scholarly journals RNA dimerization plays a role in ribosomal frameshifting of the SARS coronavirus

2012 ◽  
Vol 41 (4) ◽  
pp. 2594-2608 ◽  
Author(s):  
Daniella Ishimaru ◽  
Ewan P. Plant ◽  
Amy C. Sims ◽  
Boyd L. Yount ◽  
Braden M. Roth ◽  
...  
Keyword(s):  
Messenger Rna ◽  
Palindromic Sequence ◽  
Sars Coronavirus ◽  
Stem Loop ◽  
Ribosomal Frameshifting ◽  
Rna Dimerization ◽  
Cellular Environment ◽  
Vitro Analysis ◽  
And Function

Abstract Messenger RNA encoded signals that are involved in programmed -1 ribosomal frameshifting (-1 PRF) are typically two-stemmed hairpin (H)-type pseudoknots (pks). We previously described an unusual three-stemmed pseudoknot from the severe acute respiratory syndrome (SARS) coronavirus (CoV) that stimulated -1 PRF. The conserved existence of a third stem–loop suggested an important hitherto unknown function. Here we present new information describing structure and function of the third stem of the SARS pseudoknot. We uncovered RNA dimerization through a palindromic sequence embedded in the SARS-CoV Stem 3. Further in vitro analysis revealed that SARS-CoV RNA dimers assemble through ‘kissing’ loop–loop interactions. We also show that loop–loop kissing complex formation becomes more efficient at physiological temperature and in the presence of magnesium. When the palindromic sequence was mutated, in vitro RNA dimerization was abolished, and frameshifting was reduced from 15 to 5.7%. Furthermore, the inability to dimerize caused by the silent codon change in Stem 3 of SARS-CoV changed the viral growth kinetics and affected the levels of genomic and subgenomic RNA in infected cells. These results suggest that the homodimeric RNA complex formed by the SARS pseudoknot occurs in the cellular environment and that loop–loop kissing interactions involving Stem 3 modulate -1 PRF and play a role in subgenomic and full-length RNA synthesis.

Role of the 807 C/T Polymorphism of the α2 Gene in Platelet GP Ia Collagen Receptor Expression and Function

1999 ◽  
Vol 81 (06) ◽  
pp. 951-956 ◽  
Author(s):  
J. Corral ◽  
R. González-Conejero ◽  
J. Rivera ◽  
F. Ortuño ◽  
P. Aparicio ◽  
...  
Keyword(s):  
Venous Thrombosis ◽  
Cell Types ◽  
Receptor Expression ◽  
Case Control Studies ◽  
Platelet Surface ◽  
Vitro Analysis ◽  
And Function ◽  
In Vitro Analysis

SummaryThe variability of the platelet GP Ia/IIa density has been associated with the 807 C/T polymorphism (Phe 224) of the GP Ia gene in American Caucasian population. We have investigated the genotype and allelic frequencies of this polymorphism in Spanish Caucasians. The T allele was found in 35% of the 284 blood donors analyzed. We confirmed in 159 healthy subjects a significant association between the 807 C/T polymorphism and the platelet GP Ia density. The T allele correlated with high number of GP Ia molecules on platelet surface. In addition, we observed a similar association of this polymorphism with the expression of this protein in other blood cell types. The platelet responsiveness to collagen was determined by “in vitro” analysis of the platelet activation and aggregation response. We found no significant differences in these functional platelet parameters according to the 807 C/T genotype. Finally, results from 3 case/control studies involving 302 consecutive patients (101 with coronary heart disease, 104 with cerebrovascular disease and 97 with deep venous thrombosis) determined that the 807 C/T polymorphism of the GP Ia gene does not represent a risk factor for arterial or venous thrombosis.

scholarly journals Membrane-associated phase separation: organization and function emerge from a two-dimensional milieu

2021 ◽  
Author(s):  
Jonathon A Ditlev
Keyword(s):  
Phase Separation ◽  
Cell Biology ◽  
Cellular Environment ◽  
Condensate Formation ◽  
Associated Proteins ◽  
Available Technology ◽  
And Function ◽  
Surrounding Membrane

