Gotowe bibliografie tematyczne / Cellular RNAs

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Gotowa bibliografia na temat „Cellular RNAs”

Autor: Grafiati

Data publikacji: 6 września 2023

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Artykuły w czasopismach na temat "Cellular RNAs"

Hema, M., K. Gopinath i C. Kao. "Repair of the tRNA-Like CCA Sequence in a Multipartite Positive-Strand RNA Virus". Journal of Virology 79, nr 3 (1.02.2005): 1417–27. http://dx.doi.org/10.1128/jvi.79.3.1417-1427.2005.

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ABSTRACT The 3′ portions of plus-strand brome mosaic virus (BMV) RNAs mimic cellular tRNAs. Nucleotide substitutions or deletions in the 3′ CCA of the tRNA-like sequence (TLS) affect minus-strand initiation unless repaired. We observed that 2-nucleotide deletions involving the CCA 3′ sequence in one or all BMV RNAs still allowed RNA accumulation in barley protoplasts at significant levels. Alterations of CCA to GGA in only BMV RNA3 also allowed RNA accumulation at wild-type levels. However, substitutions in all three BMV RNAs severely reduced RNA accumulation, demonstrating that substitutions have different repair requirements than do small deletions. Furthermore, wild-type BMV RNA1 was required for the repair and replication of RNAs with nucleotide substitutions. Results from sequencing of progeny viral RNA from mutant input RNAs demonstrated that RNA1 did not contribute its sequence to the mutant RNAs. Instead, the repaired ends were heterogeneous, with one-third having a restored CCA and others having sequences with the only commonality being the restoration of one cytidylate. The role of BMV RNA1 in increased repair was examined.

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Pak, Julia, i Andrew Fire. "Distinct Populations of Primary and Secondary Effectors During RNAi in C. elegans". Science 315, nr 5809 (23.11.2006): 241–44. http://dx.doi.org/10.1126/science.1132839.

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RNA interference (RNAi) is a phylogenetically widespread gene-silencing process triggered by double-stranded RNA. In plants and Caenorhabditis elegans, two distinct populations of small RNAs have been proposed to participate in RNAi: “Primary siRNAs” (derived from DICER nuclease-mediated cleavage of the original trigger) and “secondary siRNAs” [additional small RNAs whose synthesis requires an RNA-directed RNA polymerase (RdRP)]. Analyzing small RNAs associated with ongoing RNAi in C. elegans, we found that secondary siRNAs constitute the vast majority. The bulk of secondary siRNAs exhibited structure and sequence indicative of a biosynthetic mode whereby each molecule derives from an independent de novo initiation by RdRP. Analysis of endogenous small RNAs indicated that a fraction derive from a biosynthetic mechanism that is similar to that of secondary siRNAs formed during RNAi, suggesting that small antisense transcripts derived from cellular messenger RNAs by RdRP activity may have key roles in cellular regulation.

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Macias, Sara, Ross A. Cordiner i Javier F. Cáceres. "Cellular functions of the microprocessor". Biochemical Society Transactions 41, nr 4 (18.07.2013): 838–43. http://dx.doi.org/10.1042/bst20130011.

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The microprocessor is a complex comprising the RNase III enzyme Drosha and the double-stranded RNA-binding protein DGCR8 (DiGeorge syndrome critical region 8 gene) that catalyses the nuclear step of miRNA (microRNA) biogenesis. DGCR8 recognizes the RNA substrate, whereas Drosha functions as an endonuclease. Recent global analyses of microprocessor and Dicer proteins have suggested novel functions for these components independent of their role in miRNA biogenesis. A HITS-CLIP (high-throughput sequencing of RNA isolated by cross-linking immunoprecipitation) experiment designed to identify novel substrates of the microprocessor revealed that this complex binds and regulates a large variety of cellular RNAs. The microprocessor-mediated cleavage of several classes of RNAs not only regulates transcript levels, but also modulates alternative splicing events, independently of miRNA function. Importantly, DGCR8 can also associate with other nucleases, suggesting the existence of alternative DGCR8 complexes that may regulate the fate of a subset of cellular RNAs. The aim of the present review is to provide an overview of the diverse functional roles of the microprocessor.

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Jiang, Di. "Cellular RNAs guide CRISPR-Cas9". Science 372, nr 6545 (27.05.2021): 929.10–929. http://dx.doi.org/10.1126/science.372.6545.929-j.

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Hopper, Anita K. "Cellular Dynamics of Small RNAs". Critical Reviews in Biochemistry and Molecular Biology 41, nr 1 (styczeń 2006): 3–19. http://dx.doi.org/10.1080/10409230500405237.

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Cooper, Daphne A., Shuvojit Banerjee, Arindam Chakrabarti, Adolfo García-Sastre, Jay R. Hesselberth, Robert H. Silverman i David J. Barton. "RNase L Targets Distinct Sites in Influenza A Virus RNAs". Journal of Virology 89, nr 5 (24.12.2014): 2764–76. http://dx.doi.org/10.1128/jvi.02953-14.

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ABSTRACTInfluenza A virus (IAV) infections are influenced by type 1 interferon-mediated antiviral defenses and by viral countermeasures to these defenses. When IAV NS1 protein is disabled, RNase L restricts virus replication; however, the RNAs targeted for cleavage by RNase L under these conditions have not been defined. In this study, we used deep-sequencing methods to identify RNase L cleavage sites within host and viral RNAs from IAV PR8ΔNS1-infected A549 cells. Short hairpin RNA knockdown of RNase L allowed us to distinguish between RNase L-dependent and RNase L-independent cleavage sites. RNase L-dependent cleavage sites were evident at discrete locations in IAV RNA segments (both positive and negative strands). Cleavage in PB2, PB1, and PA genomic RNAs suggests that viral RNPs are susceptible to cleavage by RNase L. Prominent amounts of cleavage mapped to specific regions within IAV RNAs, including some areas of increased synonymous-site conservation. Among cellular RNAs, RNase L-dependent cleavage was most frequent at precise locations in rRNAs. Our data show that RNase L targets specific sites in both host and viral RNAs to restrict influenza virus replication when NS1 protein is disabled.IMPORTANCERNase L is a critical component of interferon-regulated and double-stranded-RNA-activated antiviral host responses. We sought to determine how RNase L exerts its antiviral activity during influenza virus infection. We enhanced the antiviral activity of RNase L by disabling a viral protein, NS1, that inhibits the activation of RNase L. Then, using deep-sequencing methods, we identified the host and viral RNAs targeted by RNase L. We found that RNase L cleaved viral RNAs and rRNAs at very precise locations. The direct cleavage of IAV RNAs by RNase L highlights an intimate battle between viral RNAs and an antiviral endonuclease.

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Riddihough, Guy. "RNA editing helps identify cellular RNAs". Science Signaling 8, nr 393 (8.09.2015): ec260-ec260. http://dx.doi.org/10.1126/scisignal.aad3741.

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Yao, Run-Wen, Yang Wang i Ling-Ling Chen. "Cellular functions of long noncoding RNAs". Nature Cell Biology 21, nr 5 (maj 2019): 542–51. http://dx.doi.org/10.1038/s41556-019-0311-8.

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Riddihough, G. "RNA editing helps identify cellular RNAs". Science 349, nr 6252 (3.09.2015): 1066–68. http://dx.doi.org/10.1126/science.349.6252.1066-q.

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Xu, Ning, Bo Segerman, Xiaofu Zhou i Göran Akusjärvi. "Adenovirus Virus-Associated RNAII-Derived Small RNAs Are Efficiently Incorporated into the RNA-Induced Silencing Complex and Associate with Polyribosomes". Journal of Virology 81, nr 19 (25.07.2007): 10540–49. http://dx.doi.org/10.1128/jvi.00885-07.

