Academic literature on the topic 'Functional mRNA'
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Journal articles on the topic "Functional mRNA"
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Yu, Jia, and J. Eric Russell. "Structural and Functional Analysis of an mRNP Complex That Mediates the High Stability of Human β-Globin mRNA." Molecular and Cellular Biology 21, no. 17 (September 1, 2001): 5879–88. http://dx.doi.org/10.1128/mcb.21.17.5879-5888.2001.
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ABSTRACT Human globins are encoded by mRNAs exhibiting high stabilities in transcriptionally silenced erythrocyte progenitors. Unlike α-globin mRNA, whose stability is enhanced by assembly of a specific messenger RNP (mRNP) α complex on its 3′ untranslated region (UTR), neither the structure(s) nor the mechanism(s) that effects the high-level stability of human β-globin mRNA has been identified. The present work describes an mRNP complex assembling on the 3′ UTR of the β-globin mRNA that exhibits many of the properties of the stability-enhancing α complex. The β-globin mRNP complex is shown to contain one or more factors homologous to αCP, a 39-kDa RNA-binding protein that is integral to α-complex assembly. Sequence analysis implicates a specific 14-nucleotide pyrimidine-rich track within its 3′ UTR as the site of β-globin mRNP assembly. The importance of this track to mRNA stability is subsequently verified in vivo using mice expressing human β-globin transgenes that contain informative mutations in this region. In combination, the in vitro and in vivo analyses indicate that the high stabilities of the α- and β-globin mRNAs are maintained through related mRNP complexes that may share a common regulatory pathway.
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Alekhina, Olga, Ilya Terenin, Sergey Dmitriev, and Konstantin Vassilenko. "Functional Cyclization of Eukaryotic mRNAs." International Journal of Molecular Sciences 21, no. 5 (February 29, 2020): 1677. http://dx.doi.org/10.3390/ijms21051677.
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The closed-loop model of eukaryotic translation states that mRNA is circularized by a chain of the cap-eIF4E-eIF4G-poly(A)-binding protein (PABP)-poly(A) interactions that brings 5′ and 3′ ends together. This circularization is thought to promote the engagement of terminating ribosomes to a new round of translation at the same mRNA molecule, thus enhancing protein synthesis. Despite the general acceptance and the elegance of the hypothesis, it has never been proved experimentally. Using continuous in situ monitoring of luciferase synthesis in a mammalian in vitro system, we show here that the rate of translation initiation at capped and polyadenylated reporter mRNAs increases after the time required for the first ribosomes to complete mRNA translation. Such acceleration strictly requires the presence of a poly(A)-tail and is abrogated by the addition of poly(A) RNA fragments or m7GpppG cap analog to the translation reaction. The optimal functional interaction of mRNA termini requires 5′ untranslated region (UTR) and 3′ UTR of moderate lengths and provides stronger acceleration, thus a longer poly(A)-tail. Besides, we revealed that the inhibitory effect of the dominant negative R362Q mutant of initiation factor eIF4A diminishes in the course of translation reaction, suggesting a relaxed requirement for ATP. Taken together, our results imply that, upon the functional looping of an mRNA, the recycled ribosomes can be recruited to the start codon of the same mRNA molecule in an eIF4A-independent fashion. This non-canonical closed-loop assisted reinitiation (CLAR) mode provides efficient translation of the functionally circularized mRNAs.
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Yang, Edward, Erik van Nimwegen, Mihaela Zavolan, Nikolaus Rajewsky, Mark Schroeder, Marcelo Magnasco, and James E. Darnell. "Decay Rates of Human mRNAs: Correlation With Functional Characteristics and Sequence Attributes." Genome Research 13, no. 8 (August 2003): 1863–72. http://dx.doi.org/10.1101/gr.1272403.