Abstract Liquid‒liquid phase separation (LLPS) of biomolecules has emerged as an important mechanism that contributes to cellular organization. Phase separated biomolecular condensates, or membrane-less organelles, are compartments composed of specific biomolecules without a surrounding membrane in the nucleus and cytoplasm. LLPS also occurs at membranes, where both lipids and membrane-associated proteins can de-mix to form phase separated compartments. Investigation of these membrane-associated condensates using in vitro biochemical reconstitution and cell biology has provided key insights into the role of phase separation in membrane domain formation and function. However, these studies have generally been limited by available technology to study LLPS on model membranes and the complex cellular environment that regulates condensate formation, composition, and function. Here, I briefly review our current understanding of membrane-associated condensates, establish why LLPS can be advantageous for certain membrane-associated condensates, and offer a perspective for how these condensates may be studied in the future.

scholarly journals Low c-kit expression of cultured mast cells of mi/mi genotype may be involved in their defective responses to fibroblasts that express the ligand for c-kit

Blood ◽  
1992 ◽  
Vol 80 (6) ◽  
pp. 1454-1462 ◽  
Author(s):  
Y Ebi ◽  
Y Kanakura ◽  
T Jippo-Kanemoto ◽  
T Tsujimura ◽  
T Furitsu ◽  
...  
Keyword(s):  
Mast Cell ◽  
Mast Cells ◽  
Tyrosine Phosphorylation ◽  
Messenger Rna ◽  
Tissue Type ◽  
Phenotypic Change ◽  
3T3 Fibroblasts ◽  
Mouse Mast Cell ◽  
And Function

Abstract Mutant mice of mi/mi genotype are osteopetrotic and deficient in tissue mast cells due to a defect in osteoclasts and mast cells. In an effort to further understand the mechanisms behind why mi/mi mouse-derived cultured mast cells (mi/mi-CMC) responded to interleukin-3 (IL-3), but not to the proliferative stimuli presented by fibroblasts, mi/mi-CMC and congenic normal (+/+) mouse-derived CMC (+/+-CMC), both of which expressed the phenotypic characteristics of immature mast cells, were cocultured with Swiss albino/3T3 fibroblasts in a medium containing IL- 3. In the in vitro CMC/fibroblast coculture, mi/mi-CMC did not acquire the phenotypes of connective tissue-type mast cells (CTMC), while +/+- CMC did. In addition, attachment of mi/mi-CMC to the fibroblasts was found to be significantly lower than that of +/+-CMC. Because the interaction of c-kit product with its ligand (stem cell factor [SCF]) is known to play an important role not only in proliferation and differentiation of mast cells but also in attachment of CMC to fibroblasts, the expression and function of c-kit were investigated in mi/mi-CMC and +/+-CMC. Recombinant rat SCF (rrSCF164) induced a dose- dependent proliferation of +/+-CMC. Also, rrSCF164 induced +/+-CMC to acquire the phenotypes of CTMC in the medium containing IL-3. By contrast, rrSCF164 did not stimulate the proliferation of mi/mi-CMC nor induce a phenotypic change of the cells from immature mast cells to mature, CTMC-like mast cells. Immunoblotting with antiphosphotyrosine antibody showed that rrSCF164 induced considerable tyrosine phosphorylation of 145- to 165-Kd protein, the product of c-kit, in +/+- CMC, whereas tyrosine phosphorylation of the protein was barely detectable in mi/mi-CMC. Northern blot and flow cytometry analyses showed that mi/mi-CMC expressed much less c-kit at both protein and message levels than +/+-CMC. Further, mi/mi-CMC were found to differ from +/+-CMC in the expression of mouse mast cell protease-6 (MMCP-6) and MMCP-2 messenger RNA transcripts. These results suggest that the gene product of the mi locus may be important in regulating the expression of gene products such as c-kit, and that mast cell deficiency of mi/mi mice appears to be due, at least in part, to impaired signaling through the c-kit receptor because of the low c-kit expression.

scholarly journals Transcript Lifetime Is Balanced between Stabilizing Stem-Loop Structures and Degradation-Promoting Polyadenylation in Plant Mitochondria

2001 ◽  
Vol 21 (3) ◽  
pp. 731-742 ◽  
Author(s):  
Josef Kuhn ◽  
Ulrike Tengler ◽  
Stefan Binder
Keyword(s):  
Plant Mitochondria ◽  
Rna Stability ◽  
Processing System ◽  
Stem Loop ◽  
Functional Studies ◽  
In Vitro Processing ◽  
Rapid Amplification ◽  
And Function

ABSTRACT To determine the influence of posttranscriptional modifications on 3′ end processing and RNA stability in plant mitochondria, peaatp9 and Oenothera atp1 transcripts were investigated for the presence and function of 3′ nonencoded nucleotides. A 3′ rapid amplification of cDNA ends approach initiated at oligo(dT)-adapter primers finds the expected poly(A) tails predominantly attached within the second stem or downstream of the double stem-loop structures at sites of previously mapped 3′ ends. Functional studies in a pea mitochondrial in vitro processing system reveal a rapid removal of the poly(A) tails up to termini at the stem-loop structure but little if any influence on further degradation of the RNA. In contrast 3′ poly(A) tracts at RNAs without such stem-loop structures significantly promote total degradation in vitro. To determine the in vivo identity of 3′ nonencoded nucleotides more accurately, pea atp9 transcripts were analyzed by a direct anchor primer ligation-reverse transcriptase PCR approach. This analysis identified maximally 3-nucleotide-long nonencoded extensions most frequently of adenosines combined with cytidines. Processing assays with substrates containing homopolymer stretches of different lengths showed that 10 or more adenosines accelerate RNA processivity, while 3 adenosines have no impact on RNA life span. Thus polyadenylation can generally stimulate the decay of RNAs, but processivity of degradation is almost annihilated by the stabilizing effect of the stem-loop structures. These antagonistic actions thus result in the efficient formation of 3′ processed and stable transcripts.

Clinical, Molecular, Functional, and Structural Characterization of CYP17A1 Mutations in Brazilian Patients with 17-Hydroxylase Deficiency

2020 ◽  
Vol 52 (03) ◽  
pp. 186-193 ◽  
Author(s):  
Fernanda Borchers Coeli-Lacchini ◽  
Livia M. Mermejo ◽  
Aline Faccioli Bodoni ◽  
Lucila Leico Kagohara Elias ◽  
Wilson Araújo Silva Jr ◽  
...  
Keyword(s):  
Three Dimensional ◽  
External Genitalia ◽  
Structural Modeling ◽  
Primary Amenorrhea ◽  
Compound Heterozygous ◽  
Hydroxylase Deficiency ◽  
Vitro Analysis ◽  
And Function

Abstract17-Hydroxylase-deficiency (17OHD) is a rare form of congenital adrenal hyperplasia. The aim of the work was to study clinical, biochemical, and the follow up of 17OHD patients and evaluate the function and structure of CYP17A1 mutations. Brazilian patients (three 46, XX and four 46, XY; 17±1.9 years) with combined 17-hydroxylase/17,20-lyase deficiency were evaluated. CYP17A1 gene was sequenced. Functional analysis was performed transfecting COS7 cells, which were exposed to progesterone or 17α-hydroxypregnolone substrates. Hormones were determined by RIA or LC-MS/MS. Three-dimensional structural modeling was performed by Modeller software. All patients presented prepubertal female external genitalia, primary amenorrhea, hypergonadotrophic hypogonadism, hypokalemic hypertension, decreased cortisol, and increased ACTH and corticosterone levels. Five patients presented previously described mutations: p.W406R/p.W406R, IVS2–2A>C/p.P428L, and p.P428L/p.P428L. Two patients presented the compound heterozygous p.G478S/p.I223Nfs*10 mutations, whose CYP17A1 activity and the three dimensional structural modeling are originally studied in this paper. CYP17A1 activity of p.G478S was 13 and 58% against progesterone and 17-hydroxypregnenolone, respectively. The p.I223Nfs*10 caused a truncated inactive protein. Three-dimensional p.G478S structural modeling showed different internal hydrophobic interaction with W313 and created an additional chain side contact with L476 residue. Due to the rarity of 17OHD, the long term follow up (15.3±3.1 years) of our patients will help endocrinologists on the management of patients with 17OHD. The mutation p.G478S/pI223Nfs*10 led to severe 17OHD and impaired CYP17A1 structure and function. The integration of in silico and in vitro analysis showed how the amino acid changes affected the CYP17A1 activity and contributed to clarify the molecular interactions of CYP17A1.

scholarly journals The energy landscape of −1 ribosomal frameshifting

2020 ◽  
Vol 6 (1) ◽  
pp. eaax6969 ◽  
Author(s):  
Junhong Choi ◽  
Sinéad O’Loughlin ◽  
John F. Atkins ◽  
Joseph D. Puglisi
Keyword(s):  
Single Molecule ◽  
Messenger Rna ◽  
Information Transfer ◽  
Mechanistic Model ◽  
High Rate ◽  
Coding Region ◽  
Ribosomal Frameshifting ◽  
Reading Frame

Maintenance of translational reading frame ensures the fidelity of information transfer during protein synthesis. Yet, programmed ribosomal frameshifting sequences within the coding region promote a high rate of reading frame change at predetermined sites thus enriching genomic information density. Frameshifting is typically stimulated by the presence of 3′ messenger RNA (mRNA) structures, but how these mRNA structures enhance −1 frameshifting remains debatable. Here, we apply single-molecule and ensemble approaches to formulate a mechanistic model of ribosomal −1 frameshifting. Our model suggests that the ribosome is intrinsically susceptible to frameshift before its translocation and this transient state is prolonged by the presence of a precisely positioned downstream mRNA structure. We challenged this model using temperature variation in vivo, which followed the prediction made based on in vitro results. Our results provide a quantitative framework for analyzing other frameshifting enhancers and a potential approach to control gene expression dynamically using programmed frameshifting.

Sexual dimorphism of messenger RNA isolated from neonatal rat brain

1986 ◽  
Vol 109 (1) ◽  
pp. 23-NP ◽  
Author(s):  
I. M. Adcock ◽  
B. D. Greenstein
Keyword(s):  
Sexual Dimorphism ◽  
Rat Brain ◽  
Limbic System ◽  
Messenger Rna ◽  
Neonatal Rat ◽  
Transcriptional Level ◽  
Sexually Dimorphic ◽  
Clear Cut ◽  
And Function

ABSTRACT The rat brain is sexually dimorphic with respect to structure and function, and there is evidence that these differences are effected in the fetus through changes in protein synthesis, some of which may result from the intervention of gonadal steroids. To investigate this, messenger RNA (mRNA) from the limbic system and cerebellum of neonatal rats was prepared, translated in a rabbit reticulocyte system in vitro and the products were analysed by two-dimensional electrophoresis and fluorography. Some of the results were further analysed using image analysis. There was a striking sexual dimorphism in the patterns of incorporation of [35S]methionine into proteins using mRNA from the limbic system, in that groups of proteins were apparently present in male-but not in female-derived fluorograms and vice versa. One protein, tentatively identified from its coordinates as α-tubulin, was more abundant in male-derived fluorograms. Although there were no clear-cut qualitative sex differences using mRNA derived from the cerebellum, that derived from the male cerebellum appeared to be consistently more active. These results provide direct evidence for a sexual dimorphism at the transcriptional level in the neonatal limbic system of the rat. J. Endocr. (1986) 109, 23–28

scholarly journals Regulators of Viral Frameshifting: More Than RNA Influences Translation Events

2020 ◽  
Vol 7 (1) ◽  
pp. 219-238
Author(s):  
Wesley D. Penn ◽  
Haley R. Harrington ◽  
Jonathan P. Schlebach ◽  
Suchetana Mukhopadhyay
Keyword(s):  
Messenger Rna ◽  
Protein Production ◽  
Productive Infection ◽  
Cis Elements ◽  
Stem Loop ◽  
Viral Translation ◽  
Ribosomal Frameshifting ◽  
Evolutionarily Conserved ◽  
Rna Pseudoknot ◽  
New Protein

Programmed ribosomal frameshifting (PRF) is a conserved translational recoding mechanism found in all branches of life and viruses. In bacteria, archaea, and eukaryotes PRF is used to downregulate protein production by inducing a premature termination of translation, which triggers messenger RNA (mRNA) decay. In viruses, PRF is used to drive the production of a new protein while downregulating the production of another protein, thus maintaining a stoichiometry optimal for productive infection. Traditionally, PRF motifs have been defined by the characteristics of two cis elements: a slippery heptanucleotide sequence followed by an RNA pseudoknot or stem-loop within the mRNA. Recently, additional cis and new trans elements have been identified that regulate PRF in both host and viral translation. These additional factors suggest PRF is an evolutionarily conserved process whose function and regulation we are just beginning to understand.

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