This service exclusively searches for literature that cites resources. Please be aware that the total number of searchable documents is limited to those containing RRIDs and does not include all open-access literature.
The lack of knowledge about extreme conservation in genomes remains a major gap in our understanding of the evolution of gene regulation. Here, we reveal an unexpected role of extremely conserved 5' untranslated regions (UTRs) in noncanonical translational regulation that is linked to the emergence of essential developmental features in vertebrate species. Endogenous deletion of conserved elements within these 5' UTRs decreased gene expression, and extremely conserved 5' UTRs possess cis-regulatory elements that promote cell-type-specific regulation of translation. We further developed in-cell mutate-and-map (icM2), a new methodology that maps RNA structure inside cells. Using icM2, we determined that an extremely conserved 5' UTR encodes multiple alternative structures and that each single nucleotide within the conserved element maintains the balance of alternative structures important to control the dynamic range of protein expression. These results explain how extreme sequence conservation can lead to RNA-level biological functions encoded in the untranslated regions of vertebrate genomes.
Regulatory RNA regions within a transcript, particularly in the 5' untranslated region (5'UTR), have been shown in a variety of organisms to control the expression levels of these mRNAs in response to various metabolites or environmental conditions. Considering the unique tolerance of Zymomonas mobilis to ethanol and the growing interest in engineering microbial strains with enhanced tolerance to industrial inhibitors, we searched natural cis-regulatory regions in this microorganism using transcriptomic data and bioinformatics analysis. Potential regulatory 5'UTRs were identified and filtered based on length, gene function, relative gene counts, and conservation in other organisms. An in vivo fluorescence-based screening system was developed to confirm the responsiveness of 36 5'UTR candidates to ethanol, acetate, and xylose stresses. UTR_ZMO0347 (5'UTR of gene ZMO0347 encoding the RNA binding protein Hfq) was found to down-regulate downstream gene expression under ethanol stress. Genomic deletion of UTR_ZMO0347 led to a general decrease of hfq expression at the transcript level and increased sensitivity for observed changes in Hfq expression at the protein level. The role of UTR_ZMO0347 and other 5'UTRs gives us insight into the regulatory network of Z. mobilis in response to stress and unlocks new strategies for engineering robust industrial strains as well as for harvesting novel responsive regulatory biological parts for controllable gene expression platforms in this organism.
p56lck is a new member of the src family of cellular tyrosine protein kinases. It is expressed constitutively at a low level in normal T cells and at an elevated level in the LSTRA and Thy19 Moloney murine leukemia virus-induced thymoma cell lines. It is possible that the expression of p56lck at an elevated level contributes to the transformation of these thymoma cells. The structure of the mRNAs encoding p56lck was examined by using an RNase protection assay. Both a chimeric lck mRNA containing the 5' untranslated region of Moloney virus mRNA and a normal lck mRNA were found in Thy19 and LSTRA cells. The chimeric lck transcript was 4- to 10-fold more abundant than the normal transcript. Transcription arising from a viral promoter is therefore responsible for the elevated levels of lck mRNA in these two cell lines. Surprisingly, uninfected murine T cells were also found to contain lck transcripts with differing 5' untranslated regions. One species of mRNA was colinear with the region of the chromosome just upstream of the initiation codon for p56lck. The other appeared to arise through splicing of an unidentified 5' untranslated exon to a sometimes cryptic splice acceptor just upstream of the region encoding p56lck. These data suggest that lck is expressed through the use of at least two different promoters. The promoters could be subject to different forms of regulation.
