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Ribosome profiling

Yaara Finkel, Noam Stern-Ginossar, Michal Schwartz
Definition of functional genomic elements is one of the greater challenges of the genomic era. Traditionally, putative short Open Reading Frames (sORFs) coding for less than 100 amino acids were disregarded due to computational and experimental limitations, however it has become clear over the past several years that translation of sORFs is pervasive and serves diverse functions. The development of ribosome profiling, allowing identification of translated sequences genome-wide, revealed wide-spread, previously unidentified translation events...
November 18, 2017: Proteomics
Anastasia H Potts, Christopher A Vakulskas, Archana Pannuri, Helen Yakhnin, Paul Babitzke, Tony Romeo
CsrA is a post-transcriptional regulatory protein that is widely distributed among bacteria. This protein influences bacterial lifestyle decisions by binding to the 5' untranslated and/or early coding regions of mRNA targets, causing changes in translation initiation, RNA stability, and/or transcription elongation. Here, we assess the contribution of CsrA to gene expression in Escherichia coli on a global scale. UV crosslinking immunoprecipitation and sequencing (CLIP-seq) identify RNAs that interact directly with CsrA in vivo, while ribosome profiling and RNA-seq uncover the impact of CsrA on translation, RNA abundance, and RNA stability...
November 17, 2017: Nature Communications
Volodimir Olexiouk, Wim Van Criekinge, Gerben Menschaert ( is a public repository of small open reading frames (sORFs) identified by ribosome profiling (RIBO-seq). This update elaborates on the major improvements implemented since its initial release. now additionally supports three more species (zebrafish, rat and Caenorhabditis elegans) and currently includes 78 RIBO-seq datasets, a vast increase compared to the three that were processed in the initial release. Therefore, a novel pipeline was constructed that also enables sORF detection in RIBO-seq datasets comprising solely elongating RIBO-seq data while previously, matching initiating RIBO-seq data was necessary to delineate the sORFs...
November 13, 2017: Nucleic Acids Research
Bing Bai, Ondřej Novák, Karin Ljung, Johannes Hanson, Leónie Bentsink
The importance of translational regulation during Arabidopsis seed germination has been shown previously. Here the role of transcriptional and translational regulation during seed imbibition of the very dormant DELAY OF GERMINATION 1 (DOG1) near-isogenic line was investigated. Polysome profiling was performed on dormant and after-ripened seeds imbibed for 6 and 24 h in water and in the transcription inhibitor cordycepin. Transcriptome and translatome changes were investigated. Ribosomal profiles of after-ripened seeds imbibed in cordycepin mimic those of dormant seeds...
November 15, 2017: New Phytologist
Lin-Jie Shu, Yu-Liang Yang
Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) is a reliable and rapid technique applied widely in the identification and classification of microbes. MALDI-TOF MS has been used to identify many endospore-forming Bacillus species; however, endospores affect the identification accuracy when using MALDI-TOF MS because they change the protein composition of samples. Since culture conditions directly influence endospore formation and Bacillus growth, in this study we clarified how culture conditions influence the classification of Bacillus species by using MALDI-TOF MS...
November 14, 2017: Scientific Reports
Lars B Scharff, Miriam Ehrnthaler, Marcin Janowski, Liam H Childs, Claudia Hasse, Jürgen Gremmels, Stephanie Ruf, Reimo Zoschke, Ralph Bock
In prokaryotic systems, the translation initiation of many, though not all, mRNAs depends on interaction between a sequence element upstream of the start codon (the Shine-Dalgarno sequence, SD) and a complementary sequence in the 3' end of the 16S ribosomal RNA (anti-Shine-Dalgarno sequence, aSD). Although many chloroplast mRNAs harbor putative SDs in their 5' untranslated regions and the aSD displays strong conservation, the functional relevance of SD-aSD interactions in plastid translation is unclear. Here, by generating transplastomic tobacco (Nicotiana tabacum) mutants with point mutations in the aSD coupled with genome-wide analysis of translation by ribosome profiling, we provide a global picture of SD-dependent translation in plastids...
November 13, 2017: Plant Cell
Lijuan Wang, Mengyi Zhu, Qingbo Zhang, Xu Zhang, Panlei Yang, Zihui Liu, Yun Deng, Yiguang Zhu, Xueshi Huang, Li Han, Shengqing Li, Jing He
A non-ribosomal peptide synthetase (NRPS) gene cluster (sfa) was identified in Streptomyces thioluteus to direct the biosynthesis of the diisonitrile antibiotic SF2768. Its biosynthetic pathway was reasonably proposed based on bioinformatics analysis, metabolic profiles of mutants and the elucidation of the intermediate and shunt product structures. Bioinformatics-based alignment found a putative ATP-binding cassette (ABC) transporter related to iron import within the biosynthetic gene cluster, which implied that the product might be a siderophore...
