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Methods: a Companion to Methods in Enzymology

Ahalya Balachandran, Alan Cochrane
The control of RNA processing plays an important role in the nature and quantity of protein generated from mammalian genes. Consequently, efforts to manipulate RNA processing have the capacity to significantly impact gene function. Although multiple strategies have been developed to alter splice site selection using oligonucleotide occlusion of splice sites or splicing regulatory elements, systemic delivery of such agents remains problematic. Outlined in this chapter is a protocol to screen for small molecule inhibitors of HIV-1 Gag expression that have been subsequently determined to modulate viral RNA processing...
June 17, 2017: Methods: a Companion to Methods in Enzymology
Xianjun Shen, Yao Chen, Xingpeng Jiang, Xiaohua Hu, Tingting He, Jincai Yang
As we all know, the microbiota show remarkable variability within individuals. At the same time, those microorganisms living in the human body play a very important role in our health and disease, so the identification of the relationships between microbes and diseases will contribute to better understanding of microbes interactions, mechanism of functions. However, the microbial data which are obtained through the related technical sequencing is too much, but the known associations between the diseases and microbes are very less...
June 15, 2017: Methods: a Companion to Methods in Enzymology
Masato Kato, Yi Lin, Steven L McKnight
Low-complexity (LC) sequences, typically believed to be incapable of assuming structural order, are abundant constituents of the proteomes of all eukaryotic organisms. These sequences have emerged as critical components for formation of meso-scaled, sub-cellular organelles not invested by surrounding membranes, exemplified by RNA granules. We have observed that LC domains of many RNA binding proteins known to be constituents of RNA granules readily form labile cross-β polymers under physiological conditions...
June 14, 2017: Methods: a Companion to Methods in Enzymology
Nirmal Mazumder, Gitanjal Deka, Wei-Wen Wu, Ankur Gogoi, Guan-Yu Zhuo, Fu-Jen Kao
Second harmonic (SH) microscopy has proven to be a powerful imaging modality over the past years due to its intrinsic advantages as a multiphoton process with endogenous contrast specificity, which allows pinhole-less optical sectioning, non-invasive observation, deep tissue penetration, and the possibility of easier signal detection at visible wavelengths. Depending on the relative orientation between the polarization of the incoming light and the second-order susceptibility of non-centrosymmetric structures, SH microscopy provides the unique capacity to probe the absolute molecular structure of a broad variety of biological tissues without the necessity for additional labeling...
June 14, 2017: Methods: a Companion to Methods in Enzymology
Karl E Bauer, Michael A Kiebler, Inmaculada Segura
In polarized cells, such as neurons, the synthesis of an mRNA does not ensure its proper cellular expression. Most mature transcripts require the association with RNA-binding proteins, resulting in the formation of RNA granules, which are then transported within the cytoplasm along the cytoskeleton and delivered to their proper subcellular locations, where they can be locally translated. Here we review current microscopy methods that have been developed to visualize RNA granule formation, transport and translation at the single cell level with a special emphasis on the MS2 and SunTag systems...
June 14, 2017: Methods: a Companion to Methods in Enzymology
Cong Liu, Xujun Wang, Georgi Z Genchev, Hui Lu
Motivation New developments in high-throughput genomic technologies have enabled the measurement of diverse types of omics biomarkers in a cost-efficient and clinically-feasible manner. Developing computational methods and tools for analysis and translation of such genomic data into clinically-relevant information is an ongoing and active area of investigation. For example, several studies have utilized an unsupervised learning framework to cluster patients by integrating omics data. Despite such recent advances, predicting cancer prognosis using integrated omics biomarkers remains a challenge...
June 13, 2017: Methods: a Companion to Methods in Enzymology
Michael W Webster, James A W Stowell, Terence T L Tang, Lori A Passmore
Poly(A) tails are found at the 3' end of almost every eukaryotic mRNA and are important for the stability of mRNAs and their translation into proteins. Thus, removal of the poly(A) tail, a process called deadenylation, is critical for regulation of gene expression. Most deadenylation enzymes are components of large multi-protein complexes. Here, we describe an in vitro deadenylation assay developed to study the exonucleolytic activities of the multi-protein Ccr4-Not and Pan2-Pan3 complexes. We discuss how this assay can be used with short synthetic RNAs, as well as longer RNA substrates generated using in vitro transcription...
