Academic literature on the topic 'Specific protein/RNA recognition'
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Journal articles on the topic "Specific protein/RNA recognition"
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Stolarski, Ryszard. "Thermodynamics of specific protein-RNA interactions." Acta Biochimica Polonica 50, no. 2 (June 30, 2003): 297–318. http://dx.doi.org/10.18388/abp.2003_3688.
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Description of the recognition specificity between proteins and nucleic acids at the level of molecular interactions is one of the most challenging tasks in biophysics. It is key to understanding the course and control of gene expression and to the application of the thus acquired knowledge in chemotherapy. This review presents experimental results of thermodynamic studies and a discussion of the role of thermodynamics in formation and stability of functional protein-RNA complexes, with a special attention to the interactions involving mRNA 5' cap and cap-binding proteins in the initiation of protein biosynthesis in the eukaryotic cell. A theoretical framework for analysis of the thermodynamic parameters of protein-nucleic acid association is also briefly surveyed. Overshadowed by more spectacular achievements in structural studies, the thermodynamic investigations are of equal importance for full comprehension of biopolymers' activity in a quantitative way. In this regard, thermodynamics gives a direct insight into the energetic and entropic characteristics of complex macromolecular systems in their natural environment, aqueous solution, and thus complements the structural view derived from X-ray crystallography and multidimensional NMR. Further development of the thermodynamic approach toward interpretation of recognition and binding specificity in terms of molecular biophysics requires more profound contribution from statistical mechanics.
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Shen, Cuicui, Xiang Wang, Yexing Liu, Quanxiu Li, Zhao Yang, Nieng Yan, Tingting Zou, and Ping Yin. "Specific RNA Recognition by Designer Pentatricopeptide Repeat Protein." Molecular Plant 8, no. 4 (April 2015): 667–70. http://dx.doi.org/10.1016/j.molp.2015.01.001.
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Qian, Kaiyue, Mengyu Li, Junchao Wang, Min Zhang, and Mingzhu Wang. "Structural basis for mRNA recognition by human RBM38." Biochemical Journal 477, no. 1 (January 10, 2020): 161–72. http://dx.doi.org/10.1042/bcj20190652.
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RNA-binding protein RBM38 was reported to bind the mRNA of several p53-related genes through its RRM domain and to up-regulate or down-regulate protein translation by increasing mRNA stability or recruitment of other effector proteins. The recognition mechanism, however, for RNA-binding of RBM38 remains unclear. Here, we report the crystal structure of the RRM domain of human RBM38 in complex with a single-stranded RNA. Our structural and biological results revealed that RBM38 recognizes G(U/C/A)GUG sequence single-stranded RNA in a sequence-specific and structure-specific manner. Two phenylalanine stacked with bases of RNA were crucial for RNA binding, and a series of hydrogen bonds between the base atoms of RNA and main-chain or side-chain atoms of RBM38 determine the sequence-specific recognition. Our results revealed the RNA-recognition mechanism of human RBM38 and provided structural information for understanding the RNA-binding property of RBM38.
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Spiridonova, V. A. "Molecular recognition elements - DNA/RNA-aptamers to proteins." Biomeditsinskaya Khimiya 56, no. 6 (2010): 639–56. http://dx.doi.org/10.18097/pbmc20105606639.
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In this review summarizes data on DNA/RNA aptamers - a novel class of molecular recognition elements. Special attention is paid to the aptamers to proteins involved into pathogenesis of wide spread human diseases. These include aptamers to serine protease, to cytokines/growth factors, to influenza viral protein, nucleic acid binding proteins. Strong and specific binding for a given protein target of aptamers make them an attractive class of direct protein inhibitors. They can inhibit pathogenic proteins and it is becoming clear that aptamers have the potential to be a new and effective class of therapeutic molecules.
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Pérez-Cano, Laura, and Juan Fernández-Recio. "Dissection and prediction of RNA-binding sites on proteins." BioMolecular Concepts 1, no. 5-6 (December 1, 2010): 345–55. http://dx.doi.org/10.1515/bmc.2010.037.
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AbstractRNA-binding proteins are involved in many important regulatory processes in cells and their study is essential for a complete understanding of living organisms. They show a large variability from both structural and functional points of view. However, several recent studies performed on protein-RNA crystal structures have revealed interesting common properties. RNA-binding sites usually constitute patches of positively charged or polar residues that make most of the specific and non-specific contacts with RNA. Negatively charged or aliphatic residues are less frequent at protein-RNA interfaces, although they can also be found either forming aliphatic and positive-negative pairs in protein RNA-binding sites or contacting RNA through their main chains. Aromatic residues found within these interfaces are usually involved in specific base recognition at RNA single-strand regions. This specific recognition, in combination with structural complementarity, represents the key source for specificity in protein-RNA association. From all this knowledge, a variety of computational methods for prediction of RNA-binding sites have been developed based either on protein sequence or on protein structure. Some reported methods are really successful in the identification of RNA-binding proteins or the prediction of RNA-binding sites. Given the growing interest in the field, all these studies and prediction methods will undoubtedly contribute to the identification and comprehension of protein-RNA interactions.
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Haynes, S. R., M. T. Cooper, S. Pype, and D. T. Stolow. "Involvement of a tissue-specific RNA recognition motif protein in Drosophila spermatogenesis." Molecular and Cellular Biology 17, no. 5 (May 1997): 2708–15. http://dx.doi.org/10.1128/mcb.17.5.2708.
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RNA binding proteins mediate posttranscriptional regulation of gene expression via their roles in nuclear and cytoplasmic mRNA metabolism. Many of the proteins involved in these processes have a common RNA binding domain, the RNA recognition motif (RRM). We have characterized the Testis-specific RRM protein gene (Tsr), which plays an important role in spermatogenesis in Drosophila melanogaster. Disruption of Tsr led to a dramatic reduction in male fertility due to the production of spermatids with abnormalities in mitochondrial morphogenesis. Tsr is located on the third chromosome at 87F, adjacent to the nuclear pre-mRNA binding protein gene Hrb87F. A 1.7-kb Tsr transcript was expressed exclusively in the male germ line. It encoded a protein containing two RRMs similar to those found in HRB87F as well as a unique C-terminal domain. TSR protein was located in the cytoplasm of spermatocytes and young spermatids but was absent from mature sperm. The cellular proteins expressed in premeiotic primary spermatocytes from Tsr mutant and wild-type males were assessed by two-dimensional gel electrophoresis. Lack of TSR resulted in the premature expression of a few proteins prior to meiosis; this was abolished by a transgenic copy of Tsr. These data demonstrate that TSR negatively regulated the expression of some testis proteins and, in combination with its expression pattern and subcellular localization, suggest that TSR regulates the stability or translatability of some mRNAs during spermatogenesis.
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Shi, H., B. E. Hoffman, and J. T. Lis. "A specific RNA hairpin loop structure binds the RNA recognition motifs of the Drosophila SR protein B52." Molecular and Cellular Biology 17, no. 5 (May 1997): 2649–57. http://dx.doi.org/10.1128/mcb.17.5.2649.
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B52, also known as SRp55, is a member of the Drosophila melanogaster SR protein family, a group of nuclear proteins that are both essential splicing factors and specific splicing regulators. Like most SR proteins, B52 contains two RNA recognition motifs in the N terminus and a C-terminal domain rich in serine-arginine dipeptide repeats. Since B52 is an essential protein and is expected to play a role in splicing a subset of Drosophila pre-mRNAs, its function is likely to be mediated by specific interactions with RNA. To investigate the RNA-binding specificity of B52, we isolated B52-binding RNAs by selection and amplification from a pool of random RNA sequences by using full-length B52 protein as the target. These RNAs contained a conserved consensus motif that constitutes the core of a secondary structural element predicted by energy minimization. Deletion and substitution mutations defined the B52-binding site on these RNAs as a hairpin loop structure covering about 20 nucleotides, which was confirmed by structure-specific enzymatic probing. Finally, we demonstrated that both RNA recognition motifs of B52 are required for RNA binding, while the RS domain is not involved in this interaction.
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Kress, Tracy L., Young J. Yoon, and Kimberly L. Mowry. "Nuclear RNP complex assembly initiates cytoplasmic RNA localization." Journal of Cell Biology 165, no. 2 (April 19, 2004): 203–11. http://dx.doi.org/10.1083/jcb.200309145.
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Cytoplasmic localization of mRNAs is a widespread mechanism for generating cell polarity and can provide the basis for patterning during embryonic development. A prominent example of this is localization of maternal mRNAs in Xenopus oocytes, a process requiring recognition of essential RNA sequences by protein components of the localization machinery. However, it is not yet clear how and when such protein factors associate with localized RNAs to carry out RNA transport. To trace the RNA–protein interactions that mediate RNA localization, we analyzed RNP complexes from the nucleus and cytoplasm. We find that an early step in the localization pathway is recognition of localized RNAs by specific RNA-binding proteins in the nucleus. After transport into the cytoplasm, the RNP complex is remodeled and additional transport factors are recruited. These results suggest that cytoplasmic RNA localization initiates in the nucleus and that binding of specific RNA-binding proteins in the nucleus may act to target RNAs to their appropriate destinations in the cytoplasm.
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Narayanan, Krishna, Chun-Jen Chen, Junko Maeda, and Shinji Makino. "Nucleocapsid-Independent Specific Viral RNA Packaging via Viral Envelope Protein and Viral RNA Signal." Journal of Virology 77, no. 5 (March 1, 2003): 2922–27. http://dx.doi.org/10.1128/jvi.77.5.2922-2927.2003.
