scholarly journals Structural Plasticity and Rapid Evolution in a Viral RNA Revealed by In Vivo Genetic Selection

2008 ◽  
Vol 83 (2) ◽  
pp. 927-939 ◽  
Author(s):  
Rong Guo ◽  
Wai Lin ◽  
Jiuchun Zhang ◽  
Anne E. Simon ◽  
David B. Kushner
Keyword(s):  
Sequence Similarity ◽  
Rapid Evolution ◽  
Genetic Selection ◽  
Upstream Sequence ◽  
Turnip Crinkle Virus ◽  
In Planta ◽  
High Sequence Similarity ◽  
Stem Loop ◽  
Satellite Rnas

ABSTRACT Satellite RNAs usually lack substantial homology with their helper viruses. The 356-nucleotide satC of Turnip crinkle virus (TCV) is unusual in that its 3′-half shares high sequence similarity with the TCV 3′ end. Computer modeling, structure probing, and/or compensatory mutagenesis identified four hairpins and three pseudoknots in this TCV region that participate in replication and/or translation. Two hairpins and two pseudoknots have been confirmed as important for satC replication. One portion of the related 3′ end of satC that remains poorly characterized corresponds to juxtaposed TCV hairpins H4a and H4b and pseudoknot ψ3, which are required for the TCV-specific requirement of translation (V. A. Stupina et al., RNA 14:2379-2393, 2008). Replacement of satC H4a with randomized sequence and scoring for fitness in plants by in vivo genetic selection (SELEX) resulted in winning sequences that contain an H4a-like stem-loop, which can have additional upstream sequence composing a portion of the stem. SELEX of the combined H4a and H4b region in satC generated three distinct groups of winning sequences. One group models into two stem-loops similar to H4a and H4b of TCV. However, the selected sequences in the other two groups model into single hairpins. Evolution of these single-hairpin SELEX winners in plants resulted in satC that can accumulate to wild-type (wt) levels in protoplasts but remain less fit in planta when competed against wt satC. These data indicate that two highly distinct RNA conformations in the H4a and H4b region can mediate satC fitness in protoplasts.

scholarly journals The yeast SEN3 gene encodes a regulatory subunit of the 26S proteasome complex required for ubiquitin-dependent protein degradation in vivo.

1995 ◽  
Vol 15 (11) ◽  
pp. 6311-6321 ◽  
Author(s):  
D J DeMarini ◽  
F R Papa ◽  
S Swaminathan ◽  
D Ursic ◽  
T P Rasmussen ◽  
...  
Keyword(s):  
Sequence Similarity ◽  
Genetic Selection ◽  
26S Proteasome ◽  
Yeast Cells ◽  
Regulatory Subunit ◽  
20S Proteasome ◽  
Amino Terminal ◽  
A Subunit

The yeast Sen1 protein was discovered by virtue of its role in tRNA splicing in vitro. To help determine the role of Sen1 in vivo, we attempted to overexpress the protein in yeast cells. However, cells with a high-copy SEN1-bearing plasmid, although expressing elevated amounts of SEN1 mRNA, show little increase in the level of the encoded protein, indicating that a posttranscriptional mechanism limits SEN1 expression. This control depends on an amino-terminal element of Sen1. Using a genetic selection for mutants with increased expression of Sen1-derived fusion proteins, we identified mutations in a novel gene, designated SEN3. SEN3 is essential and encodes a 945-residue protein with sequence similarity to a subunit of an activator of the 20S proteasome from bovine erythrocytes, called PA700. Earlier work indicated that the 20S proteasome associates with a multisubunit regulatory factor, resulting in a 26S proteasome complex that degrades substrates of the ubiquitin system. Mutant sen3-1 cells have severe defects in the degradation of such substrates and accumulate ubiquitin-protein conjugates. Most importantly, we show biochemically that Sen3 is a subunit of the 26S proteasome. These data provide evidence for the involvement of the 26S proteasome in the degradation of ubiquitinated proteins in vivo and for a close relationship between PA700 and the regulatory complexes within the 26S proteasome, and they directly demonstrate that Sen3 is a component of the yeast 26S proteasome.

