Prediction of guide strand of microRNAs from its sequence and secondary structure

<p>Abstract</p> <p>Background</p> <p>MicroRNAs (miRNAs) are produced by the sequential processing of a long hairpin RNA transcript by Drosha and Dicer, an RNase III enzymes, and form transitory small RNA duplexes. One strand of the duplex, which incorporates into RNA-induced silencing complex (RISC) and silences the gene expression is called guide strand, or miRNA; while the other strand of duplex is degraded and called the passenger strand, or miRNA*. Predicting the guide strand of miRNA is important for better understanding the RNA interference pathways.</p> <p>Results</p> <p>This paper describes support vector machine (SVM) models developed for predicting the guide strands of miRNAs. All models were trained and tested on a dataset consisting of 329 miRNA and 329 miRNA* pairs using five fold cross validation technique. Firstly, models were developed using mono-, di-, and tri-nucleotide composition of miRNA strands and achieved the highest accuracies of 0.588, 0.638 and 0.596 respectively. Secondly, models were developed using split nucleotide composition and achieved maximum accuracies of 0.553, 0.641 and 0.602 for mono-, di-, and tri-nucleotide respectively. Thirdly, models were developed using binary pattern and achieved the highest accuracy of 0.708. Furthermore, when integrating the secondary structure features with binary pattern, an accuracy of 0.719 was seen. Finally, hybrid models were developed by combining various features and achieved maximum accuracy of 0.799 with sensitivity 0.781 and specificity 0.818. Moreover, the performance of this model was tested on an independent dataset that achieved an accuracy of 0.80. In addition, we also compared the performance of our method with various siRNA-designing methods on miRNA and siRNA datasets.</p> <p>Conclusion</p> <p>In this study, first time a method has been developed to predict guide miRNA strands, of miRNA duplex. This study demonstrates that guide and passenger strand of miRNA precursors can be distinguished using their nucleotide sequence and secondary structure. This method will be useful in understanding microRNA processing and can be implemented in RNA silencing technology to improve the biological and clinical research. A web server has been developed based on SVM models described in this study <url>http://crdd.osdd.net:8081/RISCbinder/</url>.</p

Identification of NAD interacting residues in proteins

Background: Small molecular cofactors or ligands play a crucial role in the proper functioning of cells. Accurate annotation of their target proteins and binding sites is required for the complete understanding of reaction mechanisms. Nicotinamide adenine dinucleotide (NAD+ or NAD) is one of the most commonly used organic cofactors in living cells, which plays a critical role in cellular metabolism, storage and regulatory processes. In the past, several NAD binding proteins (NADBP) have been reported in the literature, which are responsible for a wide-range of activities in the cell. Attempts have been made to derive a rule for the binding of NAD+ to its target proteins. However, so far an efficient model could not be derived due to the time consuming process of structure determination, and limitations of similarity based approaches. Thus a sequence and non-similarity based method is needed to characterize the NAD binding sites to help in the annotation. In this study attempts have been made to predict NAD binding proteins and their interacting residues (NIRs) from amino acid sequence using bioinformatics tools. Results: We extracted 1556 proteins chains from 555 NAD binding proteins whose structure is available in Protein Data Bank. Then we removed all redundant protein chains and finally obtained 195 non-redundant NAD binding protein chains, where no two chains have more than 40% sequence identity. In this study all models were developed and evaluated using five-fold cross validation technique on the above dataset of 195 NAD binding proteins. While certain type of residues are preferred (e.g. Gly, Tyr, Thr, His) in NAD interaction, residues like Ala, Glu, Leu, Lys are not preferred. A support vector machine (SVM) based method has been developed using various window lengths of amino acid sequence for predicting NAD interacting residues and obtained maximum Matthew's correlation coefficient (MCC) 0.47 with accuracy 74.13% at window length 17. We also developed a SVM based method using evolutionary information in the form of position specific scoring matrix (PSSM) and obtained maximum MCC 0.75 with accuracy 87.25%. Conclusion: For the first time a sequence-based method has been developed for the prediction of NAD binding proteins and their interacting residues, in the absence of any prior structural information. The present model will aid in the understanding of NAD+ dependent mechanisms of action in the cell. To provide service to the scientific community, we have developed a user-friendly web server, which is available from URL http://www.imtech.res.in/raghava/nadbinder/

Designing of highly effective complementary and mismatch siRNAs for silencing a gene

In past, numerous methods have been developed for predicting efficacy of short interfering RNA (siRNA). However these methods have been developed for predicting efficacy of fully complementary siRNA against a gene. Best of author's knowledge no method has been developed for predicting efficacy of mismatch siRNA against a gene. In this study, a systematic attempt has been made to identify highly effective complementary as well as mismatch siRNAs for silencing a gene. Support vector machine (SVM) based models have been developed for predicting efficacy of siRNAs using composition, binary and hybrid pattern siRNAs. We achieved maximum correlation 0.67 between predicted and actual efficacy of siRNAs using hybrid model. All models were trained and tested on a dataset of 2182 siRNAs and performance was evaluated using five-fold cross validation techniques. The performance of our method desiRm is comparable to other well-known methods. In this study, first time attempt has been made to design mutant siRNAs (mismatch siRNAs). In this approach we mutated a given siRNA on all possible sites/positions with all possible nucleotides. Efficacy of each mutated siRNA is predicted using our method desiRm. It is well known from literature that mismatches between siRNA and target affects the silencing efficacy. Thus we have incorporated the rules derived from base mismatches experimental data to find out over all efficacy of mutated or mismatch siRNAs. Finally we developed a webserver, desiRm (http://www.imtech.res.in/raghava/desirm/) for designing highly effective siRNA for silencing a gene. This tool will be helpful to design siRNA to degrade disease isoform of heterozygous single nucleotide polymorphism gene without depleting the wild type protein

FLOating-Window Projective Separator (FloWPS): A Data Trimming Tool for Support Vector Machines (SVM) to Improve Robustness of the Classifier

Here, we propose a heuristic technique of data trimming for SVM termed FLOating Window Projective Separator (FloWPS), tailored for personalized predictions based on molecular data. This procedure can operate with high throughput genetic datasets like gene expression or mutation profiles. Its application prevents SVM from extrapolation by excluding non-informative features. FloWPS requires training on the data for the individuals with known clinical outcomes to create a clinically relevant classifier. The genetic profiles linked with the outcomes are broken as usual into the training and validation datasets. The unique property of FloWPS is that irrelevant features in validation dataset that don’t have significant number of neighboring hits in the training dataset are removed from further analyses. Next, similarly to the k nearest neighbors (kNN) method, for each point of a validation dataset, FloWPS takes into account only the proximal points of the training dataset. Thus, for every point of a validation dataset, the training dataset is adjusted to form a floating window. FloWPS performance was tested on ten gene expression datasets for 992 cancer patients either responding or not on the different types of chemotherapy. We experimentally confirmed by leave-one-out cross-validation that FloWPS enables to significantly increase quality of a classifier built based on the classical SVM in most of the applications, particularly for polynomial kernels

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

Potential of Muller glia-derived extracellular vesicles for retinal neuroprotection

Müller glia cells retain progenitor-like characteristics in the adult human eye and can partially restore visual function upon intravitreal transplantation into animal models of glaucoma. Based on observations that this effect does not depend on cell replacement of the degenerated retina, efficacy is thought to depend upon the cell transfer of neuroprotective agents to retinal ganglion cells (RGC). In an attempt to identify candidate molecules, this work first characterised the ability of Müller glia to synthesise and secrete a range of neurotrophic growth factors. Since certain pro-inflammatory cytokines are known to be upregulated in the glaucomatous eye, this study also investigated the modulatory effects of exogenous cytokines on the expression of neurotrophin genes and proteins. Recently it has been demonstrated that cells can communicate through the release of nano-sized membrane-bound organelles called extracellular vesicles (EV). These contain bioactive molecules that induce functional changes when internalised by recipient cells. Small and large sub-populations of EV (sEV and lEV) were purified from Müller glia cultures and their contents were characterised, revealing the enrichment of characteristic proteins and several RNA species. Of particular interest are the findings of small non-coding microRNAs (miRNA) present in the sEV, that directly regulate gene expression through silencing of mRNA. Sequencing of miRNAs recovered from small EV subsets indicated preferential enrichment with transcripts that target genes involved in cell growth and survival, including PTEN, the master inhibitor of the AKT/mTOR pathway. On this basis, a putative mechanism for the neural pro-survival effects induced by neurotrophic factors previously ascribed to Müller glia was suggested. Labelling of EV with a lipophilic dye confirmed the direct internalisation of these vesicles into primary RGC in culture. Since vesicle uptake has been shown to be cell type specific, it seems likely that Müller glia derived EV express membrane molecules that facilitate this internalisation, making them appealing for use as vehicles for the delivery of neuroprotective molecules to retinal neurons. Finally, Müller EV function was tested in vivo using a NMDA-model of RGC depletion. Adult rats receiving intravitreal delivery of 3x109 small EV showed significantly improvement in RGC function when compared to vehicle control, as determined by the negative scotopic threshold response (nSTR) of the dark-adapted electroretinogram (ERG). Taken together, the data presented in this thesis suggests that EV represent a significant part of the neuroprotective Müller cell signalling activity, likely in addition to the release of neurotrophic growth factors. EV enriched in neuroprotective molecules may represent a more stable, and less immunogenic alternative to whole cell transplantation, particularly given their minute size and low toxicity profiles. This work provides new insight into the neuroprotective secretome of Müller glia, adding further support to previous studies demonstrating their potential utility in a cell-based glaucoma therapy

Pulmonary arterial hypertension: role of miRNAs in animal models and pathological samples