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ABSTRACT For any of the enveloped RNA viruses studied to date, recognition of a specific RNA packaging signal by the virus's nucleocapsid (N) protein is the first step described in the process of viral RNA packaging. In the murine coronavirus a selective interaction between the viral transmembrane envelope protein M and the viral ribonucleoprotein complex, composed of N protein and viral RNA containing a short cis-acting RNA element, the packaging signal, determines the selective RNA packaging into virus particles. In this report we show that expressed coronavirus envelope protein M specifically interacted with coexpressed noncoronavirus RNA transcripts containing the short viral packaging signal in the absence of coronavirus N protein. Furthermore, this M protein-packaging signal interaction led to specific packaging of the packaging signal-containing RNA transcripts into coronavirus-like particles in the absence of N protein. These findings not only highlight a novel RNA packaging mechanism for an enveloped virus, where the specific RNA packaging can occur without the core or N protein, but also point to a new, biologically important general model of precise and selective interaction between transmembrane proteins and specific RNA elements.
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Schneemann, Anette, and Dawn Marshall. "Specific Encapsidation of Nodavirus RNAs Is Mediated through the C Terminus of Capsid Precursor Protein Alpha." Journal of Virology 72, no. 11 (November 1, 1998): 8738–46. http://dx.doi.org/10.1128/jvi.72.11.8738-8746.1998.
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ABSTRACT Flock house virus (FHV) is a small icosahedral insect virus with a bipartite, messenger-sense RNA genome. Its T=3 icosahedral capsid is initially assembled from 180 subunits of a single type of coat protein, capsid precursor protein alpha (407 amino acids). Following assembly, the precursor particles undergo a maturation step in which the alpha subunits autocatalytically cleave between Asn363 and Ala364. This cleavage generates mature coat proteins beta (363 residues) and gamma (44 residues) and is required for acquisition of virion infectivity. The X-ray structure of mature FHV shows that gamma peptides located at the fivefold axes of the virion form a pentameric helical bundle, and it has been suggested that this bundle plays a role in release of viral RNA during FHV uncoating. To provide experimental support for this hypothesis, we generated mutant coat proteins that carried deletions in the gamma region of precursor protein alpha. Surprisingly, we found that these mutations interfered with specific recognition and packaging of viral RNA during assembly. The resulting particles contained large amounts of cellular RNAs and varying amounts of the viral RNAs. Single-site amino acid substitution mutants showed that three phenylalanines located at positions 402, 405, and 407 of coat precursor protein alpha were critically important for specific recognition of the FHV genome. Thus, in addition to its hypothesized role in uncoating and RNA delivery, the C-terminal region of coat protein alpha plays a significant role in recognition of FHV RNA during assembly. A possible link between these two functions is discussed.
Dissertations / Theses on the topic "Specific protein/RNA recognition"
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Miranda, Rafael. "Sequence Specific RNA Recognition by Pentatricopeptide Repeat Proteins: Beyond the PPR Code." Thesis, University of Oregon, 2018. http://hdl.handle.net/1794/23135.
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Pentatricopeptide repeat (PPR) proteins are helical-repeat proteins that bind RNAs through a simple 1-repeat:1-nucleotide manner. Nucleotide specificity is determined by an amino acid code, the PPR code. This modular interaction mode, predictable code for nucleotide specificity, and simple repeating architecture make them a promising scaffold for engineering proteins to bind custom RNA sequences and binding site prediction of native PPR proteins. Despite these features, the alignments of the binding sites of well-characterized PPR proteins to the predicted binding sites often have mismatches and discontinuities, suggesting a tolerance for mismatches. In order to maximize the ability to predict the binding sites of native PPR proteins and effectively generate designer PPR proteins with predictable specificity, it will be important to address how affinity and specificity is distributed across a PPR tract. I developed a high- throughput bind-n-seq technique to rapidly and thoroughly address these questions. The affinity and specificity of the native PPR protein, PPR10 was determined using bind-n- seq. The results demonstrate that not all of PPR10’s repeats contribute equally to binding affinity, and there were sequence specific interactions that could not be explained by the PPR code, suggesting alternate modes of nucleotide recognition. A similar analysis of four different designer PPR proteins showed that they recognize RNA according to the code and lacked any alternate modes of nucleotide recognition, implying that the non- canonical sequence specific interactions represent idiosyncratic features of PPR10. This analysis also showed that N-terminal and purine specifying repeats have greater contributions to binding affinity, and that longer scaffolds have a greater tolerance for mismatches. Together, these findings highlight the challenges for binding site prediction and present implications for the design of PPR proteins with minimum off-target binding.
This dissertation contains previously published and unpublished co-authored material.10000-01-01
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Zanier, Katia. "Regulation of histone gene expression : solution structure determination by NMR of the 3' histone mRNA hairpin and implications for specific protein-RNA recognition." Thesis, University of London, 2002. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.269984.
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Ramos, Andres. "NMR studies of specificity in RNA-protein recognition." Thesis, University of Cambridge, 1998. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.625038.
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Chan, Yin-tung Crystal, and 陳燕彤. "Demonstration of specific physical interaction between CHOP mRNA and intracellular proteins." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2011. http://hub.hku.hk/bib/B47169369.
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The ability of a cell to respond precisely to environmental stress depends on the expression of a large number of genes in a finely coordinated manner. One of such genes is CHOP that encodes the CCAAT/Enhancer-Binding Protein Homologous Protein. CHOP is usually expressed to mediate apoptosis under the condition of excessive stress. The expression of CHOP therefore has to be stringently regulated as its expression will determine the fate of a cell under stress. The expression of many genes is regulated at the posttranscriptional level through the metabolism of their mRNA, such as maturation, transport, storage, and degradation of mRNA. Many metabolic processes of mRNA are known to be mediated by RNA-binding proteins that specifically interact with the mRNA. RNA-binding proteins that interact with the CHOP mRNA have until present not been identified. The aim of this study is to investigate what proteins may bind specifically to CHOP mRNA. The study will enable further understanding regarding how the expression of CHOP is regulated in cellular stress response.
Proteins extracted from HeLa cells were incubated with a 335bp [3H]-labelled CHOP RNA probe that spans over a part of the coding region and the 3’UTR of CHOP mRNA. Sucrose density gradient ultracentrifugation revealed that after incubation with proteins extracted from HeLa cells, the sedimentation rate of the [3H]-CHOP RNA probe was significantly higher than that of the free [3H]-RNA probe. The formation of heavy molecular complexes involving the [3H]-CHOP RNA probe was therefore suggested. However, no increase in sedimentation rate of the [3H]-CHOP RNA probe was observed in the presence of an excess of unlabelled CHOP RNA probe. Similar observations were made when the experiments were performed using proteins isolated from cells treated with As2O3.
Two putative sequence elements, the Adenylate-Uridylate-Rich Element (ARE) and the Putative Regulatory Element (PRE) located respectively in the 3’UTR and coding region of the CHOP mRNA were then examined for their involvement in RNA-protein interaction. The deletion of ARE and/or PRE, from the [3H]-CHOP RNA probe had little effect on the binding of the RNA probe to the HeLa cell proteins. Consistently, unlabelled CHOP RNA probes with the same deletions were only slightly weaker in competing with the intact [3H]-CHOP RNA probe to bind to HeLa cell proteins. Human Antigen R (HuR) was identified by Western blot analysis to be present in the proteins that were obtained by pull-down assays using biotinylated CHOP RNA as a probe. The deletion of ARE and/or PRE resulted in a slight reduction of HuR obtained by pull down assays.
This study provides the first evidence that physical binding interaction occurs between intracellular RNA-binding proteins and CHOP mRNA. More importantly, one such protein is HuR. Data suggest that HuR binding to the CHOP mRNA is mediated by sequences in the CHOP mRNA other than ARE and PRE.published_or_final_version
Biochemistry
Master
Master of Philosophy
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Davies, Holly Gibs. "MSY4, a sequence-specific RNA binding protein expressed during mouse spermatogenesis /." Thesis, Connect to this title online; UW restricted, 2000. http://hdl.handle.net/1773/10307.
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Giorgini, Flaviano. "Functional analysis of the murine sequence-specific RNA binding protein MSY4 /." Thesis, Connect to this title online; UW restricted, 2002. http://hdl.handle.net/1773/10293.
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Young, David James, and n/a. "The recognition of stop codons by the decoding release factors." University of Otago. Department of Biochemistry, 2009. http://adt.otago.ac.nz./public/adt-NZDU20090603.104834.
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Termination of protein synthesis involves the recognition of one of three stop codons (UAG, UAA or UGA) and hydrolysis of the nascent polypeptide chain from the peptidyl-tRNA on the ribosome. Unlike sense codons, which are decoded by aminoacyl-tRNAs, stop codons are decoded by proteins known as release factors. The decoding release factors occupy the same site as aminoacyl-tRNA, interacting directly with the stop codon at the decoding centre and inducing peptidyl-tRNA hydrolysis at the peptidyl transferase centre. Eubacteria have two codon-specific decoding release factors - RF1, which recognizes UAG and UAA, and RF2, which recognizes UGA and UAA. Biochemical studies identified two tripeptide 'anticodon' motifs, PXT in RF1 and SPF in RF2, which structural studies have shown occur in exposed loops (anticodon loops) on the surface of the proteins. Structures of isolated release factors show a compact 'closed' conformation whereas structures of release factors bound to the ribosome show them to be in a highly extended 'open' conformation. This suggests that a large conformational change in the release factor must take place upon or before binding to the ribosome. This transition has been invoked as a mechanism for how translational fidelity is maintained (Rawat et al, 2003), however, small angle X-ray scattering data from E. coli RF1 suggest the decoding release factors are also in the open conformation in solution challenging this mechanism.
