGLA expression plasmid in the presence or absence of miR-212. Expression of bovine FIGLA mRNA was significantly reduced in the presence of mir-212 compared to control cells transfected with FIGLA construct alone. In summary, our data establish miR-212 as a potential post-transcriptional regulator of FIGLA during the maternal-to-embryonic transition in bovine embryos. Future studies aim to determine if miR-212 mimic can inhibit endogenous FIGLA expression in bovine embryos and its effect on subsequent embryonic development.

Interferência por RNA: uma nova alternativa para terapia nas doenças reumáticas

RNA interference (RNAi) is a post-transcriptional gene silencing mechanism preserved during evolution. This mechanism, recently described, is mediated by small double-stranded RNAs (dsRNAs) that can specifically recognize a target mRNA sequence and mediate its cleavage or translational repression. The use of RNAi as a tool for gene therapy has been extensively studied, especially in viral infections, cancer, inherited genetic disorders, cardiovascular and rheumatic diseases. Together with data from human genome, the knowledge of gene silencing mediated by RNAi could allow a functional determination of virtually any cell expressed gene and its involvement in cellular functioning and homeostasis. Several in vitro and in vivo therapeutic studies with autoimmune disease animal models have been carried out with promising results. The pathways of tolerance breakage and inflammation are potential targets for RNAi therapy in inflammatory autoimmune diseases. This review will present the basic principles of RNAi and discuss several aspects of RNAi-based therapeutic approaches, from in vitro tool design and target identification to in vivo pre-clinical drug delivery, and tests of autoimmune diseases in human cells and animal models. Finally, this review will present some recent clinical experience with RNAi-based therapy.

Use of molecular beacons to image effects of titanium surface microstructure on β1 integrin expression in live osteoblast-like cells

This study used molecular beacon technology to examine substrate-dependent changes in integrin subunit expression in living cells. Molecular beacons are oligonucleotide probes that can be delivered into live cells to allow for real-time imaging of mRNA. They have a stem-loop hairpin structure with a fluorophore-quencher pair, which opens when bound to the target mRNA sequence, resulting in a fluorescent signal upon excitation. A novel molecular beacon that is specific to the β1 integrin subunit mRNA was developed and used to image osteoblast-like MG63 cells in vitro on both glass and titanium surfaces of varying roughness. Specificity was verified by comparing the molecular beacon signal intensities to real-time PCR results in both wild-type cells and cells with shRNA knockdown of β1 integrin mRNA. The molecular beacon was able to detect changes due to both surface microtopography and silencing of the mRNA target. The results showed that effects of the substrate on β1 mRNA noted previously in confluent cultures were evident in pre-confluent cells as well, supporting the hypothesis that β1 integrin pairs are important in proliferation as well as differentiation of osteoblasts. This technique overcomes the limitations of traditional gene assays (PCR, immunofluorescence) by allowing for the real-time measurement and tracking of specific mRNAs in individual live cells prior to confluence.

Hairpin DNA-functionalized gold colloids for the imaging of mRNA in live cells.

A strategy is presented for the live cell imaging of messenger RNA using hairpin DNA-functionalized gold nanoparticles (hAuNP). hAuNP improve upon technologies for studying RNA trafficking by their efficient internalization within live cells without transfection reagents, improved resistance to DNase degradation, low cytotoxicity, and the incorporation of hairpin DNA molecular beacons to confer high specificity and sensitivity to the target mRNA sequence. Furthermore, the targeted nanoparticle-beacon construct, once bound to the target mRNA sequence, remains hybridized to the target, enabling spatial and temporal studies of RNA trafficking and downstream analysis. Targeted hAuNP exhibited high specificity for glyceraldehyde 3-phosphate dehydrogenase (GADPH) mRNA in live normal HEp-2 cells and respiratory syncytial virus (RSV) mRNA in live RSV-infected HEp-2 cells with high target to background ratios. Multiplexed fluorescence imaging of distinct mRNAs in live cells and simultaneous imaging of mRNAs with immunofluorescently stained protein targets in fixed cells was enabled by appropriate selection of molecular beacon fluorophores. Pharmacologic analysis suggested that hAuNP were internalized within cells via membrane-nanoparticle interactions. hAuNP are a promising approach for the real-time analysis of mRNA transport and processing in live cells for elucidation of biological processes and disease pathogenesis.