scholarly journals Insights into Transcriptional Repression of the Homologous Toxin-Antitoxin Cassettes yefM-yoeB and axe-txe

2020 ◽  
Vol 21 (23) ◽  
pp. 9062
Author(s):  
Barbara Kędzierska ◽  
Katarzyna Potrykus ◽  
Agnieszka Szalewska-Pałasz ◽  
Beata Wodzikowska
Keyword(s):  
Transcriptional Repression ◽  
Transcription Initiation ◽  
Sequence Similarity ◽  
In Vitro Transcription ◽  
High Sequence Similarity ◽  
Sequence Composition ◽  
Repressor Complex ◽  
Transcriptional Fusions

Transcriptional repression is a mechanism which enables effective gene expression switch off. The activity of most of type II toxin-antitoxin (TA) cassettes is controlled in this way. These cassettes undergo negative autoregulation by the TA protein complex which binds to the promoter/operator sequence and blocks transcription initiation of the TA operon. Precise and tight control of this process is vital to avoid uncontrolled expression of the toxin component. Here, we employed a series of in vivo and in vitro experiments to establish the molecular basis for previously observed differences in transcriptional activity and repression levels of the pyy and pat promoters which control expression of two homologous TA systems, YefM-YoeB and Axe-Txe, respectively. Transcriptional fusions of promoters with a lux reporter, together with in vitro transcription, EMSA and footprinting assays revealed that: (1) the different sequence composition of the −35 promoter element is responsible for substantial divergence in strengths of the promoters; (2) variations in repression result from the TA repressor complex acting at different steps in the transcription initiation process; (3) transcription from an additional promoter upstream of pat also contributes to the observed inefficient repression of axe-txe module. This study provides evidence that even closely related TA cassettes with high sequence similarity in the promoter/operator region may employ diverse mechanisms for transcriptional regulation of their genes.

scholarly journals Cucumber gibberellin 1-oxidase/desaturase initiates novel gibberellin catabolic pathways

2020 ◽  
Vol 295 (25) ◽  
pp. 8442-8448
Author(s):  
Maria João Pimenta Lange ◽  
Manuela Szperlinski ◽  
Leon Kalix ◽  
Theo Lange
Keyword(s):  
Amino Acids ◽  
Plant Growth ◽  
Oxidase Activity ◽  
Growth And Development ◽  
Sequence Similarity ◽  
In Planta ◽  
Plant Growth And Development ◽  
High Sequence Similarity ◽  
Oxidation Activity ◽  
Catabolic Pathways

Bioactive gibberellins (GAs) are central regulators of plant growth and development, including seed development. GA homeostasis is achieved via complex biosynthetic and catabolic pathways, whose exact activities remain to be elucidated. Here, we isolated two cDNAs from mature or imbibed cucumber seeds with high sequence similarity to known GA 3-oxidases. We found that one enzyme (designated here CsGA3ox5) has GA 3-oxidation activity. However, the second enzyme (designated CsGA1ox/ds) performed multiple reactions, including 1β-oxidation and 9,11-desaturation of GAs, but was lacking the 3-oxidation activity. CsGA1ox/ds overexpression in Arabidopsis plants resulted in severely dwarfed plants that could be rescued by the exogenous application of bioactive GA4, confirming that CsGA1ox/ds catabolizes GAs. Substitution of three amino acids in CsGA1ox/ds, Phe93, Pro106, and Ser202, with those typically conserved among GA 3-oxidases, Tyr93, Met106, and Thr202, respectively, conferred GA 3-oxidase activity to CsGA1ox/ds and thereby augmented its potential to form bioactive GAs in addition to catabolic products. Accordingly, overexpression of this amino acid–modified GA1ox/ds variant in Arabidopsis accelerated plant growth and development, indicating that this enzyme variant can produce bioactive GAs in planta. Furthermore, a genetically modified GA3ox5 variant in which these three canonical GA 3-oxidase amino acids were changed to the ones present in CsGA1ox/ds was unable to convert GA9 to GA4, highlighting the importance of these three conserved amino acids for GA 3-oxidase activity.