Astroviruses are small nonenveloped single-stranded RNA viruses with a positive sense genome. They are known to cause gastrointestinal disease in a broad spectrum of species. Although astroviruses are distributed worldwide, a gap in knowledge of their biology and disease pathogenesis persists. Many positive-sense single-stranded RNA viruses show conserved and functionally important structures in their 5' and 3' untranslated regions (UTRs). However, not much is known about the role of the 5' and 3' UTRs in the viral replication of HAstV-1. We analyzed the UTRs of HAstV-1 for secondary RNA structures and mutated them, resulting in partial or total UTR deletion. We used a reverse genetic system to study the production of infectious viral particles and to quantify protein expression in the 5' and 3' UTR mutants, and we established an HAstV-1 replicon system containing two reporter cassettes in open reading frames 1a and 2, respectively. Our data show that 3' UTR deletions almost completely abolished viral protein expression and that 5' UTR deletions led to a reduction in infectious virus particles in infection experiments. This indicates that the presence of the UTRs is essential for the life cycle of HAstV-1 and opens avenues for further research.
Oestrogen receptors (ERs) are critical regulators of the behaviour of many cancers. Despite this, the roles and regulation of one of the two known ERs - ERβ- are poorly understood. This is partly because analyses have been confused by discrepancies between ERβ expression at mRNA and proteins levels, and because ERβ is expressed as several functionally distinct isoforms. We investigated human ERβ 5' untranslated regions (UTRs) and their influences on ERβ expression and function. We demonstrate that two alternative ERβ 5'UTRs have potent and differential influences on expression acting at the level of translation. We show that their influences are modulated by cellular context and in carcinogenesis, and demonstrate the contributions of both upstream open reading frames and RNA secondary structure. These regulatory mechanisms offer explanations for the non-concordance of ERβ mRNA and protein. Importantly, we also demonstrate that 5'UTRs allow the first reported mechanisms for differential regulation of the expression of the ERβ isoforms 1, 2 and 5, and thereby have critical influences on ERβ function.
The Flavivirus genus of the Flaviviridae family encompasses numerous enveloped plus-strand RNA viruses. Dengue virus (DENV), a flavivirus, is the leading cause of serious arthropod-borne disease globally. The genomes of DENV, like the genomes of yellow fever virus (YFV), West Nile fever virus (WNV), or Zika virus (ZIKV), control their translation by a 5'-terminal capping group. Three other genera of Flaviviridae are remarkable because their viruses use internal ribosomal entry sites (IRESs) to control translation, and they are not arthropod transmitted. In 2006, E. Harris' group published work suggesting that DENV RNA does not stringently need a cap for translation. They proposed that instead DENV translation is controlled by an interplay between 5' and 3' termini. Here we present evidence that the DENV or ZIKV 5' untranslated regions (5'-UTRs) alone have IRES competence. This conclusion is based, first, on the observation that uncapped monocistronic mRNAs 5' terminated with the DENV or ZIKV 5'-UTRs can efficiently direct translation of a reporter gene in BHK and C6/36 cells and second, that either 5'-UTR placed between two reporter genes can efficiently induce expression of the downstream gene in BHK cells but not in C6/36 cells. These experiments followed observations that uncapped DENV/ZIKV genomic transcripts, 5' terminated with pppAN… or GpppAN…, can initiate infections of mammalian (BHK) or mosquito (C6/36) cells. IRES competence of the 5'-UTRs of DENV/ZIKV raises many open questions regarding the biology and control, as well as the evolution, of insect-borne flaviviruses.IMPORTANCE Members of the genus Flavivirus of Flaviviridae are important human pathogens of great concern because they cause serious diseases, sometimes death, in human populations living in tropical, subtropical (dengue virus [DENV], Zika virus [ZIKV], and yellow fever virus), or moderate climates (West Nile virus). Flaviviruses are known to control their translation by a cap-dependent mechanism. We have observed, however, that the uncapped genomes of DENV or ZIKV can initiate infection of mammalian and insect cells. We provide evidence that the short 5' untranslated region (5'-UTR) of DENV or ZIKV genomes can fulfill the function of an internal ribosomal entry site (IRES). This strategy frees these organisms from the cap-dependent mechanism of gene expression at an as yet unknown stage of proliferation. The data raise new questions about the biology and evolution of flaviviruses, possibly leading to new controls of flavivirus disease.