Mora et al. (2003a) presented evidence that swapping the anticodon loop of RF2 with that of RF1 switched the stop codon specificity of the release factor. Recent structures of the decoding release factors bound to the ribosome showed that there was a second structural element of the release factor, the tip of helix α5, involved in recognition of the first base of the stop codon. The objectives of this thesis were to investigate both the anticodon loop and the helix α5 region for their roles in stop codon recognition, and to investigate whether there is a conformational change in the release factors on binding to the ribosome.
The anticodon loop was investigated using chimeras of E. coli RF1/RF2 and E. coli RF1/C. elegans mitochondrial RF1 (MRF1) within the anticodon loop. An RF1 variant containing the RF2-specific SPF tripeptide motif did not switch stop codon specificity showing that the tripeptide motifs are not sufficient determinants for the codon specificity of RF1 and RF2 as was originally proposed. Surprisingly repeating the complete swap of the RF1 anticodon loop to that of RF2 did not switch the stop codon specificity as claimed in Mora et al. (2003a). The studies in this thesis identified additional regions of the anticodon loop of the release factor that are important for stop codon recognition. Two of the RF1/RF2 anticodon loop variants produced showed altered codon specificity recognizing all three standard stop codons and the sense codon UGG. These variants provided unexpected insights into the mechanism of stop codon recognition and can explain why there are two release factors in eubacteria.
The C. elegans MRF1 contains a novel anticodon loop that is shorter and lacks the classical PXT motif. E. coli RF1/C. elegans MRF1 chimeras showed that this anticodon loop could function in E. coli RF1 and maintain the same codon specificity. While size and sequence within the loop together are important for recognition clearly there is more than one way RF1-type release factors can recognize the UAG and UAA stop codons.
Vertebrate mitochondria use four stop codons, two of the standard stop codons, UAA and UAG, and the reassigned arginine codons AGA and AGG. Two vertebrate mitochondrial release factors have been identified, mtRF1a and mtRF1 (renamed here mRF1[Canonical] and mRF1[Noncanonical]). Bioinformatic studies showed mRF1[C] had similar helix α5 and anticodon loop regions to classical RF1s. mRF1[NC] had different helix α5 and anticodon loop regions and was hypothesized to recognize the non-standard stop codons AGA and AGG. E. coli RF1/Human mRF1[NC] chimeras were constructed that showed that the helix α5 and anticodon loop regions are important for stop codon recognition. Nevertheless the chimeras showed poor activity at the AGA and AGG stop codons on E. coli 70S ribosomes suggesting that mRF1[NC] has evolved to function exclusively on 55S mitoribosomes.
A release factor variant of RF2 was designed that had the potential to trap this E. coli factor in the closed conformation in solution by disulphide bond formation. The RF2 double cysteine variant was successfully expressed and purified. The disulphide bond between the two cysteines was detected directly by mass spectrometry in a high proportion of molecules, showing the closed form of RF2 exists in solution. The RF2 closed form variant was shown to have release activity concomitant with the proportion of the open form in the RF preparation showing that the conformational change is required for normal release factor function. Preliminary binding studies have suggested that the RF2 closed form variant can bind to the ribosome. The ability of the closed form of RF2 to bind to the ribosome allowed a mechanism of translational fidelity to be proposed from the studies in this thesis; the release factor would recognize the stop codon in the decoding centre and, if cognate, the conformational change would occur allowing peptidyl-tRNA hydrolysis.
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Cattelin, Céline. "Exploration de la diversité des protéines à solénoïdes alpha, régulatrices de l'expression des gènes des organites dans les lignées eucaryotes photosynthétiques et étude de la dynamique conformationnelle des protéines à "PentatricoPeptide Repeats"." Electronic Thesis or Diss., Sorbonne université, 2023. http://www.theses.fr/2023SORUS158.
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Au sein des Archaeplastida (eucaryotes photosynthétiques ayant acquis un chloroplaste suite à une endosymbiose avec une cyanobactérie ancestrale) les génomes chloroplastiques et mitochondriaux des algues vertes et des plantes terrestres sont régulés de manière post-transcriptionnelle, principalement par des protéines à solénoïde alpha codées dans le noyau. Ces facteurs nucléaires sont composés de motifs répétés dégénérés (protéines PPR et OPR, respectivement pentatricopeptide repeat et octatricopeptide repeats) interagissant de façon spécifique avec une partie de la séquence de leur ARN cible et forment de grandes familles de paralogues. Les protéines PPR sont très abondantes chez les plantes terrestres tandis que les OPR le sont chez les algues vertes. Ces expansions différentielles, en parallèle de l'évolution du métabolisme des ARN dans les organites pourraient refléter des adaptations génétiques préservant la phototrophie dans diverses conditions et niches écologiques. Chez les autres Archaeplastida (algues rouges et Glaucophytes) et chez les eucaryotes issus d'une endosymbiose avec une microalgue ancestrale comme les Diatomées, la régulation des génomes des organites reste peu explorée. Un premier objectif de ma thèse a été de décrire la diversité et la dynamique évolutive des protéines à solénoïde alpha connues ou candidates pour la régulation de l'expression du génome des organites et ce, dans l'ensemble des eucaryotes photosynthétiques. Pour les identifier, j'ai développé une approche combinant détection d'homologie lointaine de séquence et classification indépendante de la similarité entre séquences. J'ai validé cette approche en retrouvant et complétant les familles OPR et PPR connues chez les espèces modèles Chlamydomonas reinhardtii et Arabidopsis thaliana. J'ai montré que les expansions d'OPR étaient restreintes au sein des Chlorophytes et qu'en dehors des algues vertes et des plantes terrestres, les protéines à PPR et à OPR étaient peu nombreuses, suggérant que d'autres acteurs de la régulation de l'expression des génomes des organites restent à découvrir. J'ai également identifié plusieurs dizaines d'autres familles de protéines à solénoïde alpha adressées aux organites dans tous les protéomes étudiés, certaines aux fonctions encore inconnues et dont la caractérisation expérimentale dans des organismes modèles serait pertinente. Dans un second temps, j'ai utilisé des approches de dynamique moléculaire pour mieux comprendre l'affinité et la spécificité des liaisons entre les PPR et leurs ARN cibles. J'ai notamment étudié la dynamique des motifs répétés et la géométrie des sites de liaison des nucléotides en fonction de leur position dans la séquence des motifs PPR, y compris les effets du nombre de répétitions et de la présence ou non des domaines N- et C-terminaux, en plus de l'évolution de la conformation globale de la protéine. Nos résultats suggèrent le rôle de la flexibilité des protéines PPR, tant au niveau de la protéine que du motif dans la liaison à sa cible ARN et sa pertinence pour l'affinité et la spécificité de la reconnaissance des nucléotidesIn Archaeplastida (photosynthetic eukaryotes that acquired a chloroplast following endosymbiosis with an ancestral cyanobacterium) the chloroplast and mitochondrial genomes of green algae and land plants are regulated post-transcriptionally, mainly by alpha-solenoid proteins encoded in the nucleus. These nuclear factors are composed of degenerate repeat motifs (PPR and OPR proteins, respectively pentatricopeptide repeat and octatricopeptide repeats) that interact specifically with part of their target RNA sequence and form large families of paralogs. PPR proteins are very abundant in terrestrial plants while OPRs are abundant in green algae. These differential expansions, in parallel with the evolution of RNA metabolism in organelles, may reflect genetic adaptations that preserve phototrophy under different conditions and ecological niches. In other Archaeplastids (red algae and Glaucophytes) and in eukaryotes that originate from endosymbiosis with an ancestral microalga such as the Diatoms, the regulation of organelle genomes remains poorly explored. A first objective of my thesis was to describe the diversity and evolutionary dynamics of known or candidate alpha-solenoid proteins for the regulation of organelle genome expression in all photosynthetic eukaryotes. To identify them, I developed an approach that combines distant sequence homology detection and sequence similarity independent classification. I validated this approach by finding and completing the known OPR and PPR families in the model species Chlamydomonas reinhardtii and Arabidopsis thaliana. I showed that OPR expansions were restricted within Chlorophytes and that outside of green algae and land plants, PPR and OPR proteins were few in number, suggesting that other players in the regulation of organelle genome expression remain to be discovered. I also identified several dozen other families of organelle-addressed alpha-solenoid proteins in all the proteomes studied, some of which have as yet unknown functions and whose experimental characterisation in model organisms would be relevant. In a second step, I used molecular dynamics approaches to better understand the affinity and specificity of binding between PPRs and their target RNAs. In particular, I studied the dynamics of the repeat motifs and the geometry of the nucleotide binding sites as a function of their position in the PPR motif sequence, including the effects of the number of repeats and the presence or absence of N- and C-terminal domains, in addition to the evolution of the overall conformation of the protein. Our results suggest the role of PPR protein flexibility, both at the protein and motif level, in binding to its RNA target and its relevance to the affinity and specificity of nucleotide recognition
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Bronicki, Lucas M. "Characterization of Multiple Exon 1 Variants and Neuron-specific Transcriptional Control of Mammalian HuD." Thèse, Université d'Ottawa / University of Ottawa, 2013. http://hdl.handle.net/10393/23682.
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The RNA-binding protein (RBP) and Hu/ELAV family member HuD regulates mRNA metabolism of genes that encode proteins involved in neuronal differentiation, learning and memory, and certain neurological diseases. Given the important functions of HuD in a variety of processes, we set out to characterize the 5’ genomic region of the mammalian HuD gene and determine the mechanisms that regulate its mRNA expression in neurons using P19 cells and mouse brain as models.