scholarly journals NDE1 and NDEL1: twin neurodevelopmental proteins with similar ‘nature’ but different ‘nurture’

2013 ◽  
Vol 4 (5) ◽  
pp. 447-464 ◽  
Author(s):  
Nicholas J. Bradshaw ◽  
William Hennah ◽  
Dinesh C. Soares
Keyword(s):  
Neurodevelopmental Disorders ◽  
Sequence Similarity ◽  
Review Of The Literature ◽  
Post Translational Modification ◽  
Similar Nature ◽  
High Sequence Similarity ◽  
Basic Functions ◽  
Key Aspects ◽  
Paralogous Proteins

AbstractNuclear distribution element 1 (NDE1, also known as NudE) and NDE-like 1 (NDEL1, also known as Nudel) are paralogous proteins essential for mitosis and neurodevelopment that have been implicated in psychiatric and neurodevelopmental disorders. The two proteins possess high sequence similarity and have been shown to physically interact with one another. Numerous lines of experimental evidence in vivo and in cell culture have demonstrated that these proteins share common functions, although instances of differing functions between the two have recently emerged. We review the key aspects of NDE1 and NDEL1 in terms of recent advances in structure elucidation and cellular function, with an emphasis on their differing mechanisms of post-translational modification. Based on a review of the literature and bioinformatics assessment, we advance the concept that the twin proteins NDE1 and NDEL1, while sharing a similar ‘nature’ in terms of their structure and basic functions, appear to be different in their ‘nurture’, the manner in which they are regulated both in terms of expression and of post-translational modification within the cell. These differences are likely to be of significant importance in understanding the specific roles of NDE1 and NDEL1 in neurodevelopment and disease.

scholarly journals A double-stranded RNA-inducible fish gene homologous to the murine influenza virus resistance gene Mx.

1989 ◽  
Vol 9 (7) ◽  
pp. 3117-3121 ◽  
Author(s):  
P Staeheli ◽  
Y X Yu ◽  
R Grob ◽  
O Haller
Keyword(s):  
Influenza Virus ◽  
Resistance Gene ◽  
Virus Resistance ◽  
Genomic Dna ◽  
Sequence Similarity ◽  
Perca Fluviatilis ◽  
High Sequence Similarity ◽  
Double Stranded Rna ◽  
Virus Resistance Gene

We cloned and sequenced a 2.35-kilobase EcoRI fragment of genomic DNA from a local freshwater fish (Perca fluviatilis) that strongly hybridized to probes derived from the murine influenza virus resistance gene Mx. The cloned fish DNA contained blocks of sequences related to Mx gene exons 3 to 8, which appeared to represent exons of a bona fide fish gene because they were separated by intron sequences flanked by consensus splice acceptor and donor sites. Injection of double-stranded RNA into the peritoneal cavity of trouts resulted in 5- to 10-fold elevated levels of two liver mRNAs of about 2.0 to 2.5 kilobases in length that hybridized to the cloned genomic DNA. High sequence similarity between this fish gene and the murine Mx gene, identical exon lengths, and similar inducibilities in vivo by double-stranded RNA indicate that we isolated a fragment of a fish Mx gene.

scholarly journals Downstream Sequences Influence the Choice between a Naturally Occurring Noncanonical and Closely Positioned Upstream Canonical Heptameric Fusion Motif during Bovine Coronavirus Subgenomic mRNA Synthesis