The Jingmenvirus group (JVG), with members such as Jingmen tick virus (JMTV), Alongshan virus (ALSV), Yanggou tick virus (YGTV), and Takachi virus (TAKV), is drawing attention due to evidence of it causing disease in humans and its unique genome architecture. In the current work, complete untranslated regions (UTRs) of four strains of ALSV and eight strains of YGTV were obtained. An analysis of these sequences, as well as JVG sequences from GenBank, uncovered several regions within viral UTRs that were highly conserved for all the segments and viruses. Bioinformatics predictions suggested that the UTRs of all the segments of YGTV, ALSV, and JMTV could form similar RNA structures. The most notable feature of these structures was a stable stem-loop with one (5' UTR) or two (3' UTR) AAGU tetraloops on the end of a hairpin.
The lengths of 5'UTRs of multicellular eukaryotes have been suggested to be subject to stochastic changes, with upstream start codons (uAUGs) as the major constraint to suppress 5'UTR elongation. However, this stochastic model cannot fully explain the variations in 5'UTR length. We hypothesize that the selection pressure on a combination of genomic features is also important for 5'UTR evolution. The ignorance of these features may have limited the explanatory power of the stochastic model. Furthermore, different selective constraints between vertebrates and invertebrates may lead to differences in the determinants of 5'UTR length, which have not been systematically analyzed.
The single nucleotide polymorphism rs2071746 and a (GT)n microsatellite within the human gene encoding heme oxygenase-1 (HMOX1) are associated with incidence or outcome in a variety of diseases. Most of these associations involve either release of heme or oxidative stress. Both polymorphisms are localized in the promoter region, but previously reported correlations with heme oxygenase-1 expression remain not coherent. This ambiguity suggests a more complex organization of the 5' gene region which we sought to investigate more fully. We evaluated the 5' end of HMOX1 and found a novel first exon 1a placing the two previously reported polymorphisms in intronic or exonic positions within the 5' untranslated region respectively. Expression of exon 1a can be induced in HepG2 hepatoma cells by hemin and is a repressor of heme oxygenase-1 translation as shown by luciferase reporter assays. Moreover, minigene approaches revealed that the quantitative outcome of alternative splicing within the 5' untranslated region is affected by the (GT)n microsatellite. This data supporting an extended HMOX1 gene model and provide further insights into expression regulation of heme oxygenase-1. Alternative splicing within the HMOX1 5' untranslated region contributes to translational regulation and is a mechanistic feature involved in the interplay between genetic variations, heme oxygenase-1 expression and disease outcome.
Legionella pneumophila grows within membrane-bound vacuoles in alveolar macrophages during human disease. Pathogen manipulation of the host cell is driven by bacterial proteins translocated through a type IV secretion system (T4SS). Although host protein synthesis during infection is arrested by the action of several of these translocated effectors, translation of a subset of host proteins predicted to restrict the pathogen is maintained. To identify the spectrum of host proteins selectively synthesized after L. pneumophila challenge, macrophages infected with the pathogen were allowed to incorporate the amino acid analog azidohomoalanine (AHA) during a 2-h time window, and newly synthesized macrophage proteins were isolated by orthogonal chemistry followed by mass spectrometry. Among the proteins isolated were interferon-stimulated genes as well as proteins translated from highly abundant transcripts. Surprisingly, a large number of the identified proteins were from low-abundance transcripts. These proteins were predicted to be among the most efficiently translated per unit transcript in the cell based on ribosome profiling data sets. To determine if high ribosome loading was a consequence of efficient translation initiation, the 5' untranslated regions (5' UTR) of transcripts having the highest and lowest predicted loading levels were inserted upstream of a reporter, and translation efficiency was determined in response to L. pneumophila challenge. The efficiency of reporter expression largely correlated with predicted ribosome loading and lack of secondary structure. Therefore, determinants in the 5' UTR allow selected host cell transcripts to overcome a pathogen-driven translation blockade.