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Although mRNA decay rates are a key determinant of the steady-state concentration for any given mRNA species, relatively little is known, on a population level, about what factors influence turnover rates and how these rates are integrated into cellular decisions. We decided to measure mRNA decay rates in two human cell lines with high-density oligonucleotide arrays that enable the measurement of decay rates simultaneously for thousands of mRNA species. Using existing annotation and the Gene Ontology hierarchy of biological processes, we assign mRNAs to functional classes at various levels of resolution and compare the decay rate statistics between these classes. The results show statistically significant organizational principles in the variation of decay rates among functional classes. In particular, transcription factor mRNAs have increased average decay rates compared with other transcripts and are enriched in “fast-decaying” mRNAs with half-lives <2 h. In contrast, we find that mRNAs for biosynthetic proteins have decreased average decay rates and are deficient in fast-decaying mRNAs. Our analysis of data from a previously published study of Saccharomyces cerevisiae mRNA decay shows the same functional organization of decay rates, implying that it is a general organizational scheme for eukaryotes. Additionally, we investigated the dependence of decay rates on sequence composition, that is, the presence or absence of short mRNA motifs in various regions of the mRNA transcript. Our analysis recovers the positive correlation of mRNA decay with known AU-rich mRNA motifs, but we also uncover further short mRNA motifs that show statistically significant correlation with decay. However, we also note that none of these motifs are strong predictors of mRNA decay rate, indicating that the regulation of mRNA decay is more complex and may involve the cooperative binding of several RNA-binding proteins at different sites.
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Mauger, David M., B. Joseph Cabral, Vladimir Presnyak, Stephen V. Su, David W. Reid, Brooke Goodman, Kristian Link, et al. "mRNA structure regulates protein expression through changes in functional half-life." Proceedings of the National Academy of Sciences 116, no. 48 (November 11, 2019): 24075–83. http://dx.doi.org/10.1073/pnas.1908052116.
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Messenger RNAs (mRNAs) encode information in both their primary sequence and their higher order structure. The independent contributions of factors like codon usage and secondary structure to regulating protein expression are difficult to establish as they are often highly correlated in endogenous sequences. Here, we used 2 approaches, global inclusion of modified nucleotides and rational sequence design of exogenously delivered constructs, to understand the role of mRNA secondary structure independent from codon usage. Unexpectedly, highly expressed mRNAs contained a highly structured coding sequence (CDS). Modified nucleotides that stabilize mRNA secondary structure enabled high expression across a wide variety of primary sequences. Using a set of eGFP mRNAs with independently altered codon usage and CDS structure, we find that the structure of the CDS regulates protein expression through changes in functional mRNA half-life (i.e., mRNA being actively translated). This work highlights an underappreciated role of mRNA secondary structure in the regulation of mRNA stability.
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Pilkington, Guy R., and Roy Parker. "Pat1 Contains Distinct Functional Domains That Promote P-Body Assembly and Activation of Decapping." Molecular and Cellular Biology 28, no. 4 (December 17, 2007): 1298–312. http://dx.doi.org/10.1128/mcb.00936-07.
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ABSTRACT The control of mRNA degradation and translation are important aspects of gene regulation. Recent results suggest that translation repression and mRNA decapping can be intertwined and involve the formation of a quiescent mRNP, which can accumulate in cytoplasmic foci referred to as P bodies. The Pat1 protein is a key component of this complex and an important activator of decapping, yet little is known about its function. In this work, we analyze Pat1 in Saccharomyces cerevisiae function by deletion and functional analyses. Our results identify two primary functional domains in Pat1: one promoting translation repression and P-body assembly and a second domain promoting mRNA decapping after assembly of the mRNA into a P-body mRNP. In addition, we provide evidence that Pat1 binds RNA and has numerous domain-specific interactions with mRNA decapping factors. These results indicate that Pat1 is an RNA binding protein and a multidomain protein that functions at multiple stages in the process of translation repression and mRNA decapping.
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Chang, Jeong Ho, and Liang Tong. "Structural and functional studies of the decapping exoribonucleases." Acta Crystallographica Section A Foundations and Advances 70, a1 (August 5, 2014): C1397. http://dx.doi.org/10.1107/s2053273314086021.