2001 ◽  
Vol 75 (16) ◽  
pp. 7362-7374 ◽  
Author(s):  
Aykut Ozdarendeli ◽  
Seulah Ku ◽  
Sylvie Rochat ◽  
Gwyn D. Williams ◽  
Savithra D. Senanayake ◽  
...  
Keyword(s):  
Sequence Similarity ◽  
Virus Genome ◽  
Intergenic Sequence ◽  
Computer Prediction ◽  
Bovine Coronavirus ◽  
Stem Loop ◽  
Upstream Site ◽  
Sequence Elements ◽  
Fusion Site

ABSTRACT Mechanisms leading to subgenomic mRNA (sgmRNA) synthesis in coronaviruses are poorly understood but are known to involve a heptameric signaling motif, originally called the intergenic sequence. The intergenic sequence is the presumed crossover region (fusion site) for RNA-dependent RNA polymerase (RdRp) during discontinuous transcription, a process leading to sgmRNAs that are both 5′ and 3′ coterminal. In the bovine coronavirus, the major fusion site for synthesis of mRNA 5 (GGUAGAC) does not conform to the canonical motif (UC[U,C]AAAC) at three positions (underlined), yet it lies just 14 nucleotides downstream from such a sequence (UCCAAAC). The infrequently used canonical sequence, by computer prediction, is buried within the stem of a stable hairpin (−17.2 kcal/mol). Here we document the existence of this stem by enzyme probing and examine its influence and that of neighboring sequences on the unusual choice of fusion sites by analyzing transcripts made in vivo from mutated defective interfering RNA constructs. We learned that (i) mutations that were predicted to unfold the stem-loop in various ways did not switch RdRp crossover to the upstream canonical site, (ii) a totally nonconforming downstream motif resulted in no measurable transcription from either site, (iii) the canonical upstream site does not function ectopically to lend competence to the downstream noncanonical site, and (iv) altering flanking sequences downstream of the downstream noncanonical motif in ways that diminish sequence similarity with the virus genome 5′ end caused a dramatic switch to the upstream canonical site. These results show that sequence elements downstream of the noncanonical site can dramatically influence the choice of fusion sites for synthesis of mRNA 5 and are interpreted as being most consistent with a mechanism of similarity-assisted RdRp strand switching during minus-strand synthesis.

scholarly journals Long-lasting Analgesia via Targetedin vivoEpigenetic Repression of Nav1.7

2019 ◽  
Author(s):  
Ana M. Moreno ◽  
Glaucilene F. Catroli ◽  
Fernando Alemán ◽  
Andrew Pla ◽  
Sarah A. Woller ◽  
...  
Keyword(s):  
Chronic Pain ◽  
Genome Engineering ◽  
Therapeutic Potential ◽  
Sequence Similarity ◽  
Thermal Hyperalgesia ◽  
Loss Of Function ◽  
High Sequence Similarity ◽  
Inflammatory State

ABSTRACTCurrent treatments for chronic pain rely largely on opioids despite their unwanted side effects and risk of addiction. Genetic studies have identified in humans key targets pivotal to nociceptive processing, with the voltage-gated sodium channel, NaV1.7 (SCN9A), being perhaps the most promising candidate for analgesic drug development. Specifically, a hereditary loss-of-function mutation in NaV1.7 leads to insensitivity to pain without other neurodevelopmental alterations. However, the high sequence similarity between NaVsubtypes has frustrated efforts to develop selective inhibitors. Here, we investigated targeted epigenetic repression of NaV1.7 via genome engineering approaches based on clustered regularly interspaced short palindromic repeats (CRISPR)-dCas9 and zinc finger proteins as a potential treatment for chronic pain. Towards this end, we first optimized the efficiency of NaV1.7 repressionin vitroin Neuro2A cells, and then by the lumbar intrathecal route delivered both genome-engineering platforms via adeno-associated viruses (AAVs) to assess their effects in three mouse models of pain: carrageenan-induced inflammatory pain, paclitaxel-induced neuropathic pain and BzATP-induced pain. Our results demonstrate: one, effective repression of NaV1.7 in lumbar dorsal root ganglia; two, reduced thermal hyperalgesia in the inflammatory state; three, decreased tactile allodynia in the neuropathic state; and four, no changes in normal motor function. We anticipate this genomically scarless and non-addictivepainamelioration approach enablingLong-lastingAnalgesia viaTargetedin vivoEpigeneticRepression of Nav1.7, a methodology we dubpain LATER, will have significant therapeutic potential, such as for preemptive administration in anticipation of a pain stimulus (pre-operatively), or during an established chronic pain state.One sentence summaryIn situepigenome engineering approach for genomically scarless, durable, and non-addictive management of pain.