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ABSTRACT Adenovirus type 5 encodes two highly structured short RNAs, the virus-associated (VA) RNAI and RNAII. Both are processed by Dicer into small RNAs that are incorporated into the RNA-induced silencing complex (RISC). We show here, by cloning of small RNAs, that approximately 80% of Ago2-containing RISC immunopurified from late-infected cells is associated with VA RNA-derived small RNAs (mivaRNAs). Most surprisingly, VA RNAII, which is expressed at 20-fold lower levels compared to that of VA RNAI, appears to be the preferred substrate for Dicer and accounts for approximately 60% of all small RNAs in RISC. The mivaRNAs are derived from the 3′ strand of the terminal stems of the VA RNAs, with the major fraction of VA RNAII starting at position 138. The small RNAs derived from VA RNAI were more heterogeneous in size, with the two predominant small RNAs starting at positions 137 and 138. Collectively, our results suggest that the mivaRNAs are efficiently used for RISC assembly in late-infected cells. Potentially, they function as miRNAs, regulating translation of cellular mRNAs. In support of this hypothesis, we detected a fraction of the VA RNAII-derived mivaRNAs on polyribosomes.

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Rozprawy doktorskie na temat "Cellular RNAs"

Smith, Nikki. "The role of cellular micro-RNAs in Epstein-Barr virus induced cellular transformation and oncogenesis". Thesis, University of Birmingham, 2011. http://etheses.bham.ac.uk//id/eprint/1344/.

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Micro-RNAs (miRNAs) are a class of non-coding RNA which post-transcriptionally regulate gene expression. Epstein-Barr Virus (EBV) transforms resting B-cells in vitro to establish continuously proliferating lymphoblastoid cell lines (LCLs) and is aetiologically linked to lymphomas. Little is known about the contribution of miRNAs to the transformation of B cells. We initially examined the regulation of the oncogenic miR-155, which is highly expressed in Hodgkin’s lymphoma but was reportedly absent in Burkitt’s lymphoma. We found that miR-155 was up-regulated by EBV-LMP1 expression, and that a reported defect of miR-155 processing in Burkitt’s lymphoma was a misinterpretation of data. Next, to identify cellular miRNAs and genes modulated during EBV-induced transformation, we compared the expression profiles of resting B cells and B cells either infected with EBV or stimulated to proliferate with CD40L and IL4. This revealed that a large proportion of miRNAs and genes differentially regulated by EBV and not by CD40L/IL4 were modulated by EBV interaction with its CD21 receptor complex, but these changes were maintained or amplified in LCLs; and included a set of tumour suppressor genes down-regulated by EBV. In addition, bioinformatics analysis indicated that EBV modulates the expression of multiple miRNAs predicted to target the same cellular genes.

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Kheimar, Ahmed Mahmoud Osman [Verfasser]. "Tumor promoting functions of cellular telomerase RNA and viral RNAs in herpesvirus-induced cancer formation / Ahmed Mahmoud Osman Kheimar". Berlin : Freie Universität Berlin, 2017. http://d-nb.info/1147758247/34.

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Hewitt, Rachael Nicole. "Investigation of the role of long non-coding RNAs in oncogene induced cellular senescence". Thesis, University of Glasgow, 2015. http://theses.gla.ac.uk/7143/.

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Cellular senescence is a stable arrest of cell proliferation induced by several factors such as activated oncogenes, oxidative stress and shortening of telomeres. Senescence acts as a tumour suppression mechanism to halt the progression of cancer. However, senescence may also impact negatively upon tissue regeneration, thus contributing to the effects of ageing. The eukaryotic genome is controlled by various modes of transcriptional and translational regulation. Focus has therefore centred on the role of long non- coding RNAs (lncRNAs) in regulating the genome. Accordingly, understanding how lncRNAs function to regulate the senescent genome is integral to improving our knowledge and understanding of tumour suppression and ageing. Within this study, I set out to investigate the expression of lncRNAs’ expression within models of senescence. Through a custom expression array, I have shown that expression of multiple different lncRNAs is up-regulated and down regulated in IMR90 replicative senescent fibroblasts and oncogene-induced senescent melanocytes. LncRNA expression was determined to be specific to stable senescence-associated cell arrest and predominantly within the nucleus of senescent cells. In order to examine the function of lncRNA expression in senescence, I selected lncRNA transcript ENST0000430998 (lncRNA_98) to focus my investigations upon. LncRNA_98 was robustly upregulated within multiple models of senescence and efficiently depleted using anti-sense oligonucleotide technology. Characterisation and unbiased RNA-sequencing of lncRNA_98 deficient senescent cells highlighted a list of genes that are regulated by lncRNA_98 expression in senescent cells and may regulate aspects of the senescence program. Specifically, the formation of SAHF was impeded upon depletion of lncRNA_98 expression and levels of total pRB protein expression severely decreased. Validation and recapitulation of consequences of pRB depletion was confirmed through lncRNA_98 knock-out cells generated using CRISPR technology. Surprisingly, inhibition of ATM kinase functions permitted the restoration of pRB protein levels within lncRNA_98 deficient cells. I propose that lncRNA_98 antagonizes the ability of ATM kinase to downregulate pRB expression at a post-transcriptional level, thereby potentiating senescence. Furthermore, lncRNA expression was detected within fibroblasts of old individuals and visualised within senescent melanocytes in human benign nevi, a barrier to melanoma progression. Conversely, mining of 337 TCGA primary melanoma data sets highlighted that the lncRNA_98 gene and its expression was lost from a significant proportion of melanoma samples, consistent with lncRNA_98 having a tumour suppressor functions. The data presented in this study illustrates that lncRNA_98 expression has a regulatory role over pRB expression in senescence and may regulate aspects of tumourigenesis and ageing.

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Jeon, Young-Jun. "The Roles of Non-Coding RNAs in Solid Tumors". The Ohio State University, 2015. http://rave.ohiolink.edu/etdc/view?acc_num=osu1426083854.

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Geisler, Sarah J. "Decapping of Long Noncoding RNAs Regulates Inducible Genes". Case Western Reserve University School of Graduate Studies / OhioLINK, 2012. http://rave.ohiolink.edu/etdc/view?acc_num=case1340141951.

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Wagner, Simone [Verfasser], i Bettina [Akademischer Betreuer] Kempkes. "Identification of cellular long non-coding RNAs regulated by the EBV nuclear antigen EBNA2 / Simone Wagner ; Betreuer: Bettina Kempkes". München : Universitätsbibliothek der Ludwig-Maximilians-Universität, 2019. http://d-nb.info/1199265721/34.

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Rogowski, Karol Jerzy [Verfasser]. "Identification and characterization of non-coding RNAs and their associated proteins involved in cellular stress responses / Karol Jerzy Rogowski". Berlin : Freie Universität Berlin, 2020. http://d-nb.info/1215099606/34.

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Balaratnam, Sumirtha. "BIOGENESIS AND FUNCTIONAL APPLICATIONS OF PIWI INTERACTING RNAs (piRNAs)". Kent State University / OhioLINK, 2018. http://rave.ohiolink.edu/etdc/view?acc_num=kent1531753741509242.

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Ostler, Jeffery Brent Jr. "Characterization of Pol IV and Pol V-Dependent Non-Coding RNAs Derived from aGeminivirus Genome". The Ohio State University, 2017. http://rave.ohiolink.edu/etdc/view?acc_num=osu1492698361649423.

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Navaroli, Deanna M. "Molecular Mechanisms of Endocytosis: Trafficking and Functional Requirements for the Transferrin Receptor, Small Interfering RNAs and Dopamine Transporter: A Dissertation". eScholarship@UMMS, 2004. http://escholarship.umassmed.edu/gsbs_diss/592.