Bioinformatic and 5’RACE (rapid amplification of cDNA ends) analyses of the HuD 5’ genomic flanking region identified eight conserved leader exons (E1s), two of which are novel. Expression of all E1 variants was established in differentiating P19 cells, mouse embryonic (E14.5) and adult brains. Through several complementary approaches, we determined that the abundance of HuD mRNA is predominantly under transcriptional control in differentiating neurons. Sequential deletion of the 5’ regulatory region upstream of the predominantly expressed E1c variant revealed a well-conserved 400 bp DNA region that contains five E-boxes and is capable of directing expression of HuD specifically in neurons. Using electrophoretic mobility shift assays (EMSAs), chromatin immunoprecipitations (ChIPs), and E1c 5’ regulatory region (RR) deletion and mutation analysis, we found that two of these E-boxes are targeted by neurogenin 2 (NGN2/NEUROG2) and that this mechanism is important for induction of HuD mRNA in neurons. Additional deletion and mutation of the E1c 5’ RR revealed that putative cis-acting elements for Kruppel-like factors (KLFs) and nuclear DEAF-1-related (NuDR) transcription factors also positively regulate transcription of HuD.
Together, our findings reveal that the intricate transcriptional regulation of mammalian HuD involves eight leader exons and potentially alternate promoters. We further demonstrate that transcription of HuD requires neuron-specific control by NGN2 and possibly KLF and NuDR transcription factors. To our knowledge, this is the first study to identify transcriptional events that positively regulate expression of HuD.
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Oberstrass, Florian Christophe. "Novel modes of protein-RNA recognition in post-transcriptional gene regulation studied by NMR spectroscopy." kostenfrei kostenfrei, 2007. http://e-collection.ethbib.ethz.ch/view/eth:30123.
Full textBooks on the topic "Specific protein/RNA recognition"
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Steitz, Thomas A. Structural studies of protein-nucleic acid interaction: Thesources of sequence-specific binding. Cambridge: Cambridge University Press, 1993.
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Steitz, Thomas A. Structural studies of protein-nucleic acid interaction: The sources of sequence-specific binding. New York, NY, USA: Cambridge University Press, 1993.
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Dar, Arvin Christopher. Catalytic switching and substrate recognition mechanisms of the RNA dependent protein kinase PKR. 2006.
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Recognition of Carbohydrates in Biological Systems, Part B: Specific Applications, Volume 363 (Methods in Enzymology). Academic Press, 2003.
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Adler, M. Properties and potential of protein–DNA conjugates for analytic applications. Edited by A. V. Narlikar and Y. Y. Fu. Oxford University Press, 2017. http://dx.doi.org/10.1093/oxfordhb/9780199533053.013.25.
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This article examines the properties of protein-DNA conjugates and their potential for analytic applications. It begins with a discussion of DNA as a rigid construction tool for protein networks, reducing its functionality to the molecular equivalent of a steel bar in 'large-scale' architecture. It then describes DNA functionality in protein-DNA conjugates, like specific recognition of nucleotide sequences or its unique use as an amplification template. It also considers a range of applications for protein-DNA conjugates, including the use of artificial DNA-protein nanostructures as supramolecular building blocks and DNA-antibody conjugates for ultrasensitive antigen detection. Finally, it evaluates DNA-directed immobilization of protein-DNA adaptor molecules for flexible protein arrays. It shows that protein-DNA conjugates can be used as analytical targets for challenging and calibrating the properties of high-resolution atomic force microscopy, as well as analytical reagents for ultrasensitive target detection in immuno-PCR and related techniques.
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Walsh, Bruce, and Michael Lynch. Changes in Quantitative Traits Over Time. Oxford University Press, 2018. http://dx.doi.org/10.1093/oso/9780198830870.003.0001.
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Quantitative traits—be they morphological or physiological characters, aspects of behavior, or genome-level features such as the amount of RNA or protein expression for a specific gene—usually show considerable variation within and among populations. This chapter provides a historical overview of the study of such traits and their connections with traditional and molecular population genetics, applied breeding, and evolutionary theory.
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Byrne, John H., ed. The Oxford Handbook of Invertebrate Neurobiology. Oxford University Press, 2017. http://dx.doi.org/10.1093/oxfordhb/9780190456757.001.0001.
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Invertebrates have proven to be extremely useful models for gaining insights into the neural and molecular mechanisms of sensory processing, motor control, and higher functions, such as feeding behavior, learning and memory, navigation, and social behavior. Their enormous contribution to neuroscience is due, in part, to the relative simplicity of invertebrate nervous systems and, in part, to the large cells found in some invertebrates, like mollusks. Because of the organizms’ cell size, individual neurons can be surgically removed and assayed for expression of membrane channels, levels of second messengers, protein phosphorylation, and RNA and protein synthesis. Moreover, peptides and nucleotides can be injected into individual neurons. Other invertebrate systems such as Drosophila and Caenorhabditis elegans are ideal models for genetic approaches to the exploration of neuronal function and the neuronal bases of behavior. The Oxford Handbook of Invertebrate Neurobiology reviews neurobiological phenomena, including motor pattern generation, mechanisms of synaptic transmission, and learning and memory, as well as circadian rhythms, development, regeneration, and reproduction. Species-specific behaviors are covered in chapters on the control of swimming in annelids, crustacea, and mollusks; locomotion in hexapods; and camouflage in cephalopods. A unique feature of the handbook is the coverage of social behavior and intentionality in invertebrates. These developments are contextualized in a chapter summarizing past contributions of invertebrate research as well as areas for future studies that will continue to advance the field.
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Prout, Jeremy, Tanya Jones, and Daniel Martin. Core topics in intensive care medicine. Oxford University Press, 2014. http://dx.doi.org/10.1093/med/9780199609956.003.0020.
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This chapter on aspects of intensive care for FRCA commences with the recognition of the critically ill patient, use of early warning scores, outreach teams and the systematic assessment of the acutely ill patient. Sepsis has been a recent focus area for healthcare systems; the definition of sepsis, pathophysiological processes leading to organ dysfunction and management including early goal-directed therapy, supportive techniques and the role of specific therapies such as steroids and activated protein C are included. Acute lung injury and respiratory distress syndrome with management and specific ventilatory strategies are described. Important aspects of providing sedation and analgesia on intensive care are explained as well as the complex ethical aspects of this speciality with regards to consent and end-of-life decisions
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Kirchman, David L. Elements, biochemicals, and structures of microbes. Oxford University Press, 2018. http://dx.doi.org/10.1093/oso/9780198789406.003.0002.
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Microbiologists focus on the basic biochemical make-up of microbes, such as relative amounts of protein, RNA, and DNA in cells, while ecologists and biogeochemists use elemental ratios, most notably, the ratio of carbon to nitrogen (C:N), to explore biogeochemical processes and to connect up the carbon cycle with the cycle of other elements. Microbial ecologists make use of both types of data and approaches. This chapter combines both and reviews all things, from elements to macromolecular structures, that make up bacteria and other microbes. The most commonly used elemental ratio was discovered by Alfred Redfield who concluded that microbes have a huge impact on the chemistry of the oceans because of the similarity in nitrogen-to-phosphorus ratios for organisms and nitrate-to-phosphate ratios in the deep oceans. Although statistically different, the C:N ratios in soil microbes are remarkably similar to the ratios of aquatic microbes. The chapter moves on to discussing the macromolecular composition of bacteria and other microbes. This composition gives insights into the growth state of microbes in nature. Geochemists use specific compounds, “biomarkers”, to trace sources of organic material in ecosystems. The last section of the chapter is a review of extracellular polymers, pili, and flagella, which serve a variety of functions, from propelling microbes around to keeping them stuck in one place.
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Walsh, Bruce, and Michael Lynch. Evolution and Selection of Quantitative Traits. Oxford University Press, 2018. http://dx.doi.org/10.1093/oso/9780198830870.001.0001.
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Quantitative traits—be they morphological or physiological characters, aspects of behavior, or genome-level features such as the amount of RNA or protein expression for a specific gene—usually show considerable variation within and among populations. Quantitative genetics, also referred to as the genetics of complex traits, is the study of such characters and is based on mathematical models of evolution in which many genes influence the trait and in which non-genetic factors may also be important. Evolution and Selection of Quantitative Traits presents a holistic treatment of the subject, showing the interplay between theory and data with extensive discussions on statistical issues relating to the estimation of the biologically relevant parameters for these models. Quantitative genetics is viewed as the bridge between complex mathematical models of trait evolution and real-world data, and the authors have clearly framed their treatment as such. This is the second volume in a planned trilogy that summarizes the modern field of quantitative genetics, informed by empirical observations from wide-ranging fields (agriculture, evolution, ecology, and human biology) as well as population genetics, statistical theory, mathematical modeling, genetics, and genomics. Whilst volume 1 (1998) dealt with the genetics of such traits, the main focus of volume 2 is on their evolution, with a special emphasis on detecting selection (ranging from the use of genomic and historical data through to ecological field data) and examining its consequences. This extensive work of reference is suitable for graduate level students as well as professional researchers (both empiricists and theoreticians) in the fields of evolutionary biology, genetics, and genomics. It will also be of particular relevance and use to plant and animal breeders, human geneticists, and statisticians.
Book chapters on the topic "Specific protein/RNA recognition"
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Rhodes, Daniela. "Protein-DNA Recognition." In RNA Biochemistry and Biotechnology, 123–26. Dordrecht: Springer Netherlands, 1999. http://dx.doi.org/10.1007/978-94-011-4485-8_8.
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Travers, Andrew. "DNA-protein interactions: sequence specific recognition." In DNA-Protein Interactions, 52–86. Dordrecht: Springer Netherlands, 1993. http://dx.doi.org/10.1007/978-94-011-1480-6_3.
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Marmier-Gourrier, Nathalie, Audrey Vautrin, Christiane Branlant, and Isabelle Behm-Ansmant. "Analysis of Site-Specific RNA-Protein Interactions." In Alternative pre-mRNA Splicing, 342–56. Weinheim, Germany: Wiley-VCH Verlag GmbH & Co. KGaA, 2012. http://dx.doi.org/10.1002/9783527636778.ch32.
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Zheng, Fan, and Gevorg Grigoryan. "Design of Specific Peptide–Protein Recognition." In Methods in Molecular Biology, 249–63. New York, NY: Springer New York, 2016. http://dx.doi.org/10.1007/978-1-4939-3569-7_15.
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Bell, Thomas J., and James Eberwine. "Live Cell Genomics: RNA Exon-Specific RNA-Binding Protein Isolation." In Methods in Molecular Biology, 457–68. New York, NY: Springer New York, 2015. http://dx.doi.org/10.1007/978-1-4939-2806-4_31.
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Liang, Xue-Hai, Wen Shen, and Stanley T. Crooke. "Specific Increase of Protein Levels by Enhancing Translation Using Antisense Oligonucleotides Targeting Upstream Open Frames." In RNA Activation, 129–46. Singapore: Springer Singapore, 2017. http://dx.doi.org/10.1007/978-981-10-4310-9_9.
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Draper, David E., Graeme L. Conn, Apostolos G. Gittis, Debraj Guhathakurta, Eaton E. Lattman, and Luis Reynaldo. "RNA Tertiary Structure and Protein Recognition in an L11-RNA Complex." In The Ribosome, 105–14. Washington, DC, USA: ASM Press, 2014. http://dx.doi.org/10.1128/9781555818142.ch11.
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Varani, Gabriele, Peter Bayer, Paul Cole, Andres Ramos, and Luca Varani. "RNA Structure and RNA-Protein Recognition During Regulation of Eukaryotic Gene Expression." In RNA Biochemistry and Biotechnology, 195–216. Dordrecht: Springer Netherlands, 1999. http://dx.doi.org/10.1007/978-94-011-4485-8_15.
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Munschauer, Mathias. "Revealing Cell-Type Specific Differences in Protein Occupancy on RNA." In High-Resolution Profiling of Protein-RNA Interactions, 73–88. Cham: Springer International Publishing, 2015. http://dx.doi.org/10.1007/978-3-319-16253-9_4.
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Sharma, Shalini. "Isolation of a Sequence-Specific RNA Binding Protein, Polypyrimidine Tract Binding Protein, Using RNA Affinity Chromatography." In Methods in Molecular Biology, 1–8. Totowa, NJ: Humana Press, 2008. http://dx.doi.org/10.1007/978-1-60327-475-3_1.
Full textConference papers on the topic "Specific protein/RNA recognition"
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Schwartz, B. S., and M. C. Monroe. "INCREASED SECRETION OF A FIBRINOLYTIC INHIBITOR BY HUMAN MONONUCLEAR LEUKOCYTES PARALLELS THE PR0COAGULANT RESPONSE TO SPECIFIC ANTIGEN." In XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1644384.
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The presence of fibrin is a characteristic finding of Immune mediated tissue lesions. It is known that peripheral blood mononuclear cells (PBM) express tissue factor in response to recognition of a specific protein antigen. We have found PBM secrete a plasminogen activator (PA) Inhibitor (I) in parallel to expression of tissue factor upon exposure to a sensitizing antigen. Increased PA-I can be detected by Inhibition of urokinase (UK) in an 125I-fibrin plate assay, Inhibition of 125I-plasminogen cleavage, and formation of complexes between 125I-urokinase and UK-I. PA-I secretion is dose dependent, and antigen specific, i.e. a nonsensitizing antigen does not Induce a PA-I response.The PA-I is secreted by monocytes, however the recognition of antigen is a T-cell function. Inhibition of PBM protein or RNA synthesis abrogates the PA-I response. The PA-I appears to be the type 2 Inhibitor, in that 1) it is a much more efficient Inhibitor of UK than of tissue type PA, 2) it is labile in acid and detergent, and 3) it is neutralized by IgG to human placental PA-I, but not by antiserum to PA-I of endothelial cells. It is concluded that PBM respond to a foreign stimulus by elaborating molecules that lead to both the deposition and persistence of ftbrln.
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Nguyen, Thai Huu, and Qiao Lin. "An Aptamer-Functionalized Microfluidic Platform for Biomolecular Purification and Sensing." In ASME 2009 7th International Conference on Nanochannels, Microchannels, and Minichannels. ASMEDC, 2009. http://dx.doi.org/10.1115/icnmm2009-82142.
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Aptamers are oligonucleotides (DNA or RNA) that bind to chemical and biological analyte targets via affinity interactions. Through an in vitro synthetic process, aptamers can be developed for an extremely broad spectrum of analytes, such as small molecules, proteins, cells, viruses, and bacteria. Target recognition by aptamers is highly selective, as affinity interactions result in secondary aptamer conformational structures that specifically fit the target. The aptamer-target binding is also reversible and depends strongly on external stimuli such as pH and temperature. The specificity and stimuli-responsiveness of aptamers are highly attractive to biological purification and sensing, which generally involve isolating minute quantities of targets from complex samples with non-specific molecules and impurities present at orders-of-magnitude higher concentrations. We present an aptamer-functionalized microfluidic platform that by design exploits the specificity and temperature-dependent reversibility of aptamers to enable biomolecular purification and sensing. Using the specificity of aptamers, we demonstrate highly selective capture and enrichment of biomolecules. Employing thermally induced, reversible disruption of aptamer-target binding, we accomplish isocratic elution of the captured analytes and regeneration of the aptamer surfaces, thereby eliminating the use of potentially harsh reagents. Using integrated microfluidic control, the eluted analytes are detected in a label-free fashion by mass spectrometric methods.
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Wang, Tong, Jianxin Xue, and Yi Du. "A Method For The RNA-Protein Complexes Recognition." In ICCIR 2021: 2021 International Conference on Control and Intelligent Robotics. New York, NY, USA: ACM, 2021. http://dx.doi.org/10.1145/3473714.3473756.
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Zeiler, Brian N., Burt V. Bronk, and Abraham Grossman. "Improved approach to RNA and protein recognition for pathogen detection." In AeroSense 2000, edited by Patrick J. Gardner. SPIE, 2000. http://dx.doi.org/10.1117/12.394055.
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Liu, Zhi-Ping. "Systematic identification of local structure binding motifs in protein-RNA recognition." In 2014 8th International Conference on Systems Biology (ISB). IEEE, 2014. http://dx.doi.org/10.1109/isb.2014.6990735.
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Chrysostomou, Charalambos, Huseyin Seker, Nizamettin Aydin, and Parvez I. Haris. "Complex Resonant Recognition Model in analysing Influenza a virus subtype protein sequences." In Emerging Technologies for Patient Specific Healthcare. 2010 10th IEEE International Conference on Information Technology and Applications in Biomedicine (ITAB 2010). IEEE, 2010. http://dx.doi.org/10.1109/itab.2010.5687621.
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Zheng, Ying, and Wilson S. Meng. "Polycation Coated Polymeric Particles as Vehicles of RNA Delivery Into Immune Cells." In ASME 2010 Conference on Smart Materials, Adaptive Structures and Intelligent Systems. ASMEDC, 2010. http://dx.doi.org/10.1115/smasis2010-3714.
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The purpose of this work is to develop a carrier system for delivering RNA molecules aimed to downregulate specific functions in T cells. In many forms of cancer, T cells that express the protein Forkhead Box P3 (Foxp3) are associated with cancer progression. These cells can be identified by CD4 and CD25, molecules express on the cell surface. Studies have shown that downregulation of Foxp3 can increase the ability of other immune cells to destroy tumors. A class of RNA molecules, commonly referred to as “siRNA”, bind to and degrade specific messenger RNA (mRNA) in a sequence-dependent manner such that expression of the encoded protein is terminated. Because mRNA molecules are located inside cells, a carrier system is required to facilitate the uptake of siRNA, which does not passively diffuse through the plasma membrane. To this end, nanosized polymeric particles coated with the polycation, ornithinex10-histidinex6 (or O10H6) were used to adsorb siRNA that bind to the mRNA encoding Foxp3. The RNA-loaded particles are spherical and uniform in size (normally distributed, polydispersity index = 0.072). Loading of RNA to the particles was confirmed using gel electrophoresis. RNA complexed with the particles are protected from serum destabilization: 83.1% of RNA were recovered compared to 36.1% in RNA that were not associated with the particles. Association with the particles increased the uptake of the RNA in mouse T cells from 3.2±0.2% (free RNA) to 20.1±3.9%. Specifically, uptake of the RNA in T cells that express CD4 increased from 2.7±0.2% to 27.1±1.3% when particles were employed. These differences are statistically significant in three experiments conducted (p < 0.01). Internalization of the RNA into T cells was confirmed using confocal imaging. Flow cytometric analysis showed that the particle-complexed RNA reduced the percentage of T cells that express both CD4 and CD25 in mice carrying tumors from 24.0% when free RNA molecules were used to 13.5%. In these cells, the level of Foxp3 mRNA was reduced by 30%. In conclusion, the particles facilitate the uptake of siRNA molecules into a population of T cells that is known to promote cancer growth.
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Zhao, Guijun, Matthew B. Carson, and Hui Lu. "Prediction of Specific Protein-DNA Recognition by Knowledge-based Two-body and Three-body Interaction Potentials." In 2007 29th Annual International Conference of the IEEE Engineering in Medicine and Biology Society. IEEE, 2007. http://dx.doi.org/10.1109/iembs.2007.4353467.
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"Allele specific recognition of the Magnaporthe oryzae effector AVR Pita by the unconventional rice resistance protein Ptr." In IS-MPMI Congress. IS-MPMI, 2023. http://dx.doi.org/10.1094/ismpmi-2023-34.
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Lian, Weibin, Hongzhi Xu, Jianlin Ren, Changsheng Yan, and Yifan Lian. "IDDF2020-ABS-0100 Long intergenic non-protein coding RNA 01446 facilitates the proliferation and metastasis of gastric cancer cells through interacting with the histone lysine-specific demethylase LSD1." In Abstracts of the International Digestive Disease Forum (IDDF), 22–23 November 2020, Hong Kong. BMJ Publishing Group Ltd and British Society of Gastroenterology, 2020. http://dx.doi.org/10.1136/gutjnl-2020-iddf.21.
Full textReports on the topic "Specific protein/RNA recognition"
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Loebenstein, Gad, William Dawson, and Abed Gera. Association of the IVR Gene with Virus Localization and Resistance. United States Department of Agriculture, August 1995. http://dx.doi.org/10.32747/1995.7604922.bard.
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We have reported that localization of TMV in tobacco cultivars with the N gene, is associated with a 23 K protein (IVR) that inhibited replication of several plant viruses. This protein was also found in induced resistant tissue of Nicotiana glutinosa x Nicotiana debneyi. During the present grant we found that TMV production is enhanced in protoplasts and plants of local lesion responding tobacco cultivars exposed to 35oC, parallel to an almost complete suppression of the production of IVR. We also found that IVR is associated with resistance mechanisms in pepper cultivars. We succeeded to clone the IVR gene. In the first attempt we isolated a clone - "101" which had a specific insert of 372 bp (the full length gene for the IVR protein of 23 kD should be around 700 bp). However, attempts to isolate the full length gene did not give clear cut results, and we decided not to continue with this clone. The amino acid sequence of the N-terminus of IVR was determined and an antiserum was prepared against a synthetic peptide representing amino acids residues 1-20 of IVR. Using this antiserum as well as our polyclonal antiserum to IVR a new clone NC-330 was isolated using lamba-ZAP library. This NC-330 clone has an insert of about 1 kB with an open reading frame of 596 bp. This clone had 86.6% homology with the first 15 amino acids of the N-terminal part of IVR and 61.6% homology with the first 23 amino acids of IVR. In the QIA expression system and western blotting of the expressed protein, a clear band of about 21 kD was obtained with IVR antiserum. This clone was used for transformation of Samsun tobacco plants and we have presently plantlets which were rooted on medium containing kanamycin. Hybridization with this clone was also obtained with RNA from induced resistant tissue of Samsun NN but not with RNA from healthy control tissue of Samsun NN, or infected or healthy tissue of Samsun. This further strengthens the previous data that the NC 330 clone codes for IVR. In the U.S. it was shown that IVR is induced in plants containing the N' gene when infected with mutants of TMV that elicit the HR. This is a defined system in which the elicitor is known to be due to permutations of the coat protein which can vary in elicitor strength. The objective was to understand how IVR synthesis is induced after recognition of elicitor coat protein in the signal transduction pathway that leads to HR. We developed systems to manipulate induction of IVR by modifying the elicitor and are using these elicitor molecules to isolate the corresponding plant receptor molecules. A "far-western" procedure was developed that found a protein from N' plants that specifically bind to elicitor coat proteins. This protein is being purified and sequenced. This objective has not been completed and is still in progress. We have reported that localization of TMV in tobacco cultivars with the N gene, is associated with a 23 K protein (IVR) that inhibited replication of several plant viruses. This protein was also found in induced resistant tissue of Nicotiana glutinosa x Nicotiana debneyi.
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Rafaeli, Ada, and Russell Jurenka. Molecular Characterization of PBAN G-protein Coupled Receptors in Moth Pest Species: Design of Antagonists. United States Department of Agriculture, December 2012. http://dx.doi.org/10.32747/2012.7593390.bard.
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The proposed research was directed at determining the activation/binding domains and gene regulation of the PBAN-R’s thereby providing information for the design and screening of potential PBAN-R-blockers and to indicate possible ways of preventing the process from proceeding to its completion. Our specific aims included: (1) The identification of the PBAN-R binding domain by a combination of: (a) in silico modeling studies for identifying specific amino-acid side chains that are likely to be involved in binding PBAN with the receptor and; (b) bioassays to verify the modeling studies using mutant receptors, cell lines and pheromone glands (at tissue and organism levels) against selected, designed compounds to confirm if compounds are agonists or antagonists. (2) The elucidation ofthemolecular regulationmechanisms of PBAN-R by:(a) age-dependence of gene expression; (b) the effect of hormones and; (c) PBAN-R characterization in male hair-pencil complexes. Background to the topic Insects have several closely related G protein-coupled receptors (GPCRs) belonging to the pyrokinin/PBAN family, one with the ligand pheromone biosynthesis activating neuropeptide or pyrokinin-2 and another with diapause hormone or pyrokinin-1 as a ligand. We were unable to identify the diapause hormone receptor from Helicoverpa zea despite considerable effort. A third, related receptor is activated by a product of the capa gene, periviscerokinins. The pyrokinin/PBAN family of GPCRs and their ligands has been identified in various insects, such as Drosophila, several moth species, mosquitoes, Triboliumcastaneum, Apis mellifera, Nasoniavitripennis, and Acyrthosiphon pisum. Physiological functions of pyrokinin peptides include muscle contraction, whereas PBAN regulates pheromone production in moths plus other functions indicating the pleiotropic nature of these ligands. Based on the alignment of annotated genomic sequences, the primary and secondary structures of the pyrokinin/PBAN family of receptors have similarity with the corresponding structures of the capa or periviscerokinin receptors of insects and the neuromedin U receptors found in vertebrates. Major conclusions, solutions, achievements Evolutionary trace analysisof receptor extracellular domains exhibited several class-specific amino acid residues, which could indicate putative domains for activation of these receptors by ligand recognition and binding. Through site-directed point mutations, the 3rd extracellular domain of PBAN-R was shown to be critical for ligand selection. We identified three receptors that belong to the PBAN family of GPCRs and a partial sequence for the periviscerokinin receptor from the European corn borer, Ostrinianubilalis. Functional expression studies confirmed that only the C-variant of the PBAN-R is active. We identified a non-peptide agonist that will activate the PBAN-receptor from H. zea. We determined that there is transcriptional control of the PBAN-R in two moth species during the development of the pupa to adult, and we demonstrated that this transcriptional regulation is independent of juvenile hormone biosynthesis. This transcriptional control also occurs in male hair-pencil gland complexes of both moth species indicating a regulatory role for PBAN in males. Ultimate confirmation for PBAN's function in the male tissue was revealed through knockdown of the PBAN-R using RNAi-mediated gene-silencing. Implications, both scientific and agricultural The identification of a non-peptide agonist can be exploited in the future for the design of additional compounds that will activate the receptor and to elucidate the binding properties of this receptor. The increase in expression levels of the PBAN-R transcript was delineated to occur at a critical period of 5 hours post-eclosion and its regulation can now be studied. The mysterious role of PBAN in the males was elucidated by using a combination of physiological, biochemical and molecular genetics techniques.
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Citovsky, Vitaly, and Yedidya Gafni. Suppression of RNA Silencing by TYLCV During Viral Infection. United States Department of Agriculture, December 2009. http://dx.doi.org/10.32747/2009.7592126.bard.
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The Israeli isolate of Tomato yellow leaf curl geminivirus (TYLCV-Is) is a major tomato pathogen, causing extensive (up to 100%) crop losses in Israel and in the south-eastern U.S. (e.g., Georgia, Florida). Surprisingly, however, little is known about the molecular mechanisms of TYLCV-Is interactions with tomato cells. In the current BARD project, we have identified a TYLCV-Is protein, V2, which acts as a suppressor of RNA silencing, and showed that V2 interacts with the tomato (L. esculentum) member of the SGS3 (LeSGS3) protein family known to be involved in RNA silencing. This proposal will use our data as a foundation to study one of the most intriguing, yet poorly understood, aspects of TYLCV-Is interactions with its host plants – possible involvement of the host innate immune system, i.e., RNA silencing, in plant defense against TYLCV-Is and the molecular pathway(s) by which TYLCV-Is may counter this defense. Our project sought two objectives: I. Study of the roles of RNA silencing and its suppression by V2 in TYLCV-Is infection of tomato plants. II. Study of the mechanism by which V2 suppresses RNA silencing. Our research towards these goals has produced the following main achievements: • Identification and characterization of TYLCV V2 protein as a suppressor of RNA silencing. (#1 in the list of publications). • Characterization of the V2 protein as a cytoplasmic protein interacting with the plant protein SlSGS3 and localized mainly in specific, not yet identified, bodies. (#2 in the list of publications). • Development of new tools to study subcellular localization of interacting proteins (#3 in the list of publications). • Characterization of TYLCV V2 as a F-BOX protein and its possible role in target protein(s) degradation. • Characterization of TYLCV V2 interaction with a tomato cystein protease that acts as an anti-viral agent. These research findings provided significant insights into (I) the suppression of RNA silencing executed by the TYLCV V2 protein and (II) characterization some parts of the mechanism(s) involved in this suppression. The obtained knowledge will help to develop specific strategies to attenuate TYLCV infection, for example, by blocking the activity of the viral suppressor of gene silencing thus enabling the host cell silencing machinery combat the virus.
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Eshed, Yuval, and Sarah Hake. Exploring General and Specific Regulators of Phase Transitions for Crop Improvement. United States Department of Agriculture, November 2012. http://dx.doi.org/10.32747/2012.7699851.bard.
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The transition of plants from a juvenile to adult growth phase entails a wide range of changes in growth habit, physiological competence and composition. Strikingly, most of these changes are coordinated by the expression of a single regulator, micro RNA 156 (miR156) that coordinately regulates a family of SBP genes containing a miR156 recognition site in the coding region or in their 3’ UTR. In the framework of this research, we have taken a broad taxonomic approach to examine the role of miR156 and other genetic regulators in phase change transition and its implication to plant development and crop improvement. We set to: Determine the common and unique factors that are altered upon juvenile to adult phase transition. Determine the functions of select miR156 target genes in tomato and maize, and identify those targets that mediate phase transition. Characterize the role of miR172 and its targets in tomato phase change. Determine the relationships between the various molecular circuits directing phase change. Determine the effects of regulated manipulation of phase change genes on plant architecture and if applicable, productivity. In the course of the study, a new technology for gene expression was introduced – next generation sequencing (NGS). Hence some of the original experiments that were planned with other platforms of RNA profiling, primarily Affymetrix arrays, were substituted with the new technology. Yet, not all were fully completed. Moreover, once the initial stage was completed, each group chose to focus its efforts on specific components of the phase change program. The Israeli group focused on the roles of the DELAYED SYMPODIAL TERMINATION and FALSIFLORA factors in tomato age dependent programs whereas the US group characterized in detail the role of miR156 (also termed Cg) in other grasses and in maize, its interplay with the many genes encoding miR172.
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Mawassi, Munir, and Valerian Dolja. Role of RNA Silencing Suppression in the Pathogenicity and Host Specificity of the Grapevine Virus A. United States Department of Agriculture, January 2010. http://dx.doi.org/10.32747/2010.7592114.bard.
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RNA silencing is a defense mechanism that functions against virus infection and involves sequence-specific degradation of viral RNA. Diverse RNA and DNA viruses of plants encode RNA silencing suppressors (RSSs), which, in addition to their role in viral counterdefense, were implicated in the efficient accumulation of viral RNAs, virus transport, pathogenesis, and determination of the virus host range. Despite rapidly growing understanding of the mechanisms of RNA silencing suppression, systematic analysis of the roles played by diverse RSSs in virus biology and pathology is yet to be completed. Our research was aimed at conducting such analysis for two grapevine viruses, Grapevine virus A (GVA) and Grapevine leafroll-associated virus-2 (GLRaV- 2). Our major achievements on the previous cycle of BARD funding are as follows. 1. GVA and GLRaV-2 were engineered into efficient gene expression and silencing vectors for grapevine. The efficient techniques for grapevine infection resulting in systemic expression or silencing of the recombinant genes were developed. Therefore, GVA and GLRaV-2 were rendered into powerful tools of grapevine virology and functional genomics. 2. The GVA and GLRaV-2 RSSs, p10 and p24, respectively, were identified, and their roles in viral pathogenesis were determined. In particular, we found that p10 functions in suppression and pathogenesis are genetically separable. 3. We revealed that p10 is a self-interactive protein that is targeted to the nucleus. In contrast, p24 mechanism involves binding small interfering RNAs in the cytoplasm. We have also demonstrated that p10 is relatively weak, whereas p24 is extremely strong enhancer of the viral agroinfection. 4. We found that, in addition to the dedicated RSSs, GVA and GLRaV-2 counterdefenses involve ORF1 product and leader proteases, respectively. 5. We have teamed up with Dr. Koonin and Dr. Falnes groups to study the evolution and function of the AlkB domain presents in GVA and many other plant viruses. It was demonstrated that viral AlkBs are RNA-specific demethylases thus providing critical support for the biological relevance of the novel process of AlkB-mediated RNA repair.
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Dolja, Valerian V., Amit Gal-On, and Victor Gaba. Suppression of Potyvirus Infection by a Closterovirus Protein. United States Department of Agriculture, March 2002. http://dx.doi.org/10.32747/2002.7580682.bard.
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The plant virus family Polyviridae is the largest and most destructive of all plant viruses. Despite the continuous effort to develop resistant plant varieties, there is a desperate need for novel approaches conferring wide-range potyvirus resistance. Based on experiments with the tobacco etch potyvirus (TEV)-derived gene expression vector, we suggested approach for screening of the candidate resistance genes. This approach relies on insertion of the genes into a virus vector and evaluation of the phenotypes of the resulting recombinant viruses. The genes which suppress infection by the recombinant virus are selected as candidates for engineering transgenic resistance. Our analysis of the TEV variants expressing proteins of the beet yellows closterovirus (BYV) revealed that one of those, the leader proteinase (L-Pro), strongly and specifically interfered with the hybrid TEV infection. Since closterovirus L-Pro is evolutionary related to potyviral helper component-proteinase (HC-Pro), we suggested that the L-Pro interfered with HC-Pro function via a trans-dominant inhibitory effect. Based on these findings, we proposed to test two major hypotheses. First, we suggested that L-Pro-mediated suppression of potyvirus infection is a general phenomenon effective against a range of potyviruses. The second hypothesis stated that the suppression effect can be reproduced in transgenic plants expressing L-Pro, and can be utilized for generation of resistance to potyviruses. In accord with these hypotheses, we developed two original objectives of our proposal: A) to determine the range of the closterovirus-derived suppression of potyviral infection, and B) to try and utilize the L-Pro-mediated suppression for the development of transgenic resistance to potyviruses. In the first phase of the project, we have developed all major tools and technologies required for successful completion of the proposed research. These included TEV and ZYMV vectors engineered to express several closteroviral L-Pro variants, and generation of the large collection of transgenic plants. To our satisfaction, characterization of the infection phenotypes exhibited by chimeric TEV and ZYMV variants confirmed our first hypothesis. For instance, similar to TEV-L- Pro(BYV) chimera, ZYMV-L-Pro(LIYV) chimera was debilitated in its systemic spread. In contrast, ZYMV-GUS chimera (positive control) was competent in establishing vigorous systemic infection. These and other results with chimeric viruses indicated that several closteroviral proteinases inhibit long-distance movement of the potyviruses upon co-expression in infected plants. In order to complete the second objective, we have generated ~90 tobacco lines transformed with closteroviral L-Pro variants, as well as ~100 lines transformed with BYV Hsp70-homolog (Hsp70h; a negative control). The presence and expression of the trans gene in each line was initially confirmed using RT-PCR and RNA preparations isolated from plants. However, since detection of the trans gene-specific RNA can not guarantee production of the corresponding protein, we have also generated L-Pro- and Hsp70h-specific antisera using corresponding synthetic peptides. These antisera allowed us to confirm that the transgenic plant lines produced detectable, although highly variable levels of the closterovirus antigens. In a final phase of the project, we tested susceptibility of the transgenic lines to TEV infection. To this end, we determined that the minimal dilution of the TEV inoculum that is still capable of infecting 100% of nontransgenic plants was 1:20, and used 10 plants per line (in total, ~2,000 plants). Unfortunately, none of the lines exhibited statistically significant reduction in susceptibility. Although discouraging, this outcome prompted us to expand our experimental plan and conduct additional experiments. Our aim was to test if closteroviral proteinases are capable of functioning in trans. We have developed agroinfection protocol for BYV, and tested if co- expression of the L-Pro is capable of rescuing corresponding null-mutant. The clear-cut, negative results of these experiments demonstrated that L-Pro acts only in cis, thus explaining the lack of resistance in our transgenic plants. We have also characterized a collection of the L-Pro alanine- scanning mutants and found direct genetic evidence of the requirement for L-Pro in virus systemic spread. To conclude, our research supported by BARD confirmed one but not another of our original hypotheses. Moreover, it provided an important insight into functional specialization of the viral proteinases and generated set of tools and data with which we will be able to address the molecular mechanisms by which these proteins provide a variety of critical functions during virus life cycle.
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Gafni, Yedidya, Moshe Lapidot, and Vitaly Citovsky. Dual role of the TYLCV protein V2 in suppressing the host plant defense. United States Department of Agriculture, January 2013. http://dx.doi.org/10.32747/2013.7597935.bard.
Full textAbstract:
TYLCV-Is is a major tomato pathogen, causing extensive crop losses in Israel and the U.S. We have identified a TYLCV-Is protein, V2, which acts as a suppressor of RNA silencing. Intriguingly, the counter-defense function of V2 may not be limited to silencing suppression. Our recent data suggest that V2 interacts with the tomato CYP1 protease. CYP1 belongs to the family of papain-like cysteine proteases which participate in programmed cell death (PCD) involved in plant defense against pathogens. Based on these data we proposed a model for dual action of V2 in suppressing the host antiviral defense: V2 targets SGS3 for degradation and V2 inhibits CYP1 activity. To study this we proposed to tackle three specific objectives. I. Characterize the role of V2 in SGS3 proteasomal degradation ubiquitination, II. Study the effects of V2 on CYP1 maturation, enzymatic activity, and accumulation and, III. Analyze the effects of the CYP1-V2 interaction on TYLCV-Is infection. Here we describe results from our study that support our hypothesis: the involvement of the host's innate immune system—in this case, PCD—in plant defense against TYLCV-Is. Also, we use TYLCV-Is to discover the molecular pathway(s) by which this plant virus counters this defense. Towards the end of our study we discovered an interesting involvement of the C2 protein encoded by TYLCV-Is in inducing Hypersensitive Response in N. benthamianaplants which is not the case when the whole viral genome is introduced. This might lead to a better understanding of the multiple processes involved in the way TYLCV is overcoming the defense mechanisms of the host plant cell. In a parallel research supporting the main goal described, we also investigated Agrobacteriumtumefaciens-encoded F-box protein VirF. It has been proposed that VirF targets a host protein for the UPS-mediated degradation, very much the way TYLCV V2 does. In our study, we identified one such interactor, an Arabidopsistrihelix-domain transcription factor VFP3, and further show that its very close homolog VFP5 also interacted with VirF. Interestingly, interactions of VirF with either VFP3 or VFP5 did not activate the host UPS, suggesting that VirF might play other UPS-independent roles in bacterial infection. Another target for VirF is VFP4, a transcription factor that both VirF and its plant functional homolog VBF target to degradation by UPS. Using RNA-seqtranscriptome analysis we showed that VFP4 regulates numerous plant genes involved in disease response, including responses to viral and bacterial infections. Detailed analyses of some of these genes indicated their involvement in plant protection against Agrobacterium infection. Thus, Agrobacterium may facilitate its infection by utilizing the host cell UPS to destabilize transcriptional regulators of the host disease response machinery that limits the infection.
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Avni, Adi, and Gitta L. Coaker. Proteomic investigation of a tomato receptor like protein recognizing fungal pathogens. United States Department of Agriculture, January 2015. http://dx.doi.org/10.32747/2015.7600030.bard.
Full textAbstract:
Maximizing food production with minimal negative effects on the environment remains a long-term challenge for sustainable food production. Microbial pathogens cause devastating diseases, minimizing crop losses by controlling plant diseases can contribute significantly to this goal. All plants possess an innate immune system that is activated after recognition of microbial-derived molecules. The fungal protein Eix induces defense responses in tomato and tobacco. Plants recognize Eix through a leucine-rich-repeat receptor- like-protein (LRR-RLP) termed LeEix. Despite the knowledge obtained from studies on tomato, relatively little is known about signaling initiated by RLP-type immune receptors. The focus of this grant proposal is to generate a foundational understanding of how the tomato xylanase receptor LeEix2 signals to confer defense responses. LeEix2 recognition results in pattern triggered immunity (PTI). The grant has two main aims: (1) Isolate the LeEix2 protein complex in an active and resting state; (2) Examine the biological function of the identified proteins in relation to LeEix2 signaling upon perception of the xylanase elicitor Eix. We used two separate approaches to isolate receptor interacting proteins. Transgenic tomato plants expressing LeEix2 fused to the GFP tag were used to identify complex components at a resting and activated state. LeEix2 complexes were purified by mass spectrometry and associated proteins identified by mass spectrometry. We identified novel proteins that interact with LeEix receptor by proteomics analysis. We identified two dynamin related proteins (DRPs), a coiled coil – nucleotide binding site leucine rich repeat (SlNRC4a) protein. In the second approach we used the split ubiquitin yeast two hybrid (Y2H) screen system to identified receptor-like protein kinase At5g24010-like (SlRLK-like) (Solyc01g094920.2.1) as an interactor of LeEIX2. We examined the role of SlNRC4a in plant immunity. Co-immunoprecipitation demonstrates that SlNRC4a is able to associate with different PRRs. Physiological assays with specific elicitors revealed that SlNRC4a generally alters PRR-mediated responses. SlNRC4a overexpression enhances defense responses while silencing SlNRC4 reduces plant immunity. We propose that SlNRC4a acts as a non-canonical positive regulator of immunity mediated by diverse PRRs. Thus, SlNRC4a could link both intracellular and extracellular immune perception. SlDRP2A localizes at the plasma membrane. Overexpression of SlDRP2A increases the sub-population of LeEIX2 inVHAa1 endosomes, and enhances LeEIX2- and FLS2-mediated defense. The effect of SlDRP2A on induction of plant immunity highlights the importance of endomembrane components and endocytosis in signal propagation during plant immune . The interaction of LeEIX2 with SlRLK-like was verified using co- immunoprecipitation and a bimolecular fluorescence complementation assay. The defence responses induced by EIX were markedly reduced when SlRLK-like was over-expressed, and mutation of slrlk-likeusing CRISPR/Cas9 increased EIX- induced ethylene production and SlACSgene expression in tomato. Co-expression of SlRLK-like with different RLPs and RLKs led to their degradation, apparently through an endoplasmic reticulum-associated degradation process. We provided new knowledge and expertise relevant to expression of specific be exploited to enhance immunity in crops enabling the development of novel environmentally friendly disease control strategies.
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Dubcovsky, Jorge, Tzion Fahima, Ann Blechl, and Phillip San Miguel. Validation of a candidate gene for increased grain protein content in wheat. United States Department of Agriculture, January 2007. http://dx.doi.org/10.32747/2007.7695857.bard.
Full textAbstract:
High Grain Protein Content (GPC) of wheat is important for improved nutritional value and industrial quality. However, selection for this trait is limited by our poor understanding of the genes involved in the accumulation of protein in the grain. A gene with a large effect on GPC was detected on the short arm of chromosome 6B in a Triticum turgidum ssp. dicoccoides accession from Israel (DIC, hereafter). During the previous BARD project we constructed a half-million clones Bacterial Artificial Chromosome (BAC) library of tetraploid wheat including the high GPC allele from DIC and mapped the GPC-B1 locus within a 0.3-cM interval. Our long-term goal is to provide a better understanding of the genes controlling grain protein content in wheat. The specific objectives of the current project were to: (1) complete the positional cloning of the GPC-B1 candidate gene; (2) characterize the allelic variation and (3) expression profile of the candidate gene; and (4) validate this gene by using a transgenic RNAi approach to reduce the GPC transcript levels. To achieve these goals we constructed a 245-kb physical map of the GPC-B1 region. Tetraploid and hexaploid wheat lines carrying this 245-kb DIC segment showed delayed senescence and increased GPC and grain micronutrients. The complete sequencing of this region revealed five genes. A high-resolution genetic map, based on approximately 9,000 gametes and new molecular markers enabled us to delimit the GPC-B1 locus to a 7.4-kb region. Complete linkage of the 7.4-kb region with earlier senescence and increase in GPC, Zn, and Fe concentrations in the grain suggested that GPC-B1 is a single gene with multiple pleiotropic effects. The annotation of this 7.4-kb region identified a single gene, encoding a NAC transcription factor, designated as NAM-B1. Allelic variation studies demonstrated that the ancestral wild wheat allele encodes a functional NAC transcription factor whereas modern wheat varieties carry a non-functional NAM-B1 allele. Quantitative PCR showed that transcript levels for the multiple NAMhomologues were low in flag leaves prior to anthesis, after which their levels increased significantly towards grain maturity. Reduction in RNA levels of the multiple NAMhomologues by RNA interference delayed senescence by over three weeks and reduced wheat grain protein, Zn, and Fe content by over 30%. In the transgenic RNAi plants, residual N, Zn and Fe in the dry leaves was significantly higher than in the control plants, confirming a more efficient nutrient remobilization in the presence of higher levels of GPC. The multiple pleiotropic effects of NAM genes suggest a central role for these genes as transcriptional regulators of multiple processes during leaf senescence, including nutrient remobilization to the developing grain. The cloning of GPC-B1 provides a direct link between the regulation of senescence and nutrient remobilization and an entry point to characterize the genes regulating these two processes. This may contribute to their more efficient manipulation in crops and translate into food with enhanced nutritional value. The characterization of the GPC-B1 gene will have a significant impact on wheat production in many regions of the world and will open the door for the identification of additional genes involved in the accumulation of protein in the grain.
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Savaldi-Goldstein, Sigal, and Todd C. Mockler. Precise Mapping of Growth Hormone Effects by Cell-Specific Gene Activation Response. United States Department of Agriculture, December 2012. http://dx.doi.org/10.32747/2012.7699849.bard.
Full textAbstract:
Plant yield largely depends on a complex interplay and feedback mechanisms of distinct hormonal pathways. Over the past decade great progress has been made in elucidating the global molecular mechanisms by which each hormone is produced and perceived. However, our knowledge of how interactions between hormonal pathways are spatially and temporally regulated remains rudimentary. For example, we have demonstrated that although the BR receptor BRI1 is widely expressed, the perception of BRs in epidermal cells is sufficient to control whole-organ growth. Supported by additional recent works, it is apparent that hormones are acting in selected cells of the plant body to regulate organ growth, and furthermore, that local cell-cell communication is an important mechanism. In this proposal our goals were to identify the global profile of translated genes in response to BR stimulation and depletion in specific tissues in Arabidopsis; determine the spatio-temporal dependency of BR response on auxin transport and signaling and construct an interactive public website that will provide an integrated analysis of the data set. Our technology incorporated cell-specific polysome isolation and sequencing using the Solexa technology. In the first aim, we generated and confirmed the specificity of novel transgenic lines expressing tagged ribosomal protein in various cell types in the Arabidopsis primary root. We next crossed these lines to lines with targeted expression of BRI1 in the bri1 background. All lines were treated with BRs for two time points. The RNA-seq of their corresponding immunopurified polysomal RNA is nearly completed and the bioinformatic analysis of the data set will be completed this year. Followed, we will construct an interactive public website (our third aim). In the second aim we started revealing how spatio-temporalBR activity impinges on auxin transport in the Arabidopsis primary root. We discovered the unexpected role of BRs in controlling the expression of specific auxin efflux carriers, post-transcriptionally (Hacham et al, 2012). We also showed that this regulation depends on the specific expression of BRI1 in the epidermis. This complex and long term effect of BRs on auxin transport led us to focus on high resolution analysis of the BR signaling per se. Taking together, our ongoing collaboration and synergistic expertise (hormone action and plant development (IL) and whole-genome scale data analysis (US)) enabled the establishment of a powerful system that will tell us how distinct cell types respond to local and systemic BR signal. BR research is of special agriculture importance since BR application and BR genetic modification have been shown to significantly increase crop yield and to play an important role in plant thermotolerance. Hence, our integrated dataset is valuable for improving crop traits without unwanted impairment of unrelated pathways, for example, establishing semi-dwarf stature to allow increased yield in high planting density, inducing erect leaves for better light capture and consequent biomass increase and plant resistance to abiotic stresses.