November 13, 2017: ACS Chemical Biology
Charlotte D Majerczyk
RNA sequencing (RNAseq) enables transcriptional profiling of many organisms. This chapter describes the use of RNAseq in prokaryotes to identify quorum sensing (QS)-controlled transcripts by comparing samples from QS-induced and -uninduced conditions. Briefly, each RNA sample is converted to ds-cDNA in a method that limits amplification of ribosomal RNA species. The ds-cDNA contains adapters that enable sequencing and quantification by next-generation sequencing (NGS).
2018: Methods in Molecular Biology
J Li, Z Li, S Liu, R Zia, A Liang, L Yang
The normal maturation and ovulation from ovarian follicles is important in ensuring conception and improving fertility of buffalo. The molecular regulation mechanism of buffalo follicles growth, however, remains unknown. This study analyzed the gene expression profiles associated with buffalo ovarian follicle growth. According to the analysis of RNA sequencing, 17,700 unigenes and 13,672 differentially expressed genes (DEGs) were detected. A total of 30 common DEGs were identified during four stages of follicle growth, and the expression patterns are basically synchronized, suggesting the products as a result of expressions of these genes may cooperate to regulate follicular development...
November 6, 2017: Animal Reproduction Science
Muhammad Shakeel, Alicia Rodriguez, Urfa Bin Tahir, Fengliang Jin
Whenever gene expression is being examined, it is essential that a normalization process is carried out to eliminate non-biological variations. The use of reference genes, such as glyceraldehyde-3-phosphate dehydrogenase, actin, and ribosomal protein genes, is the usual method of choice for normalizing gene expression. Although reference genes are used to normalize target gene expression, a major problem is that the stability of these genes differs among tissues, developmental stages, species, and responses to abiotic factors...
November 9, 2017: Biotechnology Letters
O V Grinchuk, S P Yenamandra, R Iyer, M Singh, H K Lee, K H Lim, P K H Chow, V A Kuznetsov
Currently, molecular markers are not used whilst determining the prognosis and treatment strategy for patients with hepatocellular carcinoma (HCC). Here, we proposed that the identification of common pro-oncogenic pathways in primary tumors (PT), and adjacent non-malignant tissues (AT), that typically used to predict HCC patient risks may result in HCC biomarker discovery. We examined the genome-wide mRNA expression profiles of paired PT and AT samples from 321 HCC patients. The workflow integrated differentially expressed gene selection, gene ontology enrichment, computational classification, survival predictions, image analysis and experimental validation methods...
November 8, 2017: Molecular Oncology
Chuanxia Zhang, Yong Qiang Wang, Guangyi Jin, Song Wu, Jun Cui, Rong-Fu Wang
Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) is a rapid, reliable and widely used method of studying gene expression profiles that requires appropriate normalization for accurate and reliable results. Reference genes are usually used to normalize mRNA levels; however, the expression levels of these reference genes may vary between cell types, developmental stages, species and experimental conditions. Therefore, a normalization strategy is an important precondition for reliable conclusions, with endogenous controls requiring determination for every experimental system...
November 2017: Oncology Letters
Carmelo Andújar, Paula Arribas, Clare Gray, Katherine Bruce, Guy Woodward, Douglas W Yu, Alfried P Vogler
Biomonitoring underpins the environmental assessment of freshwater ecosystems and guides management and conservation. Current methodology for surveys of (macro)invertebrates uses coarse taxonomic identification where species-level resolution is difficult to obtain. Next-generation sequencing of entire assemblages (metabarcoding) provides a new approach for species detection, but requires further validation. We used metabarcoding of invertebrate assemblages with two fragments of the cox1 "barcode" and partial nuclear ribosomal (SSU) genes, to assess the effects of a pesticide spill in the River Kennet (Southern England)...
November 7, 2017: Molecular Ecology
Wanting Liu, Lunping Xiang, Tingkai Zheng, Jingjie Jin, Gong Zhang
Translation is a key regulatory step, linking transcriptome and proteome. Two major methods of translatome investigations are RNC-seq (sequencing of translating mRNA) and Ribo-seq (ribosome profiling). To facilitate the investigation of translation, we built a comprehensive database TranslatomeDB ( which provides collection and integrated analysis of published and user-generated translatome sequencing data. The current version includes 2453 Ribo-seq, 10 RNC-seq and their 1394 corresponding mRNA-seq datasets in 13 species...
November 2, 2017: Nucleic Acids Research
Kyle A Cottrell, Pawel Szczesny, Sergej Djuranovic
MicroRNAs are well known regulators of mRNA stability and translation. However, the magnitude of both translational repression and mRNA decay induced by miRNA binding varies greatly between miRNA targets. This can be the result of cis and trans factors that affect miRNA binding or action. We set out to address this issue by studying how various mRNA characteristics affect miRNA-mediated repression. Using a dual luciferase reporter system, we systematically analyzed the ability of selected mRNA elements to modulate miRNA-mediated repression...
November 2, 2017: Scientific Reports
Jessna H M Yeo, Steven C L Ho, Mariati Mariati, Esther Koh, Shi Jie Tay, Susanto Woen, Peiqing Zhang, Yuansheng Yang
There are several selection markers which are suitable for generating stably transfected Chinese hamster ovary (CHO) cell lines. Due to their different modes of action, each selection marker has its own optimal selection stringency in different host cells for obtaining high productivity. Using an internal ribosome entry site (IRES)-mediated tricistronic vector and a set of IRES variants with different strengths, the expression of five antibiotics resistance genes (ARGs) in CHO-K1 cells and dihydrofolate reductase (DHFR) in CHO DG44 cells is optimized to enhance the stringency of selection for high producing cells...
September 25, 2017: Biotechnology Journal
Sondos Samandi, Annie V Roy, Vivian Delcourt, Jean-François Lucier, Jules Gagnon, Maxime C Beaudoin, Benoît Vanderperre, Marc-André Breton, Julie Motard, Jean-François Jacques, Mylène Brunelle, Isabelle Gagnon-Arsenault, Isabelle Fournier, Aïda Ouangraoua, Darel J Hunting, Alan A Cohen, Christian R Landry, Michelle S Scott, Xavier Roucou
Recent functional, proteomic and ribosome profiling studies in eukaryotes have concurrently demonstrated the translation of alternative open reading frames (altORFs) in addition to annotated protein coding sequences (CDSs). We show that a large number of small proteins could in fact be coded by these altORFs. The putative alternative proteins translated from altORFs have orthologs in many species and contain functional domains. Evolutionary analyses indicate that altORFs often show more extreme conservation patterns than their CDSs...
October 30, 2017: ELife
Eliana P Velásquez-Mejía, Jacobo de la Cuesta-Zuluaga, Juan S Escobar
Culture-independent methods have granted the possibility to study microbial diversity in great detail, but technical issues pose a threat to the accuracy of new findings. Biases introduced during DNA extraction can result in erroneous representations of the microbial community, particularly in samples with low microbial biomass. We evaluated the DNA extraction method, initial sample biomass, and reagent contamination on the assessment of the human gut microbiota. Fecal samples of 200 mg were subjected to 1:10 serial dilutions; total DNA was obtained using two commercial kits and the microbiota assessed by 16S ribosomal RNA (rRNA) gene sequencing...
October 27, 2017: Applied Microbiology and Biotechnology
Engin Kaplan, Abdullah M S Al-Hatmi, Macit Ilkit, A H G Gerrits van den Ende, Ferry Hagen, Jacques F Meis, G Sybren de Hoog
Magnusiomyces capitatus and Saprochaete clavata are members of the clade of arthroconidial yeasts that represent emerging opportunistic pulmonary pathogens in immunocompromised patients. Given that standard rDNA identification often provides confusing results, in this study, we analyzed 34 isolates with the goal of finding new genetic markers for classification using multilocus sequencing and amplified fragment length polymorphism (AFLP). Interspecific similarity using rDNA markers [internal transcribed spacer (ITS) and large subunit] was in the range of 96-99%, whereas that of protein-coding loci (Rbp2, Act, and Tef1α) was lower at 89...
October 25, 2017: Journal of Clinical Microbiology
Oana Carja, Tongji Xing, Edward W J Wallace, Joshua B Plotkin, Premal Shah
BACKGROUND: Using high-throughput sequencing to monitor translation in vivo, ribosome profiling can provide critical insights into the dynamics and regulation of protein synthesis in a cell. Since its introduction in 2009, this technique has played a key role in driving biological discovery, and yet it requires a rigorous computational toolkit for widespread adoption. DESCRIPTION: We have developed a database and a browser-based visualization tool, riboviz, that enables exploration and analysis of riboseq datasets...
October 25, 2017: BMC Bioinformatics
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