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Endocytosis is an essential function of eukaryotic cells, providing crucial nutrients and playing key roles in interactions of the plasma membrane with the environment. The classical view of the endocytic pathway, where vesicles from the plasma membrane fuse with a homogenous population of early endosomes from which cargo is sorted, has recently been challenged by the finding of multiple subpopulations of endosomes. These subpopulations vary in their content of phosphatidylinositol 3- phosphate (PI3P) and Rab binding proteins. The role of these endosomal subpopulations is unclear, as is the role of multiple PI3P effectors, which are ubiquitously expressed and highly conserved. One possibility is that the different subpopulations represent stages in the maturation of the endocytic pathway. Alternatively, endosome subpopulations may be specialized for different functions, such as preferential trafficking of specific endocytosed cargo. To determine whether specific receptors are targeted to distinct populations of endosomes, we have built a platform for total internal reflection fluorescence (TIRF) microscopy coupled with structured illumination capabilities named TESM (TIRF Epifluorescence Structured light Microscope.) In this study, TESM, along with standard biochemical and molecular biological tools, was used to analyze the dynamic distribution of two highly conserved Rab5 and PI3P effectors, EEA1 and Rabenosyn-5, and systematically study the trafficking of transferrin. Rabenosyn-5 is necessary for proper expression of the transferrin receptor as well as internalization and recycling of transferrin-transferrin receptor complexes. Results of combining TIRF with structured light Epifluorescence (SLE) indicate that the endogenous populations of EEA1 and Rabenoysn-5 are both distinct and partially overlapping. The application of antisense oligonucleotides as potential therapeutic agents requires effective methods for their delivery to the cytoplasm of target cells. In collaboration with RXi Pharmaceuticals we show the efficient cellular uptake of the antisense oligonucleotide sd-rxRNA® in the absence of delivery vehicle or protein carrier. In this study TIRF, SLE, and biochemical approaches were utilized to determine whether sd-rxRNA traffics and functions along specific endosomal pathways. Sd-rxRNA was found to traffic along the degradative pathway and require EEA1 to functionally silence its target. These new findings will help define the cellular pathways involved in RNA silencing. Neurotransmitter reuptake and reuse by neurotransmitter transport proteins is fundamental to transmitter homeostasis and synaptic signaling. In order to understand how trafficking regulates transporters in the brain and how this system may be disregulated in monoamine-related pathologies, the transporter internalization signals and their molecular partners must be defined. We utilized a yeast two-hybrid system to identify proteins that interact with the dopamine transporter (DAT) endocytic signal. The small, membrane associated, GTPase Rin was determined to specifically and functionally interact with the DAT endocytic signal, regulating constitutive and protein kinase C (PKC) – stimulated DAT endocytosis. The results presented in this study provide new insights into functions and components of endocytosis and enhance the understanding of endocytic organization.

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Książki na temat "Cellular RNAs"

Mallick, Bibekanand. Regulatory RNAs: Basics, Methods and Applications. Berlin, Heidelberg: Springer Berlin Heidelberg, 2012.

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Pitre, Liisa K. The application of RNA interference to cellular biotechnology. Sudbury, Ont: Laurentian University, 2003.

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Brasier, Allan R., Adolfo García-Sastre i Stanley M. Lemon, red. Cellular Signaling and Innate Immune Responses to RNA Virus Infections. Washington, DC, USA: ASM Press, 2008. http://dx.doi.org/10.1128/9781555815561.

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R, Brasier Allan, García-Sastre Adolfo i Lemon Stanley M, red. Cellular signaling and innate immune responses to RNA virus infections. Washington, D.C: ASM Press, 2009.

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Kekkonen, Viktoria. Characterization of bacterial RNA and DNA signalling pathways that induce cellular dysfunction. Sudbury, Ont: Laurentian University, 2006.

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Post-transcriptional regulation by STAR proteins : control of RNA metabolism in development and disease. New York: Springer Science+Business Media, LLC, 2010.

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Yechiel, Becker, i Darai Gholamreza, red. Molecular evolution of viruses-past and present: Evolution of viruses by acquisition of cellular RNA and DNA. Boston: Kluwer Academic Publishers, 2000.

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Wingender, Edgar. Gene regulation in eukaryotes. Weinheim: VCH, 1993.

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Jeffrey, Wilusz, red. Post-transcriptional gene regulation. Totowa, N.J: Humana Press, 2008.

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Farrell, Robert E. RNA methodologies: A laboratory guide for isolation and characterization. San Diego: Academic Press, 1993.

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Części książek na temat "Cellular RNAs"

Angerer, R. C., K. H. Cox i L. M. Angerer. "In Situ Hybridization to Cellular RNAs". W Genetic Engineering: Principles and Methods, 43–65. Boston, MA: Springer US, 1985. http://dx.doi.org/10.1007/978-1-4684-4973-0_3.

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Dhahbi, Joseph M. "Small Noncoding RNAs in Senescence and Aging". W Cellular Ageing and Replicative Senescence, 287–312. Cham: Springer International Publishing, 2016. http://dx.doi.org/10.1007/978-3-319-26239-0_15.

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Chorostecki, Uciel, Jesse R. Willis, Ester Saus i Toni Gabaldon. "Profiling of RNA Structure at Single-Nucleotide Resolution Using nextPARS". W Methods in Molecular Biology, 51–62. New York, NY: Springer US, 2021. http://dx.doi.org/10.1007/978-1-0716-1307-8_4.

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AbstractRNA molecules play important roles in almost every cellular process, and their functions are mediated by their sequence and structure. Determining the secondary structure of RNAs is central to understanding RNA function and evolution. RNA structure probing techniques coupled to high-throughput sequencing allow determining structural features of RNA molecules at transcriptome-wide scales. Our group recently developed a novel Illumina-based implementation of in vitro parallel probing of RNA structures called nextPARS.Here, we describe a protocol for the computation of the nextPARS scores and their use to obtain the structural profile (single- or double-stranded state) of an RNA sequence at single-nucleotide resolution.

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Páldi, Katalin, Magdalena Mosiolek i Michael D. Nodine. "Small RNA In Situ Hybridizations on Sections of Arabidopsis Embryos". W Methods in Molecular Biology, 87–99. New York, NY: Springer US, 2020. http://dx.doi.org/10.1007/978-1-0716-0342-0_7.

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AbstractSmall RNAs mediate posttranscriptional gene silencing in plants and animals. This often occurs in specific cell or tissue types and can be necessary for their differentiation. Determining small RNA (sRNA) localization patterns at cellular resolution can therefore provide information on the corresponding gene regulatory processes they are involved in. Recent improvements with in situ hybridization methods have allowed them to be applied to sRNAs. Here we describe an in situ hybridization protocol to detect sRNAs from sections of early staged Arabidopsis thaliana (Arabidopsis) embryos.

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Sharma, Sunny, i Karl-Dieter Entian. "Chemical Modifications of Ribosomal RNA". W Ribosome Biogenesis, 149–66. New York, NY: Springer US, 2022. http://dx.doi.org/10.1007/978-1-0716-2501-9_9.

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AbstractCellular RNAs in all three kingdoms of life are modified with diverse chemical modifications. These chemical modifications expand the topological repertoire of RNAs, and fine-tune their functions. Ribosomal RNA in yeast contains more than 100 chemically modified residues in the functionally crucial and evolutionary conserved regions. The chemical modifications in the rRNA are of three types—methylation of the ribose sugars at the C2-positionAbstract (Nm), isomerization of uridines to pseudouridines (Ψ), and base modifications such as (methylation (mN), acetylation (acN), and aminocarboxypropylation (acpN)). The modifications profile of the yeast rRNA has been recently completed, providing an excellent platform to analyze the function of these modifications in RNA metabolism and in cellular physiology. Remarkably, majority of the rRNA modifications and the enzymatic machineries discovered in yeast are highly conserved in eukaryotes including humans. Mutations in factors involved in rRNA modification are linked to several rare severe human diseases (e.g., X-linked Dyskeratosis congenita, the Bowen–Conradi syndrome and the William–Beuren disease). In this chapter, we summarize all rRNA modifications and the corresponding enzymatic machineries of the budding yeast.

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Maréchal-Drouard, Laurence, Ian Small, Anne Cosset, Anne-Marie Duchêne, Raman Kumar, Hakim Mireau, Nemo Peeters i in. "Import and editing of plant mitochondrial transfer RNAs". W Cellular Integration of Signalling Pathways in Plant Development, 113–24. Berlin, Heidelberg: Springer Berlin Heidelberg, 1998. http://dx.doi.org/10.1007/978-3-642-72117-5_11.

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Yang, Jun, Peter Watzinger i Sunny Sharma. "Mapping of the Chemical Modifications of rRNAs". W Ribosome Biogenesis, 181–97. New York, NY: Springer US, 2022. http://dx.doi.org/10.1007/978-1-0716-2501-9_11.

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AbstractCellular RNAs, both coding and noncoding, contain several chemical modifications. Both ribose sugars and nitrogenous bases are targeted for these chemical additions. These modifications are believed to expand the topological potential of RNA molecules by bringing chemical diversity to otherwise limited repertoire. Here, using ribosomal RNA of yeast as an example, a detailed protocol for systematically mapping various chemical modifications to a single nucleotide resolution by a combination of Mung bean nuclease protection assay and RP-HPLC is provided. Molar levels are also calculated for each modification using their UV (254 nm) molar response factors that can be used for determining the amount of modifications at different residues in other RNA molecules. The chemical nature, their precise location and quantification of modifications will facilitate understanding the precise role of these chemical modifications in cellular physiology.

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Panigaj, Martin, Marina A. Dobrovolskaia i Kirill A. Afonin. "Viral Noncoding RNAs in Modulating Cellular Defense and Their Potential for RNA Nanotechnology". W RNA Nanotechnology and Therapeutics, 239–50. Wyd. 2. Boca Raton: CRC Press, 2022. http://dx.doi.org/10.1201/9781003001560-30.

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Agris, Paul F., Andra Boak, Joseph W. Basler, Catherine Van Voorn, Christine Smith i Morris Reichlin. "Analysis of Cellular Senescence through Detection and Assessment of RNAs and Proteins Important to Gene Expression: Transfer RNAs and Autoimmune Antigens". W Advances in Experimental Medicine and Biology, 509–39. Boston, MA: Springer US, 1985. http://dx.doi.org/10.1007/978-1-4684-7853-2_26.

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Dadafarin, Sina, Michelle Carnazza, Humayun K. Islam, Augustine Moscatello, Raj K. Tiwari i Jan Geliebter. "Noncoding RNAs in Papillary Thyroid Cancer: Interaction with Cancer-Associated Fibroblasts (CAFs) in the Tumor Microenvironment (TME) and Regulators of Differentiation and Lymph Node Metastasis". W Tumor Microenvironment: Cellular, Metabolic and Immunologic Interactions, 145–55. Cham: Springer International Publishing, 2021. http://dx.doi.org/10.1007/978-3-030-83282-7_7.

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Streszczenia konferencji na temat "Cellular RNAs"

Steitz, Joan A., Nara Lee, Walter N. Moss, Therese A. Yario, Anna Vilborg, Maria C. Passarelli i Kazimierz T. Tycowski. "Abstract IA14: Noncoding RNAs of viral and cellular origin: Links to oncogenesis". W Abstracts: AACR Special Conference on Noncoding RNAs and Cancer: Mechanisms to Medicines; December 4-7, 2015; Boston, MA. American Association for Cancer Research, 2016. http://dx.doi.org/10.1158/1538-7445.nonrna15-ia14.

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Devulder, J., J. Baker, L. Donnelly i P. J. Barnes. "Extracellular Vesicles Produced by Bronchial Epithelial Cells in Response to Oxidative Stress Contain Micro-RNAs Associated with Cellular Senescence". W American Thoracic Society 2020 International Conference, May 15-20, 2020 - Philadelphia, PA. American Thoracic Society, 2020. http://dx.doi.org/10.1164/ajrccm-conference.2020.201.1_meetingabstracts.a4081.

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Manriquez, Rogelio Montiel. "Abstract 3574: Identification of long non-coding RNAs involved in chromosomal instability in a cellular model of prostate cancer". W Proceedings: AACR Annual Meeting 2019; March 29-April 3, 2019; Atlanta, GA. American Association for Cancer Research, 2019. http://dx.doi.org/10.1158/1538-7445.am2019-3574.

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Kornev, A. A., V. V. Vysochinskaya, N. A. Knyazev, A. K. Emel'yanov i A. A. Bogdanov. "Transfection of human peripheral blood T-lymphocytes with synthetic small interfering RNAs: selection of an effective technique". W Global science. Development and novelty. L-Journal, 2020. http://dx.doi.org/10.18411/gsdn-25-12-2020-04.

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It has been shown that the state of the human immune system and its activity determines the development, course and prognosis of many oncological diseases. To date, various immunotherapeutic approaches have been developed, of which the most promising is adoptive cell therapy (ACT). An increase in the effectiveness of this type of therapy could be achieved by using synthetic small interfering RNAs (siRNAs) to suppress the expression of key immune checkpoint (ICI) genes (PD1, CTL4) in T-lymphocytes, which are involved in cellular immunosuppression. However, there is a problem of selecting an effective method for delivering such siRNAs to T-lymphocytes. In the present study, we developed a synthetic siRNA specific to the mRNA sequence of the PD1 gene and evaluated the efficiency of its delivery to activated human T-lymphocytes using chemically modified siRNA, histone H1, – 18 – Global science. Development and novelty and the liposomal agent HiperFect. Ex vivo activated T-lymphocytes from healthy donors were used. The efficiency of siRNA transfection into cells and its confirmation was assessed using flow cytofluorometry and confocal microscopy. As a result of the study, a high (51%) efficiency of transfection into these cells using chemically modified "self-delivering" siRNA was shown. For other methods of delivery of miRNA to T-lymphocytes, low efficiency is shown. Thus, our data suggest that of the three approaches used in this work for miRNAs delivery to activated T-lymphocytes, the most effective is the use of “self-delivering” chemically modified cholesterol miRNA.

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Adamek, Maksimiljan. "Molecular Grammar of RNA-binding Protein Interactions in Formation and Function of Ribonucleoprotein Complexes". W Socratic Lectures 8. University of Lubljana Press, 2023. http://dx.doi.org/10.55295/psl.2023.ii15.

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Ribonucleoproteins (RNPs) are macromolecular assemblies of proteins along RNA molecules to carry out specialized cellular processes. Understanding how RNA binding proteins (RBPs) and RNA sequences determine the interactions to form RNPs and ultimately steer biomolecular processes remains poorly understood. There is a mounting evidence that RNP assembly de-pends on the formation of a network of transient, multivalent RBP RNA and RBP RBP interac-tions, particularly between tyrosine residues from intrinsically disordered domains and argi-nine residues from RNA-binding domains of RBPs. Furthermore, RBPs, especially their intrin-sically disordered regions, are hotspots for posttranslational modification (PTM) sites. Alt-hough PTMs have been well catalogued, little is known about how these modifications regulate RNP assembly and function. Some initial studies introduced the concept of the so-called phos-pho-switch, in which RBPs require phosphorylation for condensation of larger RNP complexes, but it remains unclear how this contributes to the protein function and the pattern of selective protein binding to RNA molecules. This short review will take a look at what is currently known in the field of RNPs, their interactions, and the phase-separated biomolecular conden-sates, which are intimately connected to RNPs and are important for several key cell processes. Keywords: Ribonucleoproteins; RNA binding proteins; Multivalency; Intrinsically disordered proteins; Posttranslational modifications

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Chatterjee, Sudipta, Nitai P. Bhattacharyya, Dipanjan Bhattacharjee, Sanchaita Misra i Alakendu Ghosh. "SAT0638 INCREASED LEVELS OF CIRCULATING EXTRA CELLULAR LONG NON-CODING RNAS MALAT1, MEG3 AND NEAT1 IN PATIENTS WITH RHEUMATOID ARTHRITIS AND THEIR IMPACT ON DISEASE ACTIVITY MEASURES". W Annual European Congress of Rheumatology, EULAR 2019, Madrid, 12–15 June 2019. BMJ Publishing Group Ltd and European League Against Rheumatism, 2019. http://dx.doi.org/10.1136/annrheumdis-2019-eular.6797.

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Chen, Cheng, Houqiang Li, Xiaobo Zhou i Stephen Wong. "GRAPH CUT BASED ACTIVE CONTOUR FOR AUTOMATED CELLULAR IMAGE SEGMENTATION IN HIGH THROUGHPUT RNA INTERFACE (RNAi) SCREENING". W 2007 4th IEEE International Symposium on Biomedical Imaging: From Nano to Macro. IEEE, 2007. http://dx.doi.org/10.1109/isbi.2007.356790.

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Shikata, Tetsuo, Toshihiko Shiraishi, Kumiko Tanaka, Shin Morishita i Ryohei Takeuchi. "Effects of Amplitude and Frequency of Vibration Stimulation on Cultured Osteoblasts". W ASME 2007 International Design Engineering Technical Conferences and Computers and Information in Engineering Conference. ASMEDC, 2007. http://dx.doi.org/10.1115/detc2007-34949.

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Mechanical stimulation to bones affects osteogenesis such as decrease of bone mass of astronauts under zero gravity, walking rehabilitation to bone fracture and fracture repair with ultrasound devices. Bone cells have been reported to sense and response to mechanical stimulation at cellular level morphologically and metabolically. In the view of mechanical vibrations, bone cells are deformed according to mechanical stimulation and their mechanical characteristics. Recently, it was reported that viscoelasticity of cells was measured using tensile and creep tests and that there was likely natural frequency and nonlinearity of cells in the sense of structural dynamics. It suggests that there is effective frequency and amplitude of mechanical stimulation on osteogenesis by bone cells. In this study, sinusoidal inertia force was applied to cultured osteoblasts, MC3T3-E1, and effects of frequency and acceleration amplitude of mechanical vibration on the cells were investigated in respect of cell proliferation, cell morphology, bone matrix generation and alkaline phosphatase (ALP) gene expression. After the cells were cultured in culture plates in a CO2 incubator for one day and adhered on the cultured plane, vibrating groups of the culture plates were set on an aluminum plate attached to a exciter and cultured under sinusoidal excitation in another incubator separated from non-vibrating groups of the culture plates. Acceleration amplitude and frequency were set to several kinds of conditions. The time evolution of cell density was obtained by counting the number of cells with a hemocytometer. The cell morphology was observed with a phase contrast microscope. Calcium salts generated by the cells were observed by being stained with alizarin red S solution and their images were captured with a CCD camera. The vibrating groups for the cell proliferation and the calcium salts staining were sinusoidally excited for 24 hours a day during 28-day cultivation. Gene expression of ALP was measured by a real-time RT-PCR method. After the vibrating groups for the PCR were excited for 6 hours, the total RNAs were extracted. After reverse transcription, real-time RT-PCR was performed. Gene expression for ALP and a housekeeping gene were determined simultaneously for each sample. Gene levels in each sample were normalized to the measured housekeeping gene levels. As a result, it is shown that saturate cell density becomes high and bone matrix generation is promoted by applying mechanical vibration and that there may be some peaks to frequency and a certain threshold value to acceleration amplitude of mechanical vibration for saturation cell density and bone matrix generation.

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Holmes, Ian. "Cellular Automata Simulation of RNA Self-Replicators". W The 2020 Conference on Artificial Life. Cambridge, MA: MIT Press, 2020. http://dx.doi.org/10.1162/isal_a_00242.

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Raporty organizacyjne na temat "Cellular RNAs"

Lers, Amnon, i Pamela J. Green. Analysis of Small RNAs Associated with Plant Senescence. United States Department of Agriculture, marzec 2013. http://dx.doi.org/10.32747/2013.7593393.bard.

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Senescence is an agriculturally significant process due to its negative impact to crop yield and postharvest quality. The genetic regulatory systems controlling senescence induction and progress respond to both developmental and environmental stress signals and involve numerous gene expression changes. Knowledge about the key molecular factors which control senescence is very limited. MicroRNAs (miRNAs) are a class of small RNAs which typically function by guiding cleavage of target messenger RNAs. They have been shown to play major roles in a variety of plant processes including development, responses to environmental stresses, and senescence. The long-term goal of this work is to elucidate roles of small RNAs associated with plant senescence. The hypothesis underlying this research is that miRNA-mediated regulation makes important contributions to the senescence process in plants. Specific, original research objectives included: 1) Profiling of small RNAs from senescing plants; 2) Data Analysis and public access via a user-friendly web interface; 3) Validation of senescence-associated miRNAs and target RNAs; 4) Development of transgenic plants for functional analysis of miRNAs in Arabidopsis. Major revisions made in the research compared to the original work plan included 1) Exclusion of the planned work with tomato as recommended by the BARD review panel; 2) Performing miRNA study also in senescing Arabidopsis siliques, in addition to senescing leaves. To identify senescenceregulation of miRNAs in Arabidopsis thaliana, eight small RNA libraries were constructed and sequenced at four different stages of development and senescence from both leaves and siliques, resulting in more than 200 million genome-matched sequences. Parallel Analysis of RNA Ends (PARE) libraries, which enable the large-scale examination of miRNA-guided cleavage products, were also constructed and sequenced, resulting in over 750 million genome-matched sequences. These massive datasets lead to the identification of new miRNAs, as well as new regulation of known miRNAs and their target genes during senescence, many of which have established roles in nutrient responsiveness and cell structural integrity. In keeping with remobilization of nutrients thought to occur during senescence, many miRNAs and targets had opposite expression pattern changes between leaf and silique tissues during the progression of senescence. Taken together, these findings highlight the integral role that miRNAs may play in the remobilization of resources and alteration of cellular structure that is known to occur in senescence. Experiments were initiated for functional analysis of specific senescence-associated miRNAs and respective target genes. Transgenic Arabidopsis plants were generated in which miR408, found in this study to be significantly induced in leaf senescence, was over-expressed either constitutively or under a senescence-specific promoter. These plants are currently being characterized for any altered phenotypes. In addition T-DNA knock out mutants for various target genes identified in this research are being analyzed. This work provides insights about specific miRNAs that contribute to leaf and silique senescence. The knowledge generated may suggest new strategies to monitor and alter the progression of senescence in crops for agricultural improvement.

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Schuster, Gadi, i David Stern. Integrated Studies of Chloroplast Ribonucleases. United States Department of Agriculture, wrzesień 2011. http://dx.doi.org/10.32747/2011.7697125.bard.

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Gene regulation at the RNA level encompasses multiple mechanisms in prokaryotes and eukaryotes, including splicing, editing, endo- and exonucleolytic cleavage, and various phenomena related to small or interfering RNAs. Ribonucleases are key players in nearly all of these post-transcriptional mechanisms, as the catalytic agents. This proposal continued BARD-funded research into ribonuclease activities in the chloroplast, where RNase mutation or deficiency can cause metabolic defects and is often associated with plant chlorosis, embryo or seedling lethality, and/or failure to tolerate nutrient stress. The first objective of this proposal was to examined a series of point mutations in the PNPase enzyme of Arabidopsis both in vivo and in vitro. This goal is related to structure-function analysis of an enzyme whose importance in many cellular processes in prokaryotes and eukaryotes has only begun to be uncovered. PNPase substrates are mostly generated by endonucleolytic cleavages for which the catalytic enzymes remain poorly described. The second objective of the proposal was to examine two candidate enzymes, RNase E and RNase J. RNase E is well-described in bacteria but its function in plants was still unknown. We hypothesized it catalyzes endonucleolytic cleavages in both RNA maturation and decay. RNase J was recently discovered in bacteria but like RNase E, its function in plants had yet to be explored. The results of this work are described in the scientific manuscripts attached to this report. We have completed the first objective of characterizing in detail TILLING mutants of PNPase Arabidopsis plants and in parallel introducing the same amino acids changes in the protein and characterize the properties of the modified proteins in vitro. This study defined the roles for both RNase PH core domains in polyadenylation, RNA 3’-end maturation and intron degradation. The results are described in the collaborative scientific manuscript (Germain et al 2011). The second part of the project aimed at the characterization of the two endoribonucleases, RNase E and RNase J, also in this case, in vivo and in vitro. Our results described the limited role of RNase E as compared to the pronounced one of RNase J in the elimination of antisense transcripts in the chloroplast (Schein et al 2008; Sharwood et al 2011). In addition, we characterized polyadenylation in the chloroplast of the green alga Chlamydomonas reinhardtii, and in Arabidopsis (Zimmer et al 2009). Our long term collaboration enabling in vivo and in vitro analysis, capturing the expertise of the two collaborating laboratories, has resulted in a biologically significant correlation of biochemical and in planta results for conserved and indispensable ribonucleases. These new insights into chloroplast gene regulation will ultimately support plant improvement for agriculture.

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Sun, Lina, Yanan Han, Hua Wang, Huanyu Liu, Shan Liu, Hongbin Yang, Xiaoxia Ren i Ying Fang. MicroRNAs as Potential Biomarkers for the Diagnosis of Inflammatory Bowel Disease: A Systematic Review and Meta-analysis. INPLASY - International Platform of Registered Systematic Review and Meta-analysis Protocols, luty 2022. http://dx.doi.org/10.37766/inplasy2022.2.0027.

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Review question / Objective: The purpose of this systematic review was to systematically review the clinical studies regarding miRNAs as diagnostic biomarkers for inflammatory bowel disease and assess the overall diagnostic accuracy of miRNAs. Condition being studied: The symptoms of inflammatory bowel disease (IBD) are highly variable. The diagnosis of IBD must be made through medical history, physical, laboratory, radiologic, endoscopic, and histological examinations. However, these diagnostic techniques are not specific and sometimes even equivocal. Therefore, reliable biomarkers are urgently needed in the diagnosis of IBD. Several clinical and preclinical researches have shown that dysregulated microRNAs (miRNAs) play a crucial role in IBD development. miRNAs, as single-stranded noncoding RNAs that contain 22-24 nucleotides, can post-transcriptionally regulate gene expression by blocking mRNA translation or degrading target mRNAs. miRNAs are widely involved in physiological and pathological cellular processes, such as differentiation, proliferation and apoptosis. Besides, they are stable, noninvasive, and resistant to degradation by ribonucleases, making them valuable targets in the diagnosis, monitoring, prognosis, and treatment of diseases. To date, inconsistent results have been found about miRNA expression profiling in the patients with IBD. Moreover, the diagnostic accuracy of miRNAs for IBD has not been reported in any meta-analysis.

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Ghanim, Murad, Joe Cicero, Judith K. Brown i Henryk Czosnek. Dissection of Whitefly-geminivirus Interactions at the Transcriptomic, Proteomic and Cellular Levels. United States Department of Agriculture, luty 2010. http://dx.doi.org/10.32747/2010.7592654.bard.

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Our project focuses on gene expression and proteomics of the whitefly Bemisia tabaci (Gennadius) species complex in relation to the internal anatomy and localization of expressed genes and virions in the whitefly vector, which poses a major constraint to vegetable and fiber production in Israel and the USA. While many biological parameters are known for begomovirus transmission, nothing is known about vector proteins involved in the specific interactions between begomoviruses and their whitefly vectors. Identifying such proteins is expected to lead to the design of novel control methods that interfere with whitefly-mediated begomovirus transmission. The project objectives were to: 1) Perform gene expression analyses using microarrays to study the response of whiteflies (B, Q and A biotypes) to the acquisition of begomoviruses (Tomato yellow leaf curl (TYLCV) and Squash leaf curl (SLCV). 2) Construct a whitefly proteome from whole whiteflies and dissected organs after begomovirus acquisition. 3) Validate gene expression by q-RTPCR and sub-cellular localization of candidate ESTs identified in microarray and proteomic analyses. 4) Verify functionality of candidate ESTs using an RNAi approach, and to link these datasets to overall functional whitefly anatomical studies. During the first and second years biological experiments with TYLCV and SLCV acquisition and transmission were completed to verify the suitable parameters for sample collection for microarray experiments. The parameters were generally found to be similar to previously published results by our groups and others. Samples from whole whiteflies and midguts of the B, A and Q biotypes that acquired TYLCV and SLCV were collected in both the US and Israel and hybridized to B. tabaci microarray. The data we analyzed, candidate genes that respond to both viruses in the three tested biotypes were identified and their expression that included quantitative real-time PCR and co-localization was verified for HSP70 by the Israeli group. In addition, experiments were undertaken to employ in situ hybridization to localize several candidate genes (in progress) using an oligonucleotide probe to the primary endosymbiont as a positive control. A proteome and corresponding transcriptome to enable more effective protein identification of adult whiteflies was constructed by the US group. Further validation of the transmission route of begomoviruses, mainly SLCV and the involvement of the digestive and salivary systems was investigated (Cicero and Brown). Due to time and budget constraints the RNAi-mediated silencing objective to verify gene function was not accomplished as anticipated. HSP70, a strong candidate protein that showed over-expression after TYLCV and SLCV acquisition and retention by B. tabaci, and co-localization with TYLCV in the midgut, was further studies. Besides this protein, our joint research resulted in the identification of many intriguing candidate genes and proteins that will be followed up by additional experiments during our future research. To identify these proteins it was necessary to increase the number and breadth of whitefly ESTs substantially and so whitefly cDNAs from various libraries made during the project were sequenced (Sanger, 454). As a result, the proteome annotation (ID) was far more successful than in the initial attempt to identify proteins using Uniprot or translated insect ESTs from public databases. The extent of homology shared by insects in different orders was surprisingly low, underscoring the imperative need for genome and transcriptome sequencing of homopteran insects. Having increased the number of EST from the original usable 5500 generated several years ago to >600,000 (this project+NCBI data mining), we have identified about one fifth of the whitefly proteome using these new resources. Also we have created a database that links all identified whitefly proteins to the PAVEdb-ESTs in the database, resulting in a useful dataset to which additional ESTS will be added. We are optimistic about the prospect of linking the proteome ID results to the transcriptome database to enable our own and other labs the opportunity to functionally annotate not only genes and proteins involved in our area of interest (whitefly mediated transmission) but for the plethora of other functionalities that will emerge from mining and functionally annotating other key genes and gene families in whitefly metabolism, development, among others. This joint grant has resulted in the identification of numerous candidate proteins involved in begomovirus transmission by B. tabaci. A next major step will be to capitalize on validated genes/proteins to develop approaches to interfere with the virus transmission.

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Lapidot, Moshe, i Vitaly Citovsky. molecular mechanism for the Tomato yellow leaf curl virus resistance at the ty-5 locus. United States Department of Agriculture, styczeń 2016. http://dx.doi.org/10.32747/2016.7604274.bard.

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Tomato yellow leaf curl virus (TYLCV) is a major pathogen of tomato that causes extensive crop loss worldwide, including the US and Israel. Genetic resistance in the host plant is considered highly effective in the defense against viral infection in the field. Thus, the best way to reduce yield losses due to TYLCV is by breeding tomatoes resistant or tolerant to the virus. To date, only six major TYLCV-resistance loci, termed Ty-1 to Ty-6, have been characterized and mapped to the tomato genome. Among tomato TYLCV-resistant lines containing these loci, we have identified a major recessive quantitative trait locus (QTL) that was mapped to chromosome 4 and designated ty-5. Recently, we identified the gene responsible for the TYLCV resistance at the ty-5 locus as the tomato homolog of the gene encoding messenger RNA surveillance factor Pelota (Pelo). A single amino acid change in the protein is responsible for the resistant phenotype. Pelo is known to participate in the ribosome-recycling phase of protein biosynthesis. Our hypothesis was that the resistant allele of Pelo is a “loss-of-function” mutant, and inhibits or slows-down ribosome recycling. This will negatively affect viral (as well as host-plant) protein synthesis, which may result in slower infection progression. Hence we have proposed the following research objectives: Aim 1: The effect of Pelota on translation of TYLCV proteins: The goal of this objective is to test the effect Pelota may or may not have upon translation of TYLCV proteins following infection of a resistant host. Aim 2: Identify and characterize Pelota cellular localization and interaction with TYLCV proteins: The goal of this objective is to characterize the cellular localization of both Pelota alleles, the TYLCV-resistant and the susceptible allele, to see whether this localization changes following TYLCV infection, and to find out which TYLCV protein interacts with Pelota. Our results demonstrate that upon TYLCV-infection the resistant allele of pelota has a negative effect on viral replication and RNA transcription. It is also shown that pelota interacts with the viral C1 protein, which is the only viral protein essential for TYLCV replication. Following subcellular localization of C1 and Pelota it was found that both protein localize to the same subcellular compartments. This research is innovative and potentially transformative because the role of Peloin plant virus resistance is novel, and understanding its mechanism will lay the foundation for designing new antiviral protection strategies that target translation of viral proteins. BARD Report - Project 4953 Page 2

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Granot, David, i Noel Michelle Holbrook. Role of Fructokinases in the Development and Function of the Vascular System. United States Department of Agriculture, styczeń 2011. http://dx.doi.org/10.32747/2011.7592125.bard.

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Plant vascular tissues are superhighways whose development and function have profound implications for productivity, yield and stress response. Preliminary studies by the PI indicated that sugar metabolism mediated by fructokinases (FRKs) has a pronounced effect on the transport properties of the xylem. The goal of this research was to determine how the main fructokinase gene, FRK2, and the only plastidic fructokinase, FRK3, influence vascular development and physiology, emphasizing processes that occur at both the cellular and organismic level. We found that both genes are expressed in vascular tissues, but FRK3 is expressed primarily in vascular tissues of mature petioles. Vascular anatomy of plants with antisense suppression of FRK2 uncovered that FRK2 is necessary for xylem and phloem development, most likely due to its role in vascular cell-wall synthesis, and affects vascular development all over the plant. As a result, suppression of FRK2 reduced hydraulic conductivity of roots, stem and leaves and restricted sugar phloem transport. Vascular anatomy of plants with RNAi suppression of FRK3 uncovered that FRK3 is required for vascular development in mature petiole but its role is partially complemented by FRK2. Suppression of FRK3 combined with partial suppression of FRK2 had effects completely different from that of FRK2 suppression, resulting in wilting of mature leaves rather than young leaves of FRK2 suppressed plants, and decreased export of photoassimilates. This primary effect of FRK2 suppression on mature petioles had a secondary effect, reducing the hydraulic conductivity in roots and stem. The very fact that a plastidic fructokinase plays a role in vascular development is quite surprising and we are still seeking to uncover its metabolic mode-of-action. Yet, it is clear that these two fructokinases have different roles in the coordination between photosynthetic capacity and vascular development. We have started analyzing the role of the last third FRK, FRK1, and discovered that it is also expressed exclusively in vascular tissues. It appears therefore, that all FRKs studied here are involved in vascular development. An interesting unexpected outcome of this study was the connection of FRK2 with hormonal regulation of vascular development, most likely auxin. This observation together with the yet to be solved questions on the exact roles of FRK3 are the subjects of our current efforts.

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Ullman, Diane, James Moyer, Benjamin Raccah, Abed Gera, Meir Klein i Jacob Cohen. Tospoviruses Infecting Bulb Crops: Evolution, Diversity, Vector Specificity and Control. United States Department of Agriculture, wrzesień 2002. http://dx.doi.org/10.32747/2002.7695847.bard.

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Objectives. The overall goal of the proposed research was to develop a mechanistic understanding of tospovirus evolution, diversity and vector specificity that could be applied to development of novel methods for limiting virus establishment and spread. Our specific objectives were: 1) To characterize newly intercepted tospoviruses in onion, Hippeastrum and other bulb crops and compare them with the known tomato spotted wilt virus (TSWV) and its isolates; 2) To characterize intra- and interspecific variation in the virus transmission by thrips of the new and distinct tospoviruses. and, 3) To determine the basis of vector specificity using biological, cellular and molecular approaches. Background. New tospoviruses infecting bulb crops were detected in Israel and the US in the mid-90s. Their plant host ranges and relationships with thrips vectors showed they differed from the type member of the Tospovirus genus, tomato spotted wilt virus (TSWV). Outbreaks of these new viruses caused serious crop losses in both countries, and in agricultural and ornamental crops elsewhere. In the realm of plant infecting viruses, the tospoviruses (genus: Tospovirus , family: Bunyaviridae ) are among the most aggressive emerging viruses. Tospoviruses are transmitted by several species of thrips in a persistent, propagative fashion and the relationships between the viruses and their thrips vectors are often specific. With the emergence of new tospoviruses, new thrips vector/tospovirus relationships have also arisen and vector specificities have changed. There is known specificity between thrips vector species and particular tospoviruses, although the cellular and molecular bases for this specificity have been elusive. Major conclusions, solutions and achievements. We demonstrated that a new tospovirus, iris yellow spot virus (IYSV) caused "straw bleaching" in onion (Allium cepa) and lisianthus necrosis in lisianthus (Eustoma russellianum). Characterization of virus isolates revealed genetic diversity among US, Brazilian, Dutch and Israeli isolates. IYSV was not seed transmitted, and in Israel, was not located in bulbs of infected plants. In the US, infected plants were generated from infected bulbs. The relationship between IYSV and Thrips tabaci was shown to be specific. Frankliniella occidentalis, the primary vector of many other tospoviruses, did not transmit IYSV isolates in Israel or the US. Furthermore, 1': tabaci populations varied in their transmission ability. Transmission was correlated to IYSV presence in thrips salivary glands. In Israel, surveys in onion fields revealed that the onion thrips, Thrips tabaci Lindeman was the predominant species and that its incidence was strongly related to that of IYSV infection. In contrast, in the U.S., T. tabaci and F. occidentalis were present in high numbers during the times sampled. In Israel, insecticides reduced onion thrips population and caused a significant yield increase. In the US, a genetic marker system that differentiates non-thrips transmissible isolates from thrips transmissible isolate demonstrated the importance of the M RNA to thrips transmission of tospoviruses. In addition, a symbiotic Erwinia was discovered in thrips and was shown to cause significant artifacts in certain types of virus binding experiments. Implications, scientific and agricultural. Rapid emergence of distinct tospoviruses and new vector relationships is profoundly important to global agriculture. We advanced the understanding of IYSV in bulb crops and its relationships with thrips vector species. The knowledge gained provided growers with new strategies for control and new tools for studying the importance of particular viral proteins in thrips specificity and transmission efficiency.

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Porat, Ron, Gregory T. McCollum, Amnon Lers i Charles L. Guy. Identification and characterization of genes involved in the acquisition of chilling tolerance in citrus fruit. United States Department of Agriculture, grudzień 2007. http://dx.doi.org/10.32747/2007.7587727.bard.

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Citrus, like many other tropical and subtropical fruit are sensitive to chilling temperatures. However, application of a pre-storage temperature conditioning (CD) treatment at 16°C for 7 d or of a hot water brushing (HWB) treatment at 60°C for 20 sec remarkably enhances chilling tolerance and reduces the development of chilling injuries (CI) upon storage at 5°C. In the current research, we proposed to identify and characterize grapefruit genes that are induced by CD, and may contribute to the acquisition of fruit chilling tolerance, by two different molecular approaches: cDNA array analysis and PCR cDNA subtraction. In addition, following the recent development and commercialization of the new Affymetrix Citrus Genome Array, we further performed genome-wide transcript profiling analysis following exposure to CD and chilling treatments. To conduct the cDNA array analysis, we constructed cDNA libraries from the peel tissue of CD- and HWB-treated grapefruit, and performed an EST sequencing project including sequencing of 3,456 cDNAs from each library. Based on the obtained sequence information, we chose 70 stress-responsive and chilling-related genes and spotted them on nylon membranes. Following hybridization the constructed cDNA arrays with RNA probes from control and CD-treated fruit and detailed confirmations by RT-PCR analysis, we found that six genes: lipid-transfer protein, metallothionein-like protein, catalase, GTP-binding protein, Lea5, and stress-responsive zinc finger protein, showed higher transcript levels in flavedo of conditioned than in non-conditioned fruit stored at 5 ᵒC. The transcript levels of another four genes: galactinol synthase, ACC oxidase, temperature-induced lipocalin, and chilling-inducible oxygenase, increased only in control untreated fruit but not in chilling-tolerant CD-treated fruit. By PCR cDNA subtraction analysis we identified 17 new chilling-responsive and HWB- and CD-induced genes. Overall, characterization of the expression patterns of these genes as well as of 11 more stress-related genes by RNA gel blot hybridizations revealed that the HWB treatment activated mainly the expression of stress-related genes(HSP19-I, HSP19-II, dehydrin, universal stress protein, EIN2, 1,3;4-β-D-glucanase, and SOD), whereas the CD treatment activated mainly the expression of lipid modification enzymes, including fatty acid disaturase2 (FAD2) and lipid transfer protein (LTP). Genome wide transcriptional profiling analysis using the newly developed Affymetrix Citrus GeneChip® microarray (including 30,171 citrus probe sets) revealed the identification of three different chilling-related regulons: 1,345 probe sets were significantly affected by chilling in both control and CD-treated fruits (chilling-response regulon), 509 probe sets were unique to the CD-treated fruits (chilling tolerance regulon), and 417 probe sets were unique to the chilling-sensitive control fruits (chilling stress regulon). Overall, exposure to chilling led to expression governed arrest of general cellular metabolic activity, including concretive down-regulation of cell wall, pathogen defense, photosynthesis, respiration, and protein, nucleic acid and secondary metabolism. On the other hand, chilling enhanced various adaptation processes, such as changes in the expression levels of transcripts related to membranes, lipid, sterol and carbohydrate metabolism, stress stimuli, hormone biosynthesis, and modifications in DNA binding and transcription factors.