Several control mechanisms of eukaryotic gene expression target the initiation step of mRNA translation. The canonical translation initiation pathway begins with cap-dependent attachment of the small ribosomal subunit (SSU) to the messenger ribonucleic acid (mRNA) followed by an energy-dependent, sequential 'scanning' of the 5' untranslated regions (UTRs). Scanning through the 5'UTR requires the adenosine triphosphate (ATP)-dependent RNA helicase eukaryotic initiation factor (eIF) 4A and its efficiency contributes to the specific rate of protein synthesis. Thus, understanding the molecular details of the scanning mechanism remains a priority task for the field. Here, we studied the effects of inhibiting ATP-dependent translation and eIF4A in cell-free translation and reconstituted initiation reactions programmed with capped mRNAs featuring different 5'UTRs. An aptamer that blocks eIF4A in an inactive state away from mRNA inhibited translation of capped mRNA with the moderately structured β-globin sequences in the 5'UTR but not that of an mRNA with a poly(A) sequence as the 5'UTR. By contrast, the nonhydrolysable ATP analogue β,γ-imidoadenosine 5'-triphosphate (AMP-PNP) inhibited translation irrespective of the 5'UTR sequence, suggesting that complexes that contain ATP-binding proteins in their ATP-bound form can obstruct and/or actively block progression of ribosome recruitment and/or scanning on mRNA. Further, using primer extension inhibition to locate SSUs on mRNA ('toeprinting'), we identify an SSU complex which inhibits primer extension approximately eight nucleotides upstream from the usual toeprinting stop generated by SSUs positioned over the start codon. This '-8 nt toeprint' was seen with mRNA 5'UTRs of different length, sequence and structure potential. Importantly, the '-8 nt toeprint' was strongly stimulated by the presence of the cap on the mRNA, as well as the presence of eIFs 4F, 4A/4B and ATP, implying active scanning. We assembled cell-free translation reactions with capped mRNA featuring an extended 5'UTR and used cycloheximide to arrest elongating ribosomes at the start codon. Impeding scanning through the 5'UTR in this system with elevated magnesium and AMP-PNP (similar to the toeprinting conditions), we visualised assemblies consisting of several SSUs together with one full ribosome by electron microscopy, suggesting direct detection of scanning intermediates. Collectively, our data provide additional biochemical, molecular and physical evidence to underpin the scanning model of translation initiation in eukaryotes.
GR transcripts display a remarkable heterogeneity in their 5' untranslated regions (5'UTRs). These variable 5'UTRs are encoded by a series of alternative 1st exons, and together with their associated promoters they maintain tissue-specific GR expression levels. In this study we over-expressed GR transcripts containing individual 1st exons, and assessed their effect on RNA stability, 3'-splicing, translation initiation and protein isoform production. We showed that these alternative 5'UTRs influence the predicted mRNA structure and free energy, and were associated with differential levels of functional spliced mRNA. However, the 5'UTR had little influence on the relative levels of the two principal 3' splice transcripts, GR-α and -β. The overall mRNA length, the free energy of the transcript and the translational efficiency directly influenced total GR levels. However, individual N-terminal protein isoform levels appeared to depend upon elements within the 5'UTR. Membrane-GR specific labelling suggested that the mGR originates from transcripts containing exon 1D and possibly 1H, although the specific trafficking sequences or structures within these transcripts remain unidentified. The role of the alternative first exons and their associated 5'UTRs has now been expanded to translational control, influencing total GR levels, individual constituent isoform levels, as well as trafficking to the cell surface.
The growing field of plant molecular farming relies on expression vectors that allow high yields of recombinant proteins to be produced through transient gene expression. While numerous expression vectors currently exist for this purpose, there are very few examples of systematic efforts to improve upon these. Moreover, the current generation of expression systems makes use of naturally-occurring regulatory elements, typically selected from plant viruses, to maximise yields. This study aims to use rational design to generate synthetic sequences that can rival existing ones.