Pulmonary arterial hypertension (PAH) is a disease of the small pulmonary arteries (PAs), characterized by an increase in pulmonary arterial pressure and vascular remodelling leading to a progressive increase in pulmonary vascular resistance. The consequence of vascular obliteration is right heart failure and high mortality. Germline mutations in the gene coding for the bone morphogenetic protein (BMP) type-2 receptor (BMPR2), a receptor for the transforming growth factor (TGF)-beta super-family, have been identified in approximately 70% of patients with the heritable form of PAH (HPAH). Moreover, BMPR2 expression is markedly reduced in PAH cases in the absence of mutations in this gene (idiopathic PAH, IPAH). In pulmonary artery smooth muscle cells (PASMCs) mutations in BMPR2 are associated with an abnormal growth response to BMPs and TGF-beta. In endothelial cells (PAECs), these mutations increase the susceptibility of cells to apoptosis. The absence of BMPR2 mutations in some families and in the majority of IPAH cases suggests that further pathological mechanisms still need to be identified. The serotonin system has also been implicated in both experimental and human PAH. In fact, an additional genetic risk factor for the development of this pathology has been identified in the serotonin transporter (SERT), dysregulated in IPAH patients. Mice over-expressing SERT (SERT+ mice) exhibit PAH and exaggerated hypoxia-induced PAH. Although different advanced PAH therapies are currently available, they can only provide a symptomatic relief, and mortality rates remain high. Therefore, the identification of novel therapeutic approaches for the treatment of this pathology is urgently required. MicroRNAs (miRNAs) are a class of small, endogenous and non-coding RNAs able to negatively regulate gene expression by targeting specific messenger RNAs (mRNAs) and inducing their degradation or translational repression. These non-coding sequences are transcribed from endogenous loci as long precursors, converted in single-stranded molecules of approximately 20 nucleotides after a series of enzymatic maturation steps. miRNAs carry out their activity in association with the RNA-induced silencing complex (RISC), interacting with the 3’ untranslated region (3’UTR) of specific target mRNAs which they bind with imperfect complementarity. Several recent studies have assessed the direct role of miRNAs in vascular inflammation and in the development of cardiovascular pathologies. The aim of this project was to investigate the role of miRNAs in the development of PAH. In Chapter 3, two distinct and well established rat models (hypoxic and monocrotaline) of PAH were used to determine the regulation of miRNAs during disease initiation and progression. We demonstrate time and insult-dependent changes in a specific group if miRNAs and this dysregulation was also confirmed in vitro in rat and human PA cells exposed to chronic hypoxia. Moreover, the stimulation of rat cells with TGF-beta and BMP4 mimicked the alteration of miRNA expression observed in vivo. An analysis of the expression level of the main enzymes involved in miRNA maturation (i.e. Dicer, Drosha, DGCR8 and Exp5) revealed the significant down-regulation of Dicer in response to chronic hypoxia both in vivo and in vitro, suggesting that the manipulation of this enzyme could re-establish a normal miRNA expression level in pathological samples. We also identified selective targets altered in response to miRNA dysregulation, suggesting the possibility of future interventional studies. In Chapter 4 the specific role of miR-143 and miR-145 in the development of PAH was evaluated. We report the significant up-regulation of these miRNAs in WT mice exposed to chronic hypoxia and that genetic ablation of miR-145 is protective against the development of PAH (with no effects on miR-143 expression), assessed via measurement of systolic right ventricular pressure (sRVP), pulmonary vascular remodelling and right ventricular hypertrophy (RVH). miR-145 KO has also an effect on the expression of specific targets, including kruppel-like factor 4 and 5 (KLF4 and 5), which are regulators of smooth muscle proliferation and differentiation. Further, both miR-143 and miR-145 are up-regulated in mice heterozygous for a BMPR2 mutation. In human tissues we confirm the elevated expression of the miR-143/145 cluster observed in hypoxic mice in pathological samples compared with unaffected controls, suggesting a conserved regulation of these miRNAs in the two species. The study described in this chapter is the first to report a critical role for miR-145 in the development of PAH in vivo. Finally, in Chapter 5 a preliminary study focused on miR-21 regulation and function on PAH development is shown. An analysis of the expression of this miRNA in WT mice revealed its up-regulation in response to chronic hypoxia, whereas the genetic ablation of miR-21 induced an exaggerated hypoxia-induced PAH phenotype. However, the analysis of human pathological samples showed a reduced expression of this miRNA in comparison with unaffected controls, suggesting its differential regulation in hypoxic mice and patients, although the differences observed between the animal and the human pathology could be the cause of this different phenothipe. The identification of dysregulated targets in both the species will give more informations about the effect of miR-21 alteration in the development of PAH. In summary, the results presented in this thesis support a role for defined miRNAs in the development of PAH, both in animal models and patients. Whether this specific alteration of selective miRNAs can be used as a novel therapeutical approach still need to be evaluated, but represent an attractive possibility to assess in the longer term