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Epel, Bernard L., Roger N. Beachy, A. Katz, G. Kotlinzky, M. Erlanger, A. Yahalom, M. Erlanger i J. Szecsi. Isolation and Characterization of Plasmodesmata Components by Association with Tobacco Mosaic Virus Movement Proteins Fused with the Green Fluorescent Protein from Aequorea victoria. United States Department of Agriculture, wrzesień 1999. http://dx.doi.org/10.32747/1999.7573996.bard.

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The coordination and regulation of growth and development in multicellular organisms is dependent, in part, on the controlled short and long-distance transport of signaling molecule: In plants, symplastic communication is provided by trans-wall co-axial membranous tunnels termed plasmodesmata (Pd). Plant viruses spread cell-to-cell by altering Pd. This movement scenario necessitates a targeting mechanism that delivers the virus to a Pd and a transport mechanism to move the virion or viral nucleic acid through the Pd channel. The identity of host proteins with which MP interacts, the mechanism of the targeting of the MP to the Pd and biochemical information on how Pd are alter are questions which have been dealt with during this BARD project. The research objectives of the two labs were to continue their biochemical, cellular and molecular studies of Pd composition and function by employing infectious modified clones of TMV in which MP is fused with GFP. We examined Pd composition, and studied the intra- and intercellular targeting mechanism of MP during the infection cycle. Most of the goals we set for ourselves were met. The Israeli PI and collaborators (Oparka et al., 1999) demonstrated that Pd permeability is under developmental control, that Pd in sink tissues indiscriminately traffic proteins of sizes of up to 50 kDa and that during the sink to source transition there is a substantial decrease in Pd permeability. It was shown that companion cells in source phloem tissue export proteins which traffic in phloem and which unload in sink tissue and move cell to cell. The TAU group employing MP:GFP as a fluorescence probe for optimized the procedure for Pd isolation. At least two proteins kinases found to be associated with Pd isolated from source leaves of N. benthamiana, one being a calcium dependent protein kinase. A number of proteins were microsequenced and identified. Polyclonal antibodies were generated against proteins in a purified Pd fraction. A T-7 phage display library was created and used to "biopan" for Pd genes using these antibodies. Selected isolates are being sequenced. The TAU group also examined whether the subcellular targeting of MP:GFP was dependent on processes that occurred only in the presence of the virus or whether targeting was a property indigenous to MP. Mutant non-functional movement proteins were also employed to study partial reactions. Subcellular targeting and movement were shown to be properties indigenous to MP and that these processes do not require other viral elements. The data also suggest post-translational modification of MP is required before the MP can move cell to cell. The USA group monitored the development of the infection and local movement of TMV in N. benthamiana, using viral constructs expressing GFP either fused to the MP of TMV or expressing GFP as a free protein. The fusion protein and/or the free GFP were expressed from either the movement protein subgenomic promoter or from the subgenomic promoter of the coat protein. Observations supported the hypothesis that expression from the cp sgp is regulated differently than expression from the mp sgp (Szecsi et al., 1999). Using immunocytochemistry and electron microscopy, it was determined that paired wall-appressed bodies behind the leading edge of the fluorescent ring induced by TMV-(mp)-MP:GFP contain MP:GFP and the viral replicase. These data suggest that viral spread may be a consequence of the replication process. Observation point out that expression of proteins from the mp sgp is temporary regulated, and degradation of the proteins occurs rapidly or more slowly, depending on protein stability. It is suggested that the MP contains an external degradation signal that contributes to rapid degradation of the protein even if expressed from the constitutive cp sgp. Experiments conducted to determine whether the degradation of GFP and MP:GFP was regulated at the protein or RNA level, indicated that regulation was at the protein level. RNA accumulation in infected protoplast was not always in correlation with protein accumulation, indicating that other mechanisms together with RNA production determine the final intensity and stability of the fluorescent proteins.

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Christopher, David A., i Avihai Danon. Plant Adaptation to Light Stress: Genetic Regulatory Mechanisms. United States Department of Agriculture, maj 2004. http://dx.doi.org/10.32747/2004.7586534.bard.

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Original Objectives: 1. Purify and biochemically characterize RB60 orthologs in higher plant chloroplasts; 2. Clone the gene(s) encoding plant RB60 orthologs and determine their structure and expression; 3. Manipulate the expression of RB60; 4. Assay the effects of altered RB60 expression on thylakoid biogenesis and photosynthetic function in plants exposed to different light conditions. In addition, we also examined the gene structure and expression of RB60 orthologs in the non-vascular plant, Physcomitrella patens and cloned the poly(A)-binding protein orthologue (43 kDa RB47-like protein). This protein is believed to a partner that interacts with RB60 to bind to the psbA5' UTR. Thus, to obtain a comprehensive view of RB60 function requires analysis of its biochemical partners such as RB43. Background & Achievements: High levels of sunlight reduce photosynthesis in plants by damaging the photo system II reaction center (PSII) subunits, such as D1 (encoded by the chloroplast tpsbAgene). When the rate of D1 synthesis is less than the rate of photo damage, photo inhibition occurs and plant growth is decreased. Plants use light-activated translation and enhanced psbAmRNA stability to maintain D1 synthesis and replace the photo damaged 01. Despite the importance to photosynthetic capacity, these mechanisms are poorly understood in plants. One intriguing model derived from the algal chloroplast system, Chlamydomonas, implicates the role of three proteins (RB60, RB47, RB38) that bind to the psbAmRNA 5' untranslated leader (5' UTR) in the light to activate translation or enhance mRNA stability. RB60 is the key enzyme, protein D1sulfide isomerase (Pill), that regulates the psbA-RN :Binding proteins (RB's) by way of light-mediated redox potentials generated by the photosystems. However, proteins with these functions have not been described from higher plants. We provided compelling evidence for the existence of RB60, RB47 and RB38 orthologs in the vascular plant, Arabidopsis. Using gel mobility shift, Rnase protection and UV-crosslinking assays, we have shown that a dithiol redox mechanism which resembles a Pill (RB60) activity regulates the interaction of 43- and 30-kDa proteins with a thermolabile stem-loop in the 5' UTR of the psbAmRNA from Arabidopsis. We discovered, in Arabidopsis, the PD1 gene family consists of II members that differ in polypeptide length from 361 to 566 amino acids, presence of signal peptides, KDEL motifs, and the number and positions of thioredoxin domains. PD1's catalyze the reversible formation an disomerization of disulfide bonds necessary for the proper folding, assembly, activity, and secretion of numerous enzymes and structural proteins. PD1's have also evolved novel cellular redox functions, as single enzymes and as subunits of protein complexes in organelles. We provide evidence that at least one Pill is localized to the chloroplast. We have used PDI-specific polyclonal and monoclonal antisera to characterize the PD1 (55 kDa) in the chloroplast that is unevenly distributed between the stroma and pellet (containing membranes, DNA, polysomes, starch), being three-fold more abundant in the pellet phase. PD1-55 levels increase with light intensity and it assembles into a high molecular weight complex of ~230 kDa as determined on native blue gels. In vitro translation of all 11 different Pill's followed by microsomal membrane processing reactions were used to differentiate among PD1's localized in the endoplasmic reticulum or other organelles. These results will provide.1e insights into redox regulatory mechanisms involved in adaptation of the photosynthetic apparatus to light stress. Elucidating the genetic mechanisms and factors regulating chloroplast photosynthetic genes is important for developing strategies to improve photosynthetic efficiency, crop productivity and adaptation to high light environments.

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