June 13, 2017: Methods: a Companion to Methods in Enzymology
Haiying Wang, Huiru Zheng, Fiona Browne, Rainer Roehe, Richard J Dewhurst, Felix Engel, Matthias Hemmje, Xiangwu Lu, Paul Walsh
Methane is one of major contributors to global warming. The rumen microbiota is directly involved in methane production in cattle. The link between variations in rumen microbial communities and host genetics has important applications and implications in bioscience. Having the potential to reveal the full extent of microbial gene diversity and complex microbial interactions, integrated metagenomics and network analysis holds great promises in this endeavour. This study investigates the rumen microbial community in cattle through the integration of metagenomic and network-based approaches...
June 8, 2017: Methods: a Companion to Methods in Enzymology
P F Ellen Boswijk, Matthias Bauwens, Felix M Mottaghy, Joachim E Wildberger, Jan Bucerius
Atherosclerotic events are usually acute and often strike otherwise asymptomatic patients. Although multiple clinical risk factors have been associated with atherosclerosis up to date, no further individual prediction can be made as to who will acutely suffer from its consequences based on traditional imaging methods like CT, MRI or angiography, or biomarkers. Previously, non-invasive imaging with (18)F-fluorodeoxyglucose ((18)F-FDG) PET was shown to potentially fill this niche as it offers high sensitive detection of metabolic processes associated with inflammatory changes in atherosclerotic plaques...
June 7, 2017: Methods: a Companion to Methods in Enzymology
Gianfranco Politano, Federica Logrand, Mara Brancaccio, Stefano Di Carlo
In most developed countries, cardiovascular diseases are among the top causes of death and their development has been shown closely related to aging. In this context, because of their ability to pervasively influence gene networks, miRs have been found as possible key players in the development of cardiac pathologies, suggesting their potential role as therapeutic targets or diagnostic markers. Based on these assumptions, we hereby present a computational study that applies data fusion techniques coupled with network analysis theory to identify a regulatory model able to represent the relationship between key genes and miRs involved in cardiac senescence processes...
June 7, 2017: Methods: a Companion to Methods in Enzymology
Piero Sanfilippo, Pedro Miura, Eric C Lai
Alternative polyadenylation (APA) diversifies the 3' termini of a majority of mRNAs in most eukaryotes, and is consequently inferred to have substantial consequences for the utilization of post-transcriptional regulatory mechanisms. Since conventional RNA-sequencing methods do not accurately define mRNA termini, a number of protocols have been developed that permit sequencing of the 3' ends of polyadenylated transcripts (3'-seq). We present here our experimental protocol to generate 3'-seq libraries using a dT-priming approach, including extensive details on considerations that will enable successful library cloning...
June 7, 2017: Methods: a Companion to Methods in Enzymology
Gabriella Sanzò, Irene Taurino, Francesca Puppo, Riccarda Antiochia, Lo Gorton, Gabriele Favero, Franco Mazzei, Sandro Carrara, Giovanni De Micheli
In this work, we have developed for the first time a method to make novel gold and platinum hybrid bimetallic nanostructures differing in shape and size. Au-Pt nanostructures were prepared by electrodeposition in two simple steps. The first step consists of the electrodeposition of nanocoral Au onto a gold substrate using hydrogen as a dynamic template in an ammonium chloride solution. After that, the Pt nanostructures were deposited onto the nanocoral Au organized in pores. Using Pt (II) and Pt (IV), we realized nanocoral Au decorated with Pt nanospheres and nanocoral Au decorated with Pt nanoflowers, respectively...
June 6, 2017: Methods: a Companion to Methods in Enzymology
Sehi L'Yi, Daekyoung Jung, Minsik Oh, Bohyoung Kim, Robert J Freishtat, Mamta Giri, Eric Hoffman, Jinwook Seo
In this paper, we present miRTarVis+, a Web-based interactive visual analytics tool for miRNA target predictions and integrative analyses of multiple prediction results. Various microRNA (miRNA) target prediction algorithms have been developed to improve sequence-based miRNA target prediction by exploiting miRNA-mRNA expression profile data. There are also a few analytics tools to help researchers predict targets of miRNAs. However, there still is a need for improving the performance for miRNA prediction algorithms and more importantly for interactive visualization tools for an integrative analysis of multiple prediction results...
June 6, 2017: Methods: a Companion to Methods in Enzymology
Rachel Soemedi, Kamil J Cygan, Christy Rhine, David T Glidden, Allison J Taggart, Chien-Ling Lin, Alger M Fredericks, William G Fairbrother
Pre-mRNA molecules can form a variety of structures, and both secondary and tertiary structures have important effects on processing, function and stability of these molecules. The prediction of RNA secondary structure is a challenging problem and various algorithms that use minimum free energy, maximum expected accuracy and comparative evolutionary based methods have been developed to predict secondary structures. However, these tools are not perfect, and this remains an active area of research. The secondary structure of pre-mRNA molecules can have an enhancing or inhibitory effect on pre-mRNA splicing...
June 5, 2017: Methods: a Companion to Methods in Enzymology
Arda Soylev, Can Kockan, Fereydoun Hormozdiari, Can Alkan
Structural variations (SV) are broadly defined as genomic alterations that affect > 50 bp of DNA, which are shown to have significant effect on evolution and disease. The advent of high throughput sequencing (HTS) technologies and the ability to perform whole genome sequencing (WGS), makes it feasible to study these variants in depth. However, discovery of all forms of SV using WGS has proven to be challenging as the short reads produced by the predominant HTS platforms (<200bp for current technologies) and the fact that most genomes include large amounts of repeats make it very difficult to unambiguously map and accurately characterize such variants...
June 2, 2017: Methods: a Companion to Methods in Enzymology
Nicholas J McGlincy, Nicholas T Ingolia
Translation is one of the fundamental processes of life. It comprises the assembly of polypeptides whose amino acid sequence corresponds to the codon sequence of an mRNA's ORF. Translation is performed by the ribosome; therefore, in order to understand translation and its regulation we must be able to determine the numbers and locations of ribosomes on mRNAs in vivo. Furthermore, we must be able to examine their redistribution in different physiological contexts and in response to experimental manipulations...
June 1, 2017: Methods: a Companion to Methods in Enzymology
Fabio Alexis Lefebvre, Neal Cody, Louis Philip Benoit Bouvrette, Julie Bergalet, Xiaofeng Wang, Eric Lécuyer
The subcellular trafficking of RNA molecules is a conserved feature of eukaryotic cells and plays key functions in diverse processes implicating polarised cellular activities. Large-scale imaging and subcellular transcriptomic studies suggest that regulated RNA localization is a highly prevalent process that appears to be disrupted in several neuromuscular disorders. These features underline the importance and usefulness of implementing procedures to assess global transcriptome subcellular distribution properties...
June 1, 2017: Methods: a Companion to Methods in Enzymology
Sangseon Lee, Youngjune Park, Sun Kim
Pathway based analysis of high throughput transcriptome data is a widely used approach to investigate biological mechanisms. Since a pathway consists of multiple functions, the recent approach is to determine condition specific sub-pathways or subpaths. However, there are several challenges. First, few existing methods utilize explicit gene expression information from RNA-seq. More importantly, subpath activity is usually an average of statistical scores, e.g., correlations, of edges in a candidate subpath, which fails to reflect gene expression quantity information...
June 1, 2017: Methods: a Companion to Methods in Enzymology
Le Ou-Yang, Xiao-Fei Zhang, Min Wu, Xiao-Li Li
Recovering gene regulatory networks and exploring the network rewiring between two different disease states are important for revealing the mechanisms behind disease progression. The advent of high-throughput experimental techniques has enabled the possibility of inferring gene regulatory networks and differential networks using computational methods. However, most of existing differential network analysis methods are designed for single-platform data analysis and assume that differences between networks are driven by individual edges...
June 1, 2017: Methods: a Companion to Methods in Enzymology
Lihua Zhang, Rong Li, Junyi He, Qiuping Yang, Yanan Wu, Jingshan Huang, Bin Wu
Diabetic kidney disease (DKD) is a serious disease that presents a major health problem worldwide. There is a desperate need to explore novel biomarkers to further facilitate the early diagnosis and effective treatment in DKD patients, thus preventing them from developing end-stage renal disease (ESRD). However, most regulation mechanisms at the genetic level in DKD still remain unclear. In this paper, we describe our innovative methodologies that integrate biological, computational, and statistical approaches to investigate important roles performed by regulations among microRNAs (miRs), long non-coding RNAs (lncRNAs), and messenger RNAs (mRNAs) in DKD...
May 31, 2017: Methods: a Companion to Methods in Enzymology
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