Reversal of multidrug resistance by magnetic Fe3O4 nanoparticle copolymerizating daunorubicin and MDR1 shRNA expression vector in leukemia cells

In many instances, multidrug resistance (MDR) is mediated by increasing the expression at the cell surface of the MDR1 gene product, P-glycoprotein (P-gp), a 170-kD energy-dependent efflux pump. The aim of this study was to investigate the potential benefit of combination therapy with magnetic Fe(3)O(4) nanoparticle [MNP (Fe(3)O(4))] and MDR1 shRNA expression vector in K562/A02 cells. For stable reversal of "classical" MDR by short hairpin RNA (shRNA) aiming directly at the target sequence (3491-3509, 1539-1557, and 3103-3121 nucleotide) of MDR1 mRNA. PGC silencer-U6-neo-GFP-shRNA/MDR1 called PGY1-1, PGY1-2, and PGY1-3 were constructed and transfected into K562/A02 cells by lipofectamine 2000. After transfected and incubated with or without MNP (Fe(3)O(4)) for 48 hours, the transcription of MDR1 mRNA and the expression of P-gp were detected by quantitative real-time PCR and Western-blot assay respectively. Meanwhile intracellular concentration of DNR in K562/A02 cells was detected by flow cytometry (FCM). PGC silencer-U6-neo-GFP-shRNA/MDR1 was successfully constructed, which was confirmed by sequencing and PGY1-2 had the greatest MDR1 gene inhibitory ratio. Analysis of the reversal ratio of MDR, the concentration of daunorubicin (DNR) and the transcription of MDR1 gene and expression of P-gp in K562/A02 showed that combination of DNR with either MNP (Fe(3)O(4)) or PGY1-2 exerted a potent cytotoxic effect on K562/A02 cells, while combination of MNP (Fe(3)O(4)) and PGY1-2 could synergistically reverse multidrug resistance. Thus our in vitro data strongly suggested that a combination of MNP (Fe(3)O(4)) and shRNA expression vector might be a more sufficient and less toxic anti-MDR method on leukemia.

Secretory Mechanisms and Intercellular Transfer of MicroRNAs in Living Cells

The existence of circulating microRNAs (miRNAs) in the blood of cancer patients has raised the possibility that miRNAs may serve as a novel diagnostic marker. However, the secretory mechanism and biological function of extracellular miRNAs remain unclear. Here, we show that miRNAs are released through a ceramide-dependent secretory machinery and that the secretory miRNAs are transferable and functional in the recipient cells. Ceramide, whose biosynthesis is regulated by neutral sphingomyelinase 2 (nSMase2), triggers secretion of small membrane vesicles called exosomes. The decreased activity of nSMase2 with a chemical inhibitor, GW4869, and a specific small interfering RNA resulted in the reduced secretion of miRNAs. Complementarily, overexpression of nSMase2 increased extracellular amounts of miRNAs. We also revealed that the endosomal sorting complex required for transport system is unnecessary for the release of miRNAs. Furthermore, a tumor-suppressive miRNA secreted via this pathway was transported between cells and exerted gene silencing in the recipient cells, thereby leading to cell growth inhibition. Our findings shed a ray of light on the physiological relevance of secretory miRNAs.

Effect of Non-Coding RNA on Post-Transcriptional Gene Silencing of Alzheimer Disease

AbstractA large amount of hidden biological information is contained in the human genome, which is not expressed or revealed in the form of proteins; the usual end product form of gene expression. Instead, most of such information is in the form of non-coding RNAs (ncRNAs). ncRNAs correspond to genes that are transcribed, but do not get translated into proteins. This part of the genome was, till recently, considered as ‘junk’. The term ‘junk’ implied lack of any discernible function of these RNA. More than 98% of the human genomic size encompasses these non-coding RNAs. But, recent research has evidently brought out the indispensible contribution of non-coding RNA in controlling and regulating gene expression. ncRNA such as siRNAs and microRNAs have been reported to greatly help in causing post-transcriptional gene silencing (PTGS) in cells through RNA interference (RNAi) pathway. In this work, we have investigated the possibility of using siRNAs and microRNAs to aid in gene silencing of early onset Alzheimer’s disease genes. Alzheimer’s disease specific mutations and their corresponding positions in mRNA have been identified for six genes; Presenilin-1, Presenilin-2, APP (amyloid beta precursor protein), APBB3, BACE-1 and PSENEN. Small interfering RNAs (siRNAs) that can cause PTGS through RNA interference pathway have been designed. RNA analysis has been done to verify complementarity of antisense siRNA sequence with target mRNA sequence. Interaction studies have been done computationally between these antisense siRNA strands and seven Argonaute proteins. From the interaction studies, only one of the seven Argonaute proteins; 1Q8K, was found to have interaction with the siRNAs indicating the importance and uniqueness of this particular protein in RISC (RNA induced silencing complex). The interaction studies have been carried out for the microRNAs also. Out of the 700 mature human microRNAs collected, 394 microRNAs have been identified to show partial complementarity with their target sequence on PSEN-1 mRNA. Of these 394, five microRNAs have shown partial complementarity to early onset Alzheimer’s disease specific mutations in PSEN-1 mRNA. Interaction studies have been done between these microRNAs and Argonaute proteins. Thus, design, characterization and analysis of ncRNAs that contribute to post transcriptional gene silencing of Alzheimer’s disease have been achieved.

Effective inhibition of HCMV UL49 gene expression and viral replication by oligonucleotide external guide sequences and RNase P

BackgroundHuman cytomegalovirus (HCMV) is a ubiquitous herpesvirus that typically causes asymptomatic infections in healthy individuals but may lead to serious complications in newborns and immunodeficient individuals. The emergence of drug-resistant strains of HCMV has posed a need for the development of new drugs and treatment strategies. Antisense molecules are promising gene-targeting agents for specific regulation of gene expression. External guide sequences (EGSs) are oligonucleotides that consist of a sequence complementary to a target mRNA and recruit intracellular RNase P for specific degradation of the target RNA. The UL49-deletion BAC of HCMV was significantly defective in growth in human foreskin fibroblasts. Therefore, UL49 gene may serve as a potential target for novel drug development to combat HCMV infection. In this study, DNA-based EGS molecules were synthesized to target the UL49 mRNA of human cytomegalovirus (HCMV).ResultsBy cleavage activity assessing in vitro, the EGS aimed to the cleavage site 324 nt downstream from the translational initiation codon of UL49 mRNA (i.e. EGS324) was confirmed be efficient to direct human RNase P to cleave the target mRNA sequence. When EGS324 was exogenously administered into HCMV-infected human foreskin fibroblasts (HFFs), a significant reduction of ~76% in the mRNA and ~80% in the protein expression of UL49 gene, comparing with the cells transfected with control EGSs. Furthermore, a reduction of about 330-fold in HCMV growth were observed in HCMV-infected HFFs treated with the EGS.ConclusionsThese results indicated that UL49 gene was essential for replication of HCMV. Moreover, our study provides evidence that exogenous administration of a DNA-based EGS can be used as a potential therapeutic approach for inhibiting gene expression and replication of a human virus.

Antisense DNA parameters derived from next-nearest-neighbor analysis of experimental data

BackgroundThe enumeration of tetrameric and other sequence motifs that are positively or negatively correlated with in vivo antisense DNA effects has been a useful addition to the arsenal of information needed to predict effective targets for antisense DNA control of gene expression. Such retrospective information derived from in vivo cellular experiments characterizes aspects of the sequence dependence of antisense inhibition that are not predicted by nearest-neighbor (NN) thermodynamic parameters derived from in vitro experiments. However, quantitation of the antisense contributions of motifs is problematic, since individual motifs are not isolated from the effects of neighboring nucleotides, and motifs may be overlapping. These problems are circumvented by a next-nearest-neighbor (NNN) analysis of antisense DNA effects in which the overlapping nature of nearest-neighbors is taken into account.ResultsNext-nearest-neighbor triplet combinations of nucleotides are the simplest that include overlapping sequence effects and therefore can encompass interactions beyond those of nearest neighbors. We used singular value decomposition (SVD) to fit experimental data from our laboratory in which phosphorothioate-modified antisense DNAs (S-DNAs) 20 nucleotides long were used to inhibit cellular protein expression in 112 experiments involving four gene targets and two cell lines. Data were fitted using a NNN model, neglecting end effects, to derive NNN inhibition parameters that could be combined to give parameters for a set of 49 sequences that represents the inhibitory effects of all possible overlapping triplet interactions in the cellular targets of these antisense S-DNAs. We also show that parameters to describe subsets of the data, such as the mRNAs being targeted and the cell lines used, can be included in such a derivation. While NNN triplet parameters provided an adequate model to fit our data, NN doublet parameters did not.ConclusionsThe methodology presented illustrates how NNN antisense inhibitory information can be derived from in vivo cellular experiments. Subsequent calculations of the antisense inhibitory parameters for any mRNA target sequence automatically take into account the effects of all possible overlapping combinations of nearest-neighbors in the sequence. This procedure is more robust than the tallying of tetrameric motifs that have positive or negative antisense effects. The specific parameters derived in this work are limited in their applicability by the relatively small database of experiments that was used in their derivation.

P52–Bcl3 complex promotes cyclin D1 expression in BEAS-2B cells in response to low concentration arsenite

Arsenic is a well-recognized human carcinogen that causes a number of malignant diseases, including lung cancer. Previous studies have indicated that cyclin D1 is frequently over-expressed in many cancer types. It is also known that arsenite exposure enhances cyclin D1 expression, which involves NF-κB activation. However, the mechanism between cyclin D1 and the NF-κB pathway has not been well studied. This study was designed to characterize the underlying mechanism of induced cell growth and cyclin D1 expression in response to low concentration sodium arsenic (NaAsO 2) exposure through the NF-κB pathway. Cultured human bronchial epithelial cells, BEAS-2B, were exposed to low concentration sodium arsenite for the indicated durations, and cytotoxicity, gene expression, and protein activity were assessed. To profile the canonical and non-canonical NF-κB pathways involved in cell growth and cyclin D1 expression induced by low concentration arsenite, the NF-κB-specific inhibitor-phenethyl caffeate (CAPE) and NF-κB2 mRNA target sequences were used, and cyclin D1 expression in BEAS-2B cells was assessed. Our results demonstrated that exposure to low concentration arsenite enhanced BEAS-2B cells growth and cyclin D1 mRNA and protein expression. Activation and nuclear localization of p52 and Bcl3 in response to low concentration arsenite indicated that the non-canonical NF-κB pathway was involved in arsenite-induced cyclin D1 expression. Moreover, we further demonstrated that p52/Bcl3 complex formation enhanced cyclin D1 expression through the cyclin D1 gene promoter via its κB site. The up-regulation of cyclin D1 mediated by the p52–Bcl3 complex in response to low concentration arsenite might be important in assessing the health risk of low concentration arsenite and understanding the mechanisms of the harmful effects of arsenite.

Abstract LB-68: Comparative analysis of the miRNome in tumors and plasma from colorectal cancer patients

Abstract MicroRNAs (miRNAs) constitute a recently discovered class of small cellular RNAs (typically 21-23 nt) that function as post-transcriptional regulators of gene expression through binding of miRNA target sequences in mRNA 3′ untranslated regions (UTRs). Current estimates indicate that at least one third of the cellular transcriptome is regulated by miRNAs, and that each miRNA has the potential to regulate hundreds of different mRNAs. As a consequence, miRNAs have been proposed to be master regulators of cellular state. This hypothesis has been borne out by a large number of studies demonstrating a causal link between miRNA dys-regulation and numerous disease states, including a diverse array of human cancers. Furthermore, the high relative stability of miRNA in common clinical source materials (e.g. FFPE blocks, plasma, urine, saliva, etc.) and the ability of miRNA expression profiles to accurately classify discrete tissue types and disease states have positioned miRNA quantification as a promising new tool for a wide range of diagnostic applications. To facilitate discovery and clinical transfer of miRNA-based diagnostic markers, we developed a genome-wide LNA™-based miRNA q-rt-PCR platform with unparalleled sensitivity and robustness. The platform uses a universal RT system and thus allows high-throughput profiling of miRNAs from important clinical sources without the need for pre-amplification. Using this system, we profiled samples relevant to colorectal cancer: tumor samples from colorectal cancer patients, normal adjacent tissue (NAT) from the same patients, plasma from the same patients from blood drawn on the day before surgery, and plasma from matched healthy volunteers. Both principal component analysis and unsupervised hierarchical clustering of the most variable miRNAs accurately classified tumor samples away from the set of NAT samples, suggesting that the single most important distinguishing factor between the two sets of samples is related to neoplastic transformation. We also define miRNAs that are differentially expressed in the plasma of healthy volunteers versus colorectal cancer patients and we will present an analysis of the overlap between differentially expressed miRNAs in plasma and tissue. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-68.

Abstract 2066: Role of miR-182/miR-96 cluster in prostatic zinc homeostasis

Abstract Zinc has been shown to have a defensive action against the development of prostate cancer (PCa). Zinc is an essential element that is required for the activity of more than 300 enzymes, structure of proteins, and control of genetic expression. It plays an important role in cellular processes such as cell division, growth, differentiation, development, aging, and synthesis and repair of DNA, RNA, and protein. Prostatic secretary epithelial cells accumulate large amounts of zinc and have a high expression of human Zinc transporter 1 (hZIP1), the major zinc transporter in the prostate. PCa tissue exhibits lower levels of zinc and hZIP1 compared with surrounding normal-appearing areas. Furthermore, compared to Caucasian men, the prostates of African American men, a population with a disproportionately high risk of developing PCa, have decreased zinc and hZIP1. Previously, we examined several miRNAs in formalin-fixed paraffin-embedded prostate tissue from African American and Caucasian patients, of which miR-182/96 levels were higher in PCa samples and had inverse correlation with hZIP1 mRNA in Caucasian patients (Spearman rho = −0.77, p=0.009). No correlation between hZIP1 and miR-182 was found in the African-American specimens, suggesting a different mechanism for hZIP1 regulation in African-Americans. Based on previous studies, we hypothesize that miR-182/96 cluster is responsible for regulation of hZIP1 levels in PCa and prostate tissue. In this study, we examined the relationship between the levels of miR-182/96 and hZIP1 in more detail using cell culture models. We observed an inverse correlation between hZIP1 and miR-182 levels in several prostate cell cultures; primary normal epithelial cells, normal stromal cells, and prostate cancer cells (PC3 and LNCaP). MiR-182 is transcribed in a cluster with miR-96 and the miRNAs have an overlapping mRNA target sequence. As expected, miR-96 expression was identical to that of miR-182, thus also inversely correlated with hZIP1 in prostate cell cultures. Overexpression of miR-182 in both normal and LNCaP cells decreased hZIP1 mRNA levels, further implicating miR-182 as a regulator of zinc transport. Ongoing experiments focus on validating the two putative miR-182/96 binding sites in the 3′UTR of hZIP1 and the effect of miR-182 and miR-96 overexpression on zinc transport. Our results may establish a role for miR-182 in prostatic zinc homeostasis. Because zinc levels are linked to PCa risk, these miRNAs may be useful as a prognostic or diagnostic marker in prostate biopsies to identify patients with high risk of PCa recurrence and for identifying individuals for zinc replacement. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2066.

MiR-145 and miR-133a function as tumour suppressors and directly regulate FSCN1 expression in bladder cancer

Background:We have recently identified down-regulated microRNAs including miR-145 and miR-133a in bladder cancer (BC). The aim of this study is to determine the genes targeted by miR-145, which is the most down-regulated microRNA in BC.Methods:We focused on fascin homologue 1 (FSCN1) from the gene expression profile in miR-145 transfectant. The luciferase assay was used to confirm the actual binding sites of FSCN1 mRNA. Cell viability was evaluated by cell growth, wound-healing, and matrigel invasion assays. BC specimens were subjected to immunohistochemistry of FSCN1 and in situ hybridisation of miR-145.Results:The miR-133a as well as miR-145 had the target sequence of FSCN1 mRNA by the database search, and both microRNAs repressed the mRNA and protein expression of FSCN1. The luciferase assay revealed that miR-145 and miR-133a were directly bound to FSCN1 mRNA. Cell viability was significantly inhibited in miR-145, miR-133a, and si-FSCN1 transfectants. In situ hybridisation revealed that miR-145 expression was markedly repressed in the tumour lesion in which FSCN1 was strongly stained. The immunohistochemical score of FSCN1 in invasive BC (n=46) was significantly higher than in non-invasive BC (n=20) (P=0.0055).Conclusion:Tumour suppressive miR-145 and miR-133a directly control oncogenic FSCN1 in BC.

Comprehensive discovery of endogenous Argonaute binding sites in Caenorhabditis elegans

MicroRNAs (miRNAs) regulate gene expression by guiding Argonaute proteins to specific target mRNA sequences. Identification of bona fide miRNA target sites in animals is challenging because of uncertainties regarding the base-pairing requirements between miRNA and target as well as the location of functional binding sites within mRNAs. Here we present the results of a comprehensive strategy aimed at isolating endogenous mRNA target sequences bound by the Argonaute protein ALG-1 in C. elegans. Using cross-linking and ALG-1 immunoprecipitation coupled with high-throughput sequencing (CLIP-seq), we identified extensive ALG-1 interactions with specific 3' untranslated region (UTR) and coding exon sequences and discovered features that distinguish miRNA complex binding sites in 3' UTRs from those in other genic regions. Furthermore, our analyses revealed a striking enrichment of Argonaute binding sites in genes important for miRNA function, suggesting an autoregulatory role that may confer robustness to the miRNA pathway.

A Reversible Switch on Two-Dimensional Small Interfering RNA Condensation

Small interfering RNAs (siRNAs) are short (19-29bp) double stranded nucleic acids that efficiently mediate gene silencing in mammalian cells by directing the degradation of complementary target mRNA sequences. This has justified the recent development of technologies for siRNA transport into a host cell. Synthetic cationic lipid (CL) assemblies can efficiently be used to deliver siRNA, leading to highly specific gene silencing [1].

MicroRNAs and Breast Cancer

MicroRNAs are a class of small non-coding RNAs that are involved in the negative regulation of gene expression primarily through binding to the 3'untranslated region of their target mRNA. MicroRNAs have multiple mRNA targets, each of which can be regulated by multiple microRNAs making this a complex regulatory network. The identification of their importance in different types of cancer and validation of their role in many processes related to cancer progression led to their description as a novel class of oncogenes (or 'oncomirs') and tumor suppressors. As a result, a massive influx of related research articles has emerged. Understanding the role of microRNAs in normal breast and cancer development and progression is critical to fully comprehend this disease. Many insights have been gained to date and this review highlights those with well established roles in breast cancer, as well as providing a comprehensive depiction of the most recent research articles in the field of microRNAs and breast cancer. MICRORNAs: AN INTRODUCTION MicroRNAs (miRNAs) are endogenous 19-25 nucleotide RNAs that have recently emerged as a novel class of small, evolutionarily conserved gene regulatory molecules involved in many critical developmental and cellular functions (1). miRNAs base-pair with target mRNA sequences primarily in their 3' untranslated region (3'UTR). Through specific base pairing, miRNAs induce mRNA degradation, translational repression, or both depending upon the complementarity of the miRNA to its mRNA target. Each miRNA can target numerous (100-1000) mRNAs, often in combination with other miRNAs, therefore controlling complex regulatory networks. It is estimated that there are ~900 miRNAs in mammalian cells, and that at least 30% of all genes are regulated by miRNAs (2, 3). Over 10,000 identified mature miRNAs exist in species ranging from plants to humans, suggesting that miRNAs are ancient players in gene regula- tion (4). Their existence and conservation throughout species supports the concept that they perform critical functions in gene regulation (4). Indeed, the conserved evolution of both miRNAs and transcription factors highlights their impor- tance in and the complexity of gene regulation (5).

Characterization of proteins associating with 5’ terminus of PGHS-1 mRNA

Induction of Prostaglandin Endoperoxide H Synthase-1 (PGHS-1) gene has been previously documented in a few studies during events such as development and cellular differentiation. However, molecular mechanisms governing the regulation of PGHS-1 gene expression and contributing to changes in protein levels are poorly understood. Using the MEG-01 cell model of PGHS-1 gene induction, our laboratory has previously demonstrated that the 5’UTR and the first two exons of PGHS-1 mRNA had a significant impact on decreasing the translational efficiency of a reporter gene and suggested that the presence of a secondary structure is required for conservation of this activity. This 5’end of PGHS-1 mRNA sequence has also been shown to associate with nucleolin protein. In the current study, we set to investigate the protein composition of the mRNP (messenger ribonucleoprotein) associating with the 5’end of PGHS-1 mRNA and to identify its protein members. RNA/protein binding assays coupled with LC-MS analysis identified serpin B1 and NF45 (nuclear factor 45) proteins as potential members of PGHS-1 mRNP complex. Immunoprecipitation experiments using MEG-01 protein extracts validated mass spectrometry data and confirmed binding of nucleolin, serpin B1, NF45 and NF90. The RNA fraction was extracted from immunoprecipitated mRNP complexes and association of RNA binding proteins, serpin B1, NF45 and NF90, to PGHS-1 mRNA target sequence was confirmed by RT-PCR. Together these data suggest that serpin B1, NF45 and NF90 associate with PGHS-1 mRNA and can potentially participate in the formation a single or a number of PGHS-1 ribonucleoprotein complexes, through nucleolin that possibly serves as a docking base for other protein complex members.Electronic Supplementary MaterialSupplementary material is available for this article at 10.2478/s11658-010-0005-5 and is accessible for authorized users.

Cysteine modification of a specific repressor protein controls the translational status of nucleus-encoded LHCII mRNAs in Chlamydomonas

The cytosolic RNA-binding protein NAB1 represses translation of LHCII (light-harvesting complex of photosystem II) encoding mRNAs by sequestration into translationally silent mRNP complexes in the green alga Chlamydomonas reinhardtii. NAB1 contains 2 cysteine residues, Cys-181 and Cys-226, within its C-terminal RRM motif. Modification of these cysteines either by oxidation or by alkylation in vitro was accompanied by a decrease in RNA-binding affinity for the target mRNA sequence. To confirm the relevance of reversible NAB1 cysteine oxidation for the regulation of its activity in vivo, we replaced both cysteines with serines. All examined cysteine single and double mutants exhibited a reduced antenna at PSII caused by a perturbed NAB1 deactivation mechanism, with double mutations and Cys-226 single mutations causing a stronger and more distinctive phenotype compared with the Cys-181 mutation. Our data indicated that the responsible redox control mechanism is mediated by modification of single cysteines. Polysome analyses and RNA co-immunoprecipitation experiments demonstrated the interconnection of the NAB1 thiol state and its activity as a translation repressor in vivo. NAB1 is fully active in its dithiol state and is reversibly deactivated by modification of its cysteines. In summary, this work is an example that cytosolic translation of nucleus encoded photosynthetic genes is regulated via a reversible cysteine-based redox switch in a RNA-binding translation repressor protein.

The miR-200 Family: Central Player for Gain and Loss of the Epithelial Phenotype

The miR-200 Family: Central Player for Gain and Loss of the Epithelial Phenotype