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Recent studies showed that two homologous yeast proteins, Rai1 and Dxo1, function in a quality control mechanism to clear cells of incompletely 5' end-capped messenger RNAs (mRNAs). Rai1 possesses a novel decapping activity that can remove the entire cap structure dinucleotide from an mRNA. This activity is targeted preferentially towards mRNAs with unmethylated caps in contrast to the canonical decapping enzyme, Dcp2, which targets mRNAs with a methylated cap. Dxo1 also has robust decapping activity on RNAs with unmethylated caps, but it has no detectable pyrophosphohydrolase activity. Unexpectedly, we found that Dxo1 also possesses distributive, 5'-3' exoribonuclease activity, and we named Dxo1 (originally Ydr370C) for this new eukaryotic enzyme with both decapping and exonuclease activities. Studies of yeast in which both Dxo1 and Rai1 are disrupted reveal that mRNAs with incomplete caps are produced even under normal growth conditions, in sharp contrast to current understanding of the capping process. Here, we introduce that their mammalian homolog, Dom3Z (referred to as DXO), possesses pyrophosphohydrolase, decapping, and 5'-3' exoribonuclease activities. Surprisingly, we found that DXO preferentially degrades defectively capped pre-mRNAs in cells. Additional studies show that incompletely capped pre-mRNAs are inefficiently spliced at all introns, a fact that contrasts with current understanding, and are also poorly cleaved for polyadenylation. Crystal structures of DXO in complex with substrate mimic and products at a resolution of up to 1.5 Å provide elegant insights into the catalytic mechanism and molecular basis for their three apparently distinct activities. Our data reveal a pre-mRNA 5' end capping quality control mechanism in mammalian cells, indicating DXO as the central player for this mechanism, and demonstrate an unexpected intimate link between proper 5' end capping and subsequent pre-mRNA processing.
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Green-Willms, Noelle S., Thomas D. Fox, and Maria C. Costanzo. "Functional Interactions between Yeast Mitochondrial Ribosomes and mRNA 5′ Untranslated Leaders." Molecular and Cellular Biology 18, no. 4 (April 1, 1998): 1826–34. http://dx.doi.org/10.1128/mcb.18.4.1826.
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ABSTRACT Translation of mitochondrial mRNAs in Saccharomyces cerevisiae depends on mRNA-specific translational activators that recognize the 5′ untranslated leaders (5′-UTLs) of their target mRNAs. We have identified mutations in two new nuclear genes that suppress translation defects due to certain alterations in the 5′-UTLs of both the COX2 and COX3 mRNAs, indicating a general function in translational activation. One gene, MRP21, encodes a protein with a domain related to the bacterial ribosomal protein S21 and to unidentified proteins of several animals. The other gene, MRP51, encodes a novel protein whose only known homolog is encoded by an unidentified gene in S. kluyveri. Deletion of either MRP21 or MRP51 completely blocked mitochondrial gene expression. Submitochondrial fractionation showed that both Mrp21p and Mrp51p cosediment with the mitochondrial ribosomal small subunit. The suppressor mutations are missense substitutions, and those affecting Mrp21p alter the region homologous to E. coli S21, which is known to interact with mRNAs. Interactions of the suppressor mutations with leaky mitochondrial initiation codon mutations strongly suggest that the suppressors do not generally increase translational efficiency, since some alleles that strongly suppress 5′-UTL mutations fail to suppress initiation codon mutations. We propose that mitochondrial ribosomes themselves recognize a common feature of mRNA 5′-UTLs which, in conjunction with mRNA-specific translational activation, is required for organellar translation initiation.
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Yang, Feng, Yong Peng, Elizabeth L. Murray, Yuichi Otsuka, Nancy Kedersha, and Daniel R. Schoenberg. "Polysome-Bound Endonuclease PMR1 Is Targeted to Stress Granules via Stress-Specific Binding to TIA-1." Molecular and Cellular Biology 26, no. 23 (September 18, 2006): 8803–13. http://dx.doi.org/10.1128/mcb.00090-06.
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ABSTRACT The generalized process of mRNA decay involves deadenylation followed by release from translating polysomes, decapping, and exonuclease decay of the mRNA body. In contrast the mRNA endonuclease PMR1 forms a selective complex with its translating substrate mRNA, where it initiates decay by cleaving within the mRNA body. In stressed cells the phosphorylation of the α subunit of eukaryotic initiation factor 2 causes translating mRNAs to accumulate with stalled 48S subunits in large subcellular structures termed stress granules (SGs), wherein mRNAs undergo sorting for reinitiation, storage, or decay. Given the unique relationship between translation and PMR1-mediated mRNA decay, we examined the impact of stress-induced dissociation of polysomes on this process. Arsenite stress disrupts the polysome binding of PMR1 and its substrate mRNA but has no impact on the critical tyrosine phosphorylation of PMR1, its association with substrate mRNA, or its association with the functional ∼680-kDa mRNP complex in which it normally resides on polysomes. We show that arsenite stress drives PMR1 into an RNase-resistant complex with TIA-1, and we identify a distinct domain in the N terminus of PMR1 that facilitates its interaction with TIA-1. Finally, we show that arsenite promotes the delayed association of PMR1 with SGs under conditions which cause tristetraprolin and butyrate response factor 1, proteins that facilitate exonucleolytic mRNA, to exit SGs.
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GRÖLZ, Daniel, and Michael BACHMANN. "The nuclear autoantigen La/SS-associated antigen B: one gene, three functional mRNAs." Biochemical Journal 323, no. 1 (April 1, 1997): 151–58. http://dx.doi.org/10.1042/bj3230151.
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Transcription of the gene encoding for the nuclear autoantigen La resulted in three mRNA forms. A promoter switching combined with an alternative splicing pathway replaced exon 1 with either exon 1´ or exon 1´´. The exon 1´´ donor splice site was located 4 nts downstream of the exon 1´ donor splice site. All three La mRNA forms were expressed in all the tissues analysed including peripheral blood lymphocytes, liver, fetal spleen, cultured primary endothelial cells, and mouse LTA cell lines permanently transfected with the human La gene. Both the exons 1´ and 1´´ had unusual structures. They contained GC-rich regions and an oligo(U)-tail of 23 uridine residues. Moreover, they encoded for three open reading frames upstream of the La protein reading frame. In spite of this unusual structure, when exon 1´ or exon 1´´ La mRNAs were expressed in transfected mouse LTA cells, both La mRNAs were translated to nuclear La protein, indicating that all La mRNA forms are functional mRNAs.
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Hennigan, A. N., and A. Jacobson. "Functional mapping of the translation-dependent instability element of yeast MATalpha1 mRNA." Molecular and Cellular Biology 16, no. 7 (July 1996): 3833–43. http://dx.doi.org/10.1128/mcb.16.7.3833.
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The determinants of mRNA stability include specific cis-acting destabilizing sequences located within mRNA coding and noncoding regions. We have developed an approach for mapping coding-region instability sequences in unstable yeast mRNAs that exploits the link between mRNA translation and turnover and the dependence of nonsense-mediated mRNA decay on the activity of the UPF1 gene product. This approach, which involves the systematic insertion of in-frame translational termination codons into the coding sequence of a gene of interest in a upf1delta strain, differs significantly from conventional methods for mapping cis-acting elements in that it causes minimal perturbations to overall mRNA structure. Using the previously characterized MATalpha1 mRNA as a model, we have accurately localized its 65-nucleotide instability element (IE) within the protein coding region. Termination of translation 5' to this element stabilized the MATalpha1 mRNA two- to threefold relative to wild-type transcripts. Translation through the element was sufficient to restore an unstable decay phenotype, while internal termination resulted in different extents of mRNA stabilization dependent on the precise location of ribosome stalling. Detailed mutagenesis of the element's rare-codon/AU-rich sequence boundary revealed that the destabilizing activity of the MATalpha1 IE is observed when the terminal codon of the element's rare-codon interval is translated. This region of stability transition corresponds precisely to a MATalpha1 IE sequence previously shown to be complementary to 18S rRNA. Deletion of three nucleotides 3' to this sequence shifted the stability boundary one codon 5' to its wild-type location. Conversely, constructs containing an additional three nucleotides at this same location shifted the transition downstream by an equivalent sequence distance. Our results suggest a model in which the triggering of MATalpha1 mRNA destabilization results from establishment of an interaction between translating ribosomes and a downstream sequence element. Furthermore, our data provide direct molecular evidence for a relationship between mRNA turnover and mRNA translation.
Dissertations / Theses on the topic "Functional mRNA"
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Gape, Helen. "Microheterogeneity of porcine calpastatin and its functional implications." Thesis, University of Nottingham, 1998. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.267624.
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Wolf, Jana. "Structural and functional studies of mRNA PolyA tail processing complexes." Thesis, University of Cambridge, 2014. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.708435.
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Raher, Michael J. "Functional Analysis of Proteins Involved in Translational Regulation." Thesis, Boston College, 2003. http://hdl.handle.net/2345/433.
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Thesis advisor: Laura E. HakeCytoplasmic polyadenylation regulates translational activation of mRNA stored in immature Xenopus oocytes. This event is necessary for the beginning of oocyte maturation, and later for critical processes in early embryonic development. A major protein required for polyadenylation is the cytoplasmic polyadenylation element-binding protein (CPEB), which recruits a factor that promotes the interaction between Poly(A) polymerase and the end of the mRNA. Polyadenylation in turn leads to translation through interactions between CPEB and other proteins. Using a yeast two-hybrid screen, several of these proteins were identified and cloned, including two of note. X295, a zinc-finger containing novel protein, and DEK, which has significant homology with the Homo sapiens DEK involved in certain juvenile leukemias. Through the cloning of the genes encoding these proteins, transcription of mRNA, and protein overexpression in oocytes, a series of protein-protein interaction binding assays were performed. Immunoblotting of SDS-PAGE analyzed samples shows that GST-CPEB and HA-X295 interact in ovo, and suggests a possible in ovo interaction of endogenous CPEB and endogenous X295. In similar experiments, DEK and CPEB do not interact, suggesting they may not interact in ovo
Thesis (BS) — Boston College, 2003
Submitted to: Boston College. College of Arts and Sciences
Discipline: Biology
Discipline: College Honors Program
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Fu, Xiaonan. "Functional study of miRNA-mRNA interactions in malaria mosquito An. gambiae." Diss., Virginia Tech, 2018. http://hdl.handle.net/10919/96216.
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Female adults of many mosquito species possess distinct physiological features adapting to blood feeding for successful reproduction. The disease pathogens that are transmitted by mosquitoes have evolved to take advantages of the indispensable blood feedings to complete their transmission cycles and to survive attacks from the mosquito's innate immune system. Normal egg development and mosquito immunity are tightly controlled by tissue- and stage-specific gene expression and coordinated by many signal molecules in the mosquito. Understanding gene regulation affecting mosquito reproduction and malaria parasites infection is of paramount importance for developing novel malaria control strategies. A growing body of evidence indicates that microRNAs (miRNAs) are involved in egg maturation and immune reactions against invading pathogens in mosquitoes. However, the molecular mechanisms by which specific miRNAs selectively modulate reproduction and the survival of pathogens are largely unknown.
The miRNA-induced gene-silencing pathway in mosquitoes was mostly extrapolated from the studies of flies. To explore the dynamics of miRNAs in reproduction, I used small RNAs sequencing to monitor miRNAs expression and their association with Argonaute 1 (Ago1) and Argonaute 2 (Ago2) in the malaria mosquito Anopheles gambiae (An. gambiae) during the 72-h period immediately after blood feeding. I found the abundance and Ago loading of most of the mature miRNAs were relatively stable after blood ingestion. However, miRNAs of the miR-309/286/2944 cluster were considerably upregulated after blood feeding. I confirmed that miR-309 is essential for normal egg development by depletion of endogenous miR-309 with a specific antagomir. In addition, my results showed that the Ago association of some miRNAs was not proportional to their cellular abundance implying additional regulation at miRNA integration.
To investigate the functional roles of miRNAs and define context-dependent miRNA-mRNA interactions during the reproductive process, I have applied an innovative experimental approach to study miRNA-mRNA interactome. CLEAR (covalent ligation of endogenous Argonaute-bound RNAs)-CLIP can generate miRNA-mRNA chimeras from UV-irradiation stabilized Ago-miRNA-mRNA complex. My results have defined tens of thousands of miRNA-mRNA interactions in mosquitoes, including novel targets for mosquito-specific miRNAs. Verification of the predicted interactions using mRNA-seq, ribosome-profiling, and luciferase reporter assay revealed a reliable miRNA-mRNA interaction network. Based on the detected interactions, I refined the paring rules for mosquito miRNAs and illustrated the dynamic pairing between different regions of miRNAs with their targets in vivo. The miRNA-mRNA interactions were compared using this approach at multiple time points before and after blood feeding. Importantly, this study showed that the interactions were dynamic and enriched in genes that are involved in metabolisms, supporting the proposed functions of miRNAs in coordinating the gene regulation in mosquito reproduction.
Plasmodium falciparum (P. falciparum) is a major human malaria parasite. To understand the functions of miRNAs in the mosquito resistance to Plasmodium infection, we analyzed the miRNA-mRNA interactions after female mosquitoes taking a P. falciparum-infected blood meal or an uninfected blood meal. Comparison of the interactions revealed enhanced miRNA-mRNA interactions after P. falciparum infection involving a group of immunity-related genes. In summary, this study has provided a systematic view and significantly advanced our understanding of the miRNA functions in mosquito reproduction and P. falciparum infection.PHD
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Henscheid, Kristy L. "Functional conservation and RNA binding of the pre-mRNA splicing factor U2AF65 /." view abstract or download file of text, 2007. http://proquest.umi.com/pqdweb?did=1400950821&sid=5&Fmt=2&clientId=11238&RQT=309&VName=PQD.
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Thesis (Ph. D.)--University of Oregon, 2007.Typescript. Includes vita and abstract. Includes bibliographical references (leaves 129-141). Also available for download via the World Wide Web; free to University of Oregon users.
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Noerenberg, Marko. "Functional analysis of the KSHV ORF57 protein in mRNA nuclear export mechanisms." Thesis, University of Leeds, 2011. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.574517.
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Kaposi's sarcoma-associated Herpesvirus (KSHV; HHV -8) is associated with multiple malignancies, including Kaposi's sarcoma. KSHV has two distinct life cycle phases, latent persistence and lytic replication. In contrast to other oncogenic Herpesviruses, lytic replication plays an important part in tumourigenicity and pathogenesis of KSHV. Therefore, it is essential to study the molecular mechanisms which regulate lytic replication to fully understand KSHV pathogenesis. This in turn may lead to novel therapies, which could become an important strategy for the treatment of KSHV -associated diseases. Post-transcriptional regulation of RNA biogenesis is fundamental to KSHV lytic gene expression. The KSHV ORF57 protein plays an essential role in viral RNA processing, transcription, splicing, mRNA stability, nuclear mRNA export and translation. To date, it is unknown how ORF57 co-ordinates these many roles. Functional diversity of a protein can be achieved by post-translational modifications. We demonstrate that ORF57 is post-translationally methylated and inhibition of methylation has a dramatic effect on the ability of ORF57 to export intronless viral RN A out of the nucleus. This work shows that hypomethylation of ORF57 enhances its ability to bind RNA. Attempts were made to find ORF57 residues which are methylated and results identified PRMT5, a cellular protein methyltransferase as well as the putative demethylase MINA, which interact with ORF57. Furthermore, a proteomic-based approach was used to identify ce~lular proteins which are changed in their intracellular distribution or abundance upon ORF57 expression. This SILAC-based approach highlighted proteins and pathways affected by ORF57. Data showed changes in RNA processing pathways previously unknown to be affected by ORF57, e.g. mRNA polyadenylation and nucleoskeleton rearrangements. In addition, changes of putative, novel components of the human TREX complex were found. Data presented will provide a valuable resource not only to enhance our understanding of this viral pathogen but also to show new routes for drug intervention.
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Sampson, Natalie D. "Identification and functional characterisation of the novel pre-mRNA processing factor SCAF6." Thesis, University of Nottingham, 2002. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.272374.
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Muraru, Mariela I. "Functional analysis of Prp45p, a pre-mRNA splicing factor in Saccharomyces cerevisiae." Thesis, University of Edinburgh, 2003. http://hdl.handle.net/1842/15461.
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In this study we aimed to understand the role Prp45p executes within the spliceosome and the splicing mechanism. Part of this work was a detailed investigation into the relationships between the structure and the function of this protein by employing a screen for temperature-sensitive mutants. Prp45p is known to associate with the spliceosome throughout the splicing reactions but at what stage the protein is involved in spliceosome assembly i.e. pre-spliceosome, inactive or active spliceosome remained to be determined. Using a PRP45 conditionally regulated strain, it was found upon depletion of Prp45p from yeast cells and analysis of the in vivo splicing systems by native gel fractionation that the formation of the active spliceosome does not take place. This behaