High-throughput analysis of the RNA-induced silencing complex in myotonic dystrophy type 1 patients identifies the dysregulation of miR-29c and its target ASB2

Myotonic dystrophy type 1 (DM1) is a multi-systemic disorder caused by abnormally expanded stretches of CTG DNA triplets in the DMPK gene, leading to mutated-transcript RNA-toxicity. MicroRNAs (miRNAs) are short non-coding RNAs that, after maturation, are loaded onto the RISC effector complex that destabilizes target mRNAs and represses their translation. In DM1 muscle biopsies not only the expression, but also the intracellular localization of specific miRNAs is disrupted, leading to the dysregulation of the relevant mRNA targets. To investigate the functional alterations of the miRNA/target interactions in DM1, we analyzed by RNA-sequencing the RISC-associated RNAs in skeletal muscle biopsies derived from DM1 patients and matched controls. The mRNAs found deregulated in DM1 biopsies were involved in pathways and functions relevant for the disease, such as energetic metabolism, calcium signaling, muscle contraction and p53-dependent apoptosis. Bioinformatic analysis of the miRNA/mRNA interactions based on the RISC enrichment profiles, identified 24 miRNA/mRNA correlations. Following validation in 21 independent samples, we focused on the couple miR-29c/ASB2 because of the role of miR-29c in fibrosis (a feature of late-stage DM1 patients) and of ASB2 in the regulation of muscle mass. Luciferase reporter assay confirmed the direct interaction between miR-29c and ASB2. Moreover, decreased miR-29c and increased ASB2 levels were verified also in immortalized myogenic cells and primary fibroblasts, derived from biopsies of DM1 patients and controls. CRISPR/Cas9-mediated deletion of CTG expansions rescued normal miR-29c and ASB2 levels, indicating a direct link between the mutant repeats and the miRNA/target expression. In conclusion, functionally relevant miRNA/mRNA interactions were identified in skeletal muscles of DM1 patients, highlighting the dysfunction of miR-29c and ASB2

The Nefarious Nexus of Noncoding RNAs in Cancer

The past decade has witnessed enormous progress, which has seen the noncoding RNAs (ncRNAs) turn from the so called dark matter RNA to critical functional molecules, influencing most physiological processes in development and disease contexts. Many ncRNAs interact with each other and are part of networks that influence the cell transcriptome and proteome and consequently the outcome of biological processes. The regulatory circuits controlled by ncRNAs have become increasingly more relevant in cancer. Further understanding of these complex network interactions and how ncRNAs are regulated, is paving the way for the identification of better therapeutic strategies in cancer

siRNA and RNAi optimization

The discovery and examination of the posttranscriptional gene regulatory mechanism known as RNA interference (RNAi) contributed to the identification of small interfering RNA (siRNA) and the comprehension of its enormous potential for clinical purposes. Theoretically, the ability of specific target gene downregulation makes the RNAi pathway an appealing solution for several diseases. Despite numerous hurdles resulting from the inherent properties of siRNA molecule and proper delivery to the target tissue, more than 50 RNA-based drugs are currently under clinical testing. In this work, we analyze the recent literature in the optimization of siRNA molecules. In detail, we focused on describing the most recent advances of siRNA field aimed at optimize siRNA pharmacokinetic properties. Special attention has been given in describing the impact of RNA modifications in the potential off-target effects (OTEs) such as saturation of the RNAi machinery, passenger strand-mediated silencing, immunostimulation, and miRNA-like OTEs as well as to recent developments on the delivery issue. The novel delivery systems and modified siRNA provide significant steps toward the development of reliable siRNA molecules for therapeutic use. © 2016 Wiley Periodicals, Inc.The work is supported by grants of the European Commission (NMP4-LA-2011-262943, MULTIFUN), the Spanish Ministry of Economy (CTQ2014-52588-R, RTC-2014-2038-1), Generalitat de Catalunya (2014/SGR/624) and the Instituto de Salud Carlos III (CB06_01_0019) with assistance from the European Regional Development Fund.Peer reviewe

Characterisation of microRNAs in Human Stem Cells

In collaboration with David Baulcombe and Attila Molnar we have generated microRNA libraries for human embryonic stem cells (hESCs) before and after differentiation along the neuronal lineage and also from human mesenchymal stem cells (hMSCs). Both cell types are of medical importance and understanding how their proliferation and differentiation is regulated by microRNAs is also of scientific interest. The hMSC library was sequenced by 454 technology and the two subsequent hESC libraries by Solexa sequencing. Approximately a quarter of all currently known microRNAs were identified between the libraries, in addition to 3 novel microRNAs and 25 annotated piRNAs. For the hESC libraries, we verified the presence of embryonic specific microRNAs (miR-302 family) and neuronal specific microRNAs (miR-9/miR-9*), and demonstrated that expression of these miRNAs is regulated at the transcriptional level. Additionally, promoter assessments of miR-9 transcription revealed that multiple upstream regions may be important in neuronal specific expression. Almost half of all known human microRNAs are located within the introns of host genes. We used microarrays to analyse host gene expression and found that there was little correlation with microRNA expression, indicating that many microRNAs are not regulated at the transcriptional level by their host promoter. Furthermore, the expression of microRNAs from the same cluster, and also from the same hairpin precursor, did not always correlate when compared between the stem cell libraries. Taken together, this data indicates that microRNAs are regulated at a variety of levels both pre- and post-transcriptionally. Many microRNA isomers were also detected that differed in expression between human cell types, and upon differentiation of the hMSCs through the osteoblastic lineage. Interestingly, microRNAs and some of their isomers showed different affinities for Argonaute proteins in pulldown assays. We also profiled mRNAs that were immunoprecipitated with Argonaute in order to identify miRNA target

MicroRNA and Cardiac Stem Cell Therapy

Cardiac Progenitor Cells (CPCs) are multipotent cells of the myocardium. They are located inside niches of the heart muscle, can be isolated, characterized and used for cardiac regeneration in stem cell therapy. Actually, CPCs may be isolated by tissue digestion with or without cell sorting, but it is difficult to achieve the maximum level of differentiation when these cells are implanted into a damaged myocardium. The knowledge recently acquired on small molecules of non-coding RNAs, microRNA (miRNA), may improve the use of these cells in stem cell therapy. In fact, these small molecules may be attached to devices or adminstered as they are or in combination with nanoparticles in order to drive the correct differentiation of stem cells. Regarding heart regeneration, we can acquire knowledge from the role of miRNAs in heart development and use it to reprogram CPCs to gain the correct three-dimensional structure of the cardiac muscle

A-to-I RNA Editing: Current Knowledge Sources and Computational Approaches with Special Emphasis on Non-Coding RNA Molecules

RNA editing is a dynamic mechanism for gene regulation attained through the alteration of the sequence of primary RNA transcripts. A-to-I (Adenosine-to-Inosine) RNA editing, which is catalyzed by members of the Adenosine Deaminase Acting on RNA (ADAR) family of enzymes, is the most common post-transcriptional modification in humans. The ADARs bind double-stranded regions and deaminate adenosine (A) into inosine (I), which in turn is interpreted by the translation and splicing machineries as guanosine (G). In recent years, this modification has been discovered to occur not only in coding RNAs but also in non-coding RNAs (ncRNA), such as microRNAs (miRNAs), small interfering RNAs (siRNAs), transfer RNAs (tRNAs), and long non-coding RNAs (lncRNAs). This may have several consequences, such as the creation or disruption of microRNA/mRNA binding sites, and thus affect the biogenesis, stability, and target recognition properties of ncRNAs. The malfunction of the editing machinery is not surprisingly associated with various human diseases, such as neurodegenerative, cardiovascular and carcinogenic diseases.Despite the enormous efforts made so far, the real biological function of this phenomenon, as well as the features of the ADAR substrate, in particular in non-coding RNAs, has still not been fully understood. In this work we focus on the current knowledge of RNA editing on ncRNA molecules and provide a few examples of computational approaches to elucidate its biological function

Regulation of microRNA biogenesis and turnover by animals and their viruses

Item does not contain fulltextMicroRNAs (miRNAs) are a ubiquitous component of gene regulatory networks that modulate the precise amounts of proteins expressed in a cell. Despite their small size, miRNA genes contain various recognition elements that enable specificity in when, where and to what extent they are expressed. The importance of precise control of miRNA expression is underscored by functional studies in model organisms and by the association between miRNA mis-expression and disease. In the last decade, identification of the pathways by which miRNAs are produced, matured and turned-over has revealed many aspects of their biogenesis that are subject to regulation. Studies in viral systems have revealed a range of mechanisms by which viruses target these pathways through viral proteins or non-coding RNAs in order to regulate cellular gene expression. In parallel, a field of study has evolved around the activation and suppression of antiviral RNA interference (RNAi) by viruses. Virus encoded suppressors of RNAi can impact miRNA biogenesis in cases where miRNA and small interfering RNA pathways converge. Here we review the literature on the mechanisms by which miRNA biogenesis and turnover are regulated in animals and the diverse strategies that viruses use to subvert or inhibit these processes

Post-transcriptional and post-translational regulations of drought and heat response in plants: a spider’s web of mechanisms

Drought and heat tolerance are complex quantitative traits. Moreover, the adaptive significance of some stress-related traits is more related to plant survival than to agronomic performance. A web of regulatory mechanisms fine-tunes the expression of stress-related traits and integrates both environmental and developmental signals. Both post-transcriptional and post-translational modifications contribute substantially to this network with a pivotal regulatory function of the transcriptional changes related to cellular and plant stress response. Alternative splicing and RNA-mediated silencing control the amount of specific transcripts, while ubiquitin and SUMO modify activity, sub-cellular localization and half-life of proteins. Interactions across these modification mechanisms ensure temporally and spatially appropriate patterns of downstream-gene expression. For key molecular components of these regulatory mechanisms, natural genetic diversity exists among genotypes with different behavior in terms of stress tolerance, with effects upon the expression of adaptive morphological and/or physiological target traits

miR-196b target screen reveals mechanisms maintaining leukemia stemness with therapeutic potential.

We have shown that antagomiR inhibition of miRNA miR-21 and miR-196b activity is sufficient to ablate MLL-AF9 leukemia stem cells (LSC) in vivo. Here, we used an shRNA screening approach to mimic miRNA activity on experimentally verified miR-196b targets to identify functionally important and therapeutically relevant pathways downstream of oncogenic miRNA in MLL-r AML. We found Cdkn1b (p27Kip1) is a direct miR-196b target whose repression enhanced an embryonic stem cell–like signature associated with decreased leukemia latency and increased numbers of leukemia stem cells in vivo. Conversely, elevation of p27Kip1 significantly reduced MLL-r leukemia self-renewal, promoted monocytic differentiation of leukemic blasts, and induced cell death. Antagonism of miR-196b activity or pharmacologic inhibition of the Cks1-Skp2–containing SCF E3-ubiquitin ligase complex increased p27Kip1 and inhibited human AML growth. This work illustrates that understanding oncogenic miRNA target pathways can identify actionable targets in leukemia