Our ability to predict protein expression from DNA sequence alone remains poor, reflecting our limited understanding of cis-regulatory grammar and hampering the design of engineered genes for synthetic biology applications. Here, we generate a model that predicts the protein expression of the 5' untranslated region (UTR) of mRNAs in the yeast Saccharomyces cerevisiae. We constructed a library of half a million 50-nucleotide-long random 5' UTRs and assayed their activity in a massively parallel growth selection experiment. The resulting data allow us to quantify the impact on protein expression of Kozak sequence composition, upstream open reading frames (uORFs), and secondary structure. We trained a convolutional neural network (CNN) on the random library and showed that it performs well at predicting the protein expression of both a held-out set of the random 5' UTRs as well as native S. cerevisiae 5' UTRs. The model additionally was used to computationally evolve highly active 5' UTRs. We confirmed experimentally that the great majority of the evolved sequences led to higher protein expression rates than the starting sequences, demonstrating the predictive power of this model.
We previously reported a recombinant protein production system based on a geminivirus replicon that yields high levels of vaccine antigens and monoclonal antibodies in plants. The bean yellow dwarf virus (BeYDV) replicon generates massive amounts of DNA copies, which engage the plant transcription machinery. However, we noticed a disparity between transcript level and protein production, suggesting that mRNAs could be more efficiently utilized. In this study, we systematically evaluated genetic elements from human, viral, and plant sources for their potential to improve the BeYDV system. The tobacco extensin terminator enhanced transcript accumulation and protein production compared to other commonly used terminators, indicating that efficient transcript processing plays an important role in recombinant protein production. Evaluation of human-derived 5' untranslated regions (UTRs) indicated that many provided high levels of protein production, supporting their cross-kingdom function. Among the viral 5' UTRs tested, we found the greatest enhancement with the tobacco mosaic virus omega leader. An analysis of the 5' UTRs from the Arabidopsis thaliana and Nicotinana benthamiana photosystem I K genes found that they were highly active when truncated to include only the near upstream region, providing a dramatic enhancement of transgene production that exceeded that of the tobacco mosaic virus omega leader. The tobacco Rb7 matrix attachment region inserted downstream from the gene of interest provided significant enhancement, which was correlated with a reduction in plant cell death. Evaluation of Agrobacterium strains found that EHA105 enhanced protein production and reduced cell death compared to LBA4301 and GV3101. We used these improvements to produce Norwalk virus capsid protein at >20% total soluble protein, corresponding to 1.8 mg/g leaf fresh weight, more than twice the highest level ever reported in a plant system. We also produced the monoclonal antibody rituximab at 1 mg/g leaf fresh weight.
Bidirectional promoters are defined as those that regulate adjacent genes organized in a divergent fashion (head to head orientation) and separated by <1 kb. In order to dissect bidirectional promoter activity in a model plant, deletion analysis was performed for seven rice promoters using promoter-reporter gene constructs, which identified three promoters to be bidirectional. Regulatory elements located in or close to the 5'-untranslated regions (UTR) of one of the genes (divergent gene pair) were found to be responsible for their bidirectional activity. DNA footprinting analysis identified unique protein binding sites in these promoters. Deletion/alteration of these motifs resulted in significant loss of expression of the reporter genes on either side of the promoter. Changes in the motifs at both the positions resulted in a remarkable decrease in bidirectional activity of the reporter genes flanking the promoter. Based on our results, we propose a novel mechanism for the bidirectionality of rice bidirectional promoters.
