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upstream open reading frame

Chang-Ye Hui, Yan Guo, Wen Zhang, Xian-Qing Huang
Real-time quantification of recombinant proteins is important in studies on fermentation engineering, cell engineering, etc. Measurement of the expression level of heterologous proteins in bacterial fermentation broth has traditionally relied on time-consuming and labor-intensive procedures, such as polyacrylamide gel electrophoresis, immunoblot analysis, and biological activity assays. We describe a simple, fast, and high sensitive assay for detecting heterologous proteins production in bacteria either at the overall level (fluorescence spectrophotometry) or at the individual level (fluorescence microscopic image) in this study...
May 21, 2018: AMB Express
Penyu Zhou, Zhengyuan Zhai, Xiong Yao, Peihua Ma, Yanling Hao
A novel cryptic plasmid from Enterococcus durans 1-8, designated as pMK8, was sequenced and analyzed in this study. It consists of 3337 bp with a G + C content of 33.11%. Sequence analysis of pMK8 revealed three putative open reading frames (ORFs). Based on homology, two of them were identified as genes encoding replication initiation (RepC) and mobilization (Mob) protein, respectively. Sequence analysis revealed a pT181 family double-strand origin (dso) and a putative single-strand origin (sso) located upstream of the repC gene...
May 17, 2018: Current Microbiology
Izumi Aibara, Tatsuya Hirai, Koji Kasai, Junpei Takano, Hitoshi Onouchi, Satoshi Naito, Toru Fujiwara, Kyoko Miwa
Boron (B) is an essential element for plants; however, as high B concentrations are toxic, B transport must be tightly regulated. BOR1 is a borate exporter in Arabidopsis thaliana that facilitates B translocation into shoots under B deficiency conditions. When the B supply is sufficient, BOR1 expression is downregulated by selective degradation of BOR1 protein, while additional BOR1 regulatory mechanisms are proposed to exist. In this study, we identified a novel B-dependent BOR1 translational suppression mechanism...
May 4, 2018: Plant Physiology
Christine Müller, Laura M Zidek, Tobias Ackermann, Tristan de Jong, Peng Liu, Verena Kliche, Mohamad Amr Zaini, Gertrud Kortman, Liesbeth Harkema, Dineke S Verbeek, Jan P Tuckermann, Julia von Maltzahn, Alain de Bruin, Victor Guryev, Zhao-Qi Wang, Cornelis F Calkhoven
Ageing is associated with physical decline and the development of age-related diseases such as metabolic disorders and cancer. Few conditions are known that attenuate the adverse effects of ageing, including calorie restriction (CR) and reduced signalling through the mechanistic target of rapamycin complex 1 (mTORC1) pathway. Synthesis of the metabolic transcription factor C/EBPβ-LIP is stimulated by mTORC1, which critically depends on a short upstream open reading frame (uORF) in the Cebpb -mRNA. Here we describe that reduced C/EBPβ-LIP expression due to genetic ablation of the uORF delays the development of age-associated phenotypes in mice...
April 30, 2018: ELife
Mikhail M Pooggin, Lyubov A Ryabova
Viruses have compact genomes and usually translate more than one protein from polycistronic RNAs using leaky scanning, frameshifting, stop codon suppression or reinitiation mechanisms. Viral (pre-)genomic RNAs often contain long 5'-leader sequences with short upstream open reading frames (uORFs) and secondary structure elements, which control both translation initiation and replication. In plants, viral RNA and DNA are targeted by RNA interference (RNAi) generating small RNAs that silence viral gene expression, while viral proteins are recognized by innate immunity and autophagy that restrict viral infection...
2018: Frontiers in Microbiology
Courtney Whalen, Christine Tuohy, Thomas Tallo, James W Kaufman, Claire Moore, Jason N Kuehner
Termination of RNA Polymerase II (Pol II) activity serves a vital cellular function by separating ubiquitous transcription units and influencing RNA fate and function. In the yeast Saccharomyces cerevisiae , Pol II termination is carried out by cleavage and polyadenylation factor (CPF-CF) and Nrd1-Nab3-Sen1 (NNS) complexes, which operate primarily at mRNA and non-coding RNA genes, respectively. Premature Pol II termination (attenuation) contributes to gene regulation, but there is limited knowledge of its prevalence and biological significance...
April 23, 2018: G3: Genes—Genomes—Genetics
Chun Shen Lim, Samuel J T Wardell, Torsten Kleffmann, Chris M Brown
Introns in mRNA leaders are common in complex eukaryotes, but often overlooked. These introns are spliced out before translation, leaving exon-exon junctions in the mRNA leaders (leader EEJs). Our multi-omic approach shows that the number of leader EEJs inversely correlates with the main protein translation, as does the number of upstream open reading frames (uORFs). Across the five species studied, the lowest levels of translation were observed for mRNAs with both leader EEJs and uORFs (29%). This class of mRNAs also have ribosome footprints on uORFs, with strong triplet periodicity indicating uORF translation...
April 19, 2018: Nucleic Acids Research
Kevin L McKnight, Stanley M Lemon
Hepatitis A virus (HAV) is a positive-strand RNA virus classified in the genus Hepatovirus of the family Picornaviridae It is an ancient virus with a long evolutionary history and multiple features of its capsid structure, genome organization, and replication cycle that distinguish it from other mammalian picornaviruses. HAV proteins are produced by cap-independent translation of a single, long open reading frame under direction of an inefficient, upstream internal ribosome entry site (IRES). Genome replication occurs slowly and is noncytopathic, with transcription likely primed by a uridylated protein primer as in other picornaviruses...
April 2, 2018: Cold Spring Harbor Perspectives in Medicine
Jiao Ma, Alan Saghatelian, Maxim Nikolaievich Shokhirev
Proteogenomics methods have identified many non-annotated protein-coding genes in the human genome. Many of the newly discovered protein-coding genes encode peptides and small proteins, referred to collectively as microproteins. Microproteins are produced through ribosome translation of small open reading frames (smORFs). The discovery of many smORFs reveals a blind spot in traditional gene-finding algorithms for these genes. Biological studies have found roles for microproteins in cell biology and physiology, and the potential that there exists additional bioactive microproteins drives the interest in detection and discovery of these molecules...
2018: PloS One
Patrick McGillivray, Russell Ault, Mayur Pawashe, Robert Kitchen, Suganthi Balasubramanian, Mark Gerstein
Upstream open reading frames (uORFs) latent in mRNA transcripts are thought to modify translation of coding sequences by altering ribosome activity. Not all uORFs are thought to be active in such a process. To estimate the impact of uORFs on the regulation of translation in humans, we first circumscribed the universe of all possible uORFs based on coding gene sequence motifs and identified 1.3 million unique uORFs. To determine which of these are likely to be biologically relevant, we built a simple Bayesian classifier using 89 attributes of uORFs labeled as active in ribosome profiling experiments...
March 19, 2018: Nucleic Acids Research
Yair Fastman, Shany Assaraf, Miriam Rose, Elad Milrot, Katherine Basore, B Sivanandam Arasu, Sanjay A Desai, Michael Elbaum, Ron Dzikowski
Plasmodium falciparum, the causative agent of the deadliest form of human malaria, alternates expression of variable antigens, encoded by members of a multi-copy gene family named var. In var2csa, the var gene implicated in pregnancy-associated malaria, translational repression is regulated by a unique upstream open reading frame (uORF) found only in its 5' UTR. Here, we report that this translated uORF significantly alters both transcription and posttranslational protein trafficking. The parasite can alter a protein's destination without any modifications to the protein itself, but instead by an element within the 5' UTR of the transcript...
March 15, 2018: Nucleic Acids Research
Sitapriya Moorthi, Tara Ann Burns, Gui-Qin Yu, Chiara Luberto
Bcr-Abl (break-point cluster region-abelson), the oncogenic trigger of chronic myelogenous leukemia (CML), has previously been shown to up-regulate the expression and activity of sphingomyelin synthase 1 (SMS1), which contributes to the proliferation of CML cells; however, the mechanism by which this increased expression of SMS1 is mediated remains unknown. In the current study, we show that Bcr-Abl enhances the expression of SMS1 via a 30-fold up-regulation of its transcription. Of most interest, the Bcr-Abl-regulated transcription of SMS1 is initiated from a novel transcription start site (TSS) that is just upstream of the open reading frame...
March 13, 2018: FASEB Journal: Official Publication of the Federation of American Societies for Experimental Biology
Maja Bencun, Olaf Klinke, Agnes Hotz-Wagenblatt, Severina Klaus, Ming-Han Tsai, Remy Poirey, Henri-Jacques Delecluse
The Epstein-Barr virus (EBV) genome encodes several hundred transcripts. We have used ribosome profiling to characterize viral translation in infected cells and map new translation initiation sites. We show here that EBV transcripts are translated with highly variable efficiency, owing to variable transcription and translation rates, variable ribosome recruitment to the leader region and coverage by monosomes versus polysomes. Some transcripts were hardly translated, others mainly carried monosomes, showed ribosome accumulation in leader regions and most likely represent non-coding RNAs...
April 6, 2018: Nucleic Acids Research
Yi Xu, Ho-Jong Ju, Stacy DeBlasio, Elizabeth J Carino, Richard Johnson, Michael J MacCoss, Michelle Heck, W Allen Miller, Stewart M Gray
Translational readthrough of the stop codon of the capsid protein (CP) open reading frame (ORF) is used by members of the Luteoviridae to produce their minor capsid protein as a readthrough protein (RTP). The elements regulating RTP expression are not well understood, but they involve long-distance interactions between RNA domains. Using high-resolution mass spectrometry, glutamine and tyrosine were identified as the primary amino acids inserted at the stop codon of Potato leafroll virus (PLRV) CP ORF. We characterized the contributions of a cytidine-rich domain immediately downstream and a branched stem-loop structure 600 to 700 nucleotides downstream of the CP stop codon...
June 1, 2018: Journal of Virology
Shohei Kitano, Hikaru Kurasawa, Yasunori Aizawa
Transposons are major drivers of mammalian genome evolution. To obtain new insights into the contribution of transposons to the regulation of protein translation, we here examined how transposons affected the genesis and function of upstream open reading frames (uORFs), which serve as cis-acting elements to regulate translation from annotated ORFs (anORFs) located downstream of the uORFs in eukaryotic mRNAs. Among 39,786 human uORFs, 3,992 had ATG trinucleotides of a transposon origin, termed "transposon-derived upstream ATGs" or TuATGs...
April 2018: Genes to Cells: Devoted to Molecular & Cellular Mechanisms
Ronald C Wek
A central mechanism regulating translation initiation in response to environmental stress involves phosphorylation of the α subunit of eukaryotic initiation factor 2 (eIF2α). Phosphorylation of eIF2α causes inhibition of global translation, which conserves energy and facilitates reprogramming of gene expression and signaling pathways that help to restore protein homeostasis. Coincident with repression of protein synthesis, many gene transcripts involved in the stress response are not affected or are even preferentially translated in response to increased eIF2α phosphorylation by mechanisms involving upstream open reading frames (uORFs)...
February 12, 2018: Cold Spring Harbor Perspectives in Biology
Sarah M Hücker, Sonja Vanderhaeghen, Isabel Abellan-Schneyder, Romy Wecko, Svenja Simon, Siegfried Scherer, Klaus Neuhaus
BACKGROUND: Due to the DNA triplet code, it is possible that the sequences of two or more protein-coding genes overlap to a large degree. However, such non-trivial overlaps are usually excluded by genome annotation pipelines and, thus, only a few overlapping gene pairs have been described in bacteria. In contrast, transcriptome and translatome sequencing reveals many signals originated from the antisense strand of annotated genes, of which we analyzed an example gene pair in more detail...
February 12, 2018: BMC Evolutionary Biology
Dmitri E Andreev, Sergey E Dmitriev, Gary Loughran, Ilya M Terenin, Pavel V Baranov, Ivan N Shatsky
Eukaryotic cells evolved highly complex and accurate protein synthesis machinery that is finely tuned by various signaling pathways. Dysregulation of translation is a hallmark of many diseases, including cancer, and thus pharmacological approaches to modulate translation become very promising. While there has been much progress in our understanding of mammalian mRNA-specific translation control, surprisingly, relatively little is known about whether and how the protein components of the translation machinery shape translation of their own mRNAs...
April 20, 2018: Gene
Daichi Morimoto, Shigeko Kimura, Yoshihiko Sako, Takashi Yoshida
Microcystis aeruginosa forms massive blooms in eutrophic freshwaters, where it is constantly exposed to lytic cyanophages. Unlike other marine cyanobacteria, M. aeruginosa possess remarkably abundant and diverse potential antiviral defense genes. Interestingly, T4-like cyanophage Ma-LMM01, which is the sole cultured lytic cyanophage infecting M. aeruginosa , lacks the host-derived genes involved in maintaining host photosynthesis and directing host metabolism that are abundant in other marine cyanophages. Based on genomic comparisons with closely related cyanobacteria and their phages, Ma-LMM01 is predicted to employ a novel infection program that differs from that of other marine cyanophages...
2018: Frontiers in Microbiology
Julia Schulz, Nancy Mah, Martin Neuenschwander, Tabea Kischka, Richard Ratei, Peter M Schlag, Esmeralda Castaños-Vélez, Iduna Fichtner, Per-Ulf Tunn, Carsten Denkert, Oliver Klaas, Wolfgang E Berdel, Jens P von Kries, Wojciech Makalowski, Miguel A Andrade-Navarro, Achim Leutz, Klaus Wethmar
Ribosome profiling revealed widespread translational activity at upstream open reading frames (uORFs) and validated uORF-mediated translational control as a commonly repressive mechanism of gene expression. Translational activation of proto-oncogenes through loss-of-uORF mutations has been demonstrated, yet a systematic search for cancer-associated genetic alterations in uORFs is lacking. Here, we applied a PCR-based, multiplex identifier-tagged deep sequencing approach to screen 404 uORF translation initiation sites of 83 human tyrosine kinases and 49 other proto-oncogenes in 308 human malignancies...
February 5, 2018: Scientific Reports
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