scholarly journals Viral Hormones: Expanding Dimensions in Endocrinology

Endocrinology ◽  
2019 ◽  
Vol 160 (9) ◽  
pp. 2165-2179 ◽  
Author(s):  
Qian Huang ◽  
C Ronald Kahn ◽  
Emrah Altindis
Keyword(s):  
Dna Sequences ◽  
Sequence Similarity ◽  
Endocrine System ◽  
Peptide Hormones ◽  
Rna Sequences ◽  
High Sequence Similarity ◽  
In Vivo Models ◽  
Viral Mimicry

AbstractViruses have developed different mechanisms to manipulate their hosts, including the process of viral mimicry in which viruses express important host proteins. Until recently, examples of viral mimicry were limited to mimics of growth factors and immunomodulatory proteins. Using a comprehensive bioinformatics approach, we have shown that viruses possess the DNA/RNA with potential to encode 16 different peptides with high sequence similarity to human peptide hormones and metabolically important regulatory proteins. We have characterized one of these families, the viral insulin/IGF-1–like peptides (VILPs), which we identified in four members of the Iridoviridae family. VILPs can bind to human insulin and IGF-1 receptors and stimulate classic postreceptor signaling pathways. Moreover, VILPs can stimulate glucose uptake in vitro and in vivo and stimulate DNA synthesis. DNA sequences of some VILP-carrying viruses have been identified in the human enteric virome. In addition to VILPs, sequences with homology to 15 other peptide hormones or cytokines can be identified in viral DNA/RNA sequences, some with a very high identity to hormones. Recent data by others has identified a peptide that resembles and mimics α-melanocyte-stimulating hormone’s anti-inflammatory effects in in vitro and in vivo models. Taken together, these studies reveal novel mechanisms of viral and bacterial pathogenesis in which the microbe can directly target or mimic the host endocrine system. These findings also introduce the concept of a system of microbial hormones that provides new insights into the evolution of peptide hormones, as well as potential new roles of microbial hormones in health and disease.

scholarly journals cis and trans Requirements for Rolling Circle Replication of a Satellite RNA

2004 ◽  
Vol 78 (6) ◽  
pp. 3072-3082 ◽  
Author(s):  
Sang Ik Song ◽  
W. Allen Miller
Keyword(s):  
Sequence Similarity ◽  
Hammerhead Ribozyme ◽  
Helper Virus ◽  
Yellow Dwarf ◽  
Rolling Circle Replication ◽  
Satellite Rna ◽  
Rolling Circle ◽  
Satellite Rnas ◽  
Rna Accumulation

ABSTRACT Satellite RNAs usurp the replication machinery of their helper viruses, even though they bear little or no sequence similarity to the helper virus RNA. In Cereal yellow dwarf polerovirus serotype RPV (CYDV-RPV), the 322-nucleotide satellite RNA (satRPV RNA) accumulates to high levels in the presence of the CYDV-RPV helper virus. Rolling circle replication generates multimeric satRPV RNAs that self-cleave via a double-hammerhead ribozyme structure. Alternative folding inhibits formation of a hammerhead in monomeric satRPV RNA. Here we determine helper virus requirements and the effects of mutations and deletions in satRPV RNA on its replication in oat cells. Using in vivo selection of a satRPV RNA pool randomized at specific bases, we found that disruption of the base pairing necessary to form the non-self-cleaving conformation reduced satRPV RNA accumulation. Unlike other satellite RNAs, both the plus and minus strands proved to be equally infectious. Accordingly, very similar essential replication structures were identified in each strand. A different region is required only for encapsidation. The CYDV-RPV RNA-dependent RNA polymerase (open reading frames 1 and 2), when expressed from the nonhelper Barley yellow dwarf luteovirus, was capable of replicating satRPV RNA. Thus, the helper virus's polymerase is the sole determinant of the ability of a virus to replicate a rolling circle satellite RNA. We present a framework for functional domains in satRPV RNA with three types of function: (i) conformational control elements comprising an RNA switch, (ii) self-functional elements (hammerhead ribozymes), and (iii) cis-acting elements that interact with viral proteins.

scholarly journals Differential pathogenesis between Andes virus strains CHI-7913 and Chile-9717869in Syrian Hamsters

2021 ◽  
Author(s):  
Bryce M. Warner ◽  
Angela Sloan ◽  
Yvon Deschambault ◽  
Sebastian Dowhanik ◽  
Kevin Tierney ◽  
...  
Keyword(s):  
Animal Model ◽  
South America ◽  
Syrian Hamster ◽  
Sequence Similarity ◽  
Experimental Studies ◽  
Hantavirus Infection ◽  
High Sequence Similarity ◽  
Hamster Model ◽  
Syrian Hamsters

Hantavirus cardiopulmonary syndrome (HCPS) is a severe respiratory disease caused by orthohantaviruses in the Americas with a fatality rate as high as 35%. In South America, Andes orthohantavirus (Hantaviridae, Orthohantavirus, ANDV) is a major cause of HCPS, particularly in Chile and Argentina, where thousands of cases have been reported since the virus was discovered. Two strains of ANDV that are classically used for experimental studies of the virus are Chile-9717869, isolated from the natural reservoir, the long-tailed pygmy rice rat, and CHI-7913, an isolate from a lethal human case of HCPS. An important animal model for studying pathogenesis of HCPS is the lethal Syrian golden hamster model of ANDV infection. In this model, ANDV strain Chile-9717869 is uniformly lethal and has been used extensively for pathogenesis, vaccination, and therapeutic studies. Here we show that the CHI-7913 strain, despite having high sequence similarity with Chile-9717869, does not cause lethal disease in Syrian hamsters. CHI-7913, while being able to infect hamsters and replicate to moderate levels, showed a reduced ability to replicate within the tissues compared with Chile-9717869. Hamsters infected with CHI-7913 had reduced expression of cytokines IL-4, IL-6, and IFN-γ compared with Chile-9717869 infected animals, suggesting potentially limited immune-mediated pathology. These results demonstrate that certain ANDV strains may not be lethal in the classical Syrian hamster model of infection, and further exploration into the differences between lethal and non-lethal strains provide important insights into molecular determinants of pathogenic hantavirus infection. Importance: Andes orthohantavirus (ANDV) is a New World hantavirus that is a major cause of hantavirus cardiopulmonary syndrome (HCPS, also referred to as hantavirus pulmonary syndrome) in South America, particularly in Chile and Argentina. ANDV is one of the few hantaviruses for which there is a reliable animal model, the Syrian hamster model, which recapitulates important aspects of human disease. Here we infected hamsters with a human isolate of ANDV, CHI-7913, to assess its pathogenicity compared with the classical lethal Chile-9717869 strain. CHI-7913 had 22 amino acid differences compared with Chile-9717869, did not cause lethal disease in hamsters, and showed reduced ability to replicate in vivo. Our data indicate potentially important molecular signatures for pathogenesis of ANDV infection in hamsters and may lead to insights into what drives pathogenesis of certain hantaviruses in humans.

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