Virus production in the deep-sea environment has been found to be high, and viruses have been suggested to play significant roles in the overall functioning of this ecosystem. Nevertheless, little is known about these viruses, including the mechanisms that control their production, which makes them one of the least understood biological entities on Earth. Previously, we isolated the filamentous phage SW1, whose virus production and gene transcription were found to be active at low temperatures, from a deep-sea bacterium, Shewanella piezotolerans WP3. In this study, the operon structure of phage SW1 is presented, which shows two operons with exceptionally long 5' and 3' untranslated regions (UTRs). In addition, the 5'UTR was confirmed to significantly influence the RNA stability of the SW1 transcripts. Our study revealed novel regulation of the operon and led us to propose a unique regulatory mechanism for Inoviruses. This type of RNA-based regulation may represent a mechanism for significant viral production in the cold deep biosphere.
Phenazines are important compounds produced by pseudomonads and other bacteria. Two phz gene clusters called phzA1-G1 and phzA2-G2, respectively, were found in the genome of Pseudomonas sp. M18, an effective biocontrol agent, which is highly homologous to the opportunistic human pathogen P. aeruginosa PAO1, however little is known about the correlation between the expressions of two phz gene clusters.
Retinoic acid inducible gene-I (RIG-I) is a key regulator of antiviral immunity. RIG-I is generally thought to be activated by ssRNA species containing a 5'-triphosphate (PPP) group or by unphosphorylated dsRNA up to ~300 bp in length. However, it is not yet clear how changes in the length, nucleotide sequence, secondary structure, and 5' end modification affect the abilities of these ligands to bind and activate RIG-I. To further investigate these parameters in the context of naturally occurring ligands, we examined RNA sequences derived from the 5' and 3' untranslated regions (UTR) of the influenza virus NS1 gene segment. As expected, RIG-I-dependent interferon-β (IFN-β) induction by sequences from the 5' UTR of the influenza cRNA or its complement (26 nt in length) required the presence of a 5'PPP group. In contrast, activation of RIG-I by the 3' UTR cRNA sequence or its complement (172 nt) exhibited only a partial 5'PPP-dependence, as capping the 5' end or treatment with CIP showed a modest reduction in RIG-I activation. Furthermore, induction of IFN-β by a smaller, U/A-rich region within the 3' UTR was completely 5'PPP-independent. Our findings demonstrated that RNA sequence, length, and secondary structure all contributed to whether or not the 5'PPP moiety is needed for interferon induction by RIG-I.
Small insertions and deletions ("indels" with size >/= 100 bp) whose lengths are not multiples of three (non-3n) are strongly constrained and depleted in protein-coding sequences. Such a constraint has never been reported in noncoding genomic regions. In 5'untranslated regions (5'UTRs) in mammalian genomes, upstream start codons (uAUGs) and upstream open reading frames (uORFs) can regulate protein translation. The presence of non-3n indels in uORFs can potentially disrupt the functions of these regulatory elements. We thus hypothesize that natural selection disfavors non-3n indels in 5'UTRs when these regulatory elements are present.
Welcome to the FDI Lab - SciCrunch.org Resources search. From here you can search through a compilation of resources used by FDI Lab - SciCrunch.org and see how data is organized within our community.
You are currently on the Community Resources tab looking through categories and sources that FDI Lab - SciCrunch.org has compiled. You can navigate through those categories from here or change to a different tab to execute your search through. Each tab gives a different perspective on data.
If you have an account on FDI Lab - SciCrunch.org then you can log in from here to get additional features in FDI Lab - SciCrunch.org such as Collections, Saved Searches, and managing Resources.
Here is the search term that is being executed, you can type in anything you want to search for. Some tips to help searching:
You can save any searches you perform for quick access to later from here.
We recognized your search term and included synonyms and inferred terms along side your term to help get the data you are looking for.
If you are logged into FDI Lab - SciCrunch.org you can add data records to your collections to create custom spreadsheets across multiple sources of data.
Here are the facets that you can filter your papers by.
From here we'll present any options for the literature, such as exporting your current results.
If you have any further questions please check out our FAQs Page to ask questions and see our tutorials. Click this button to view this tutorial again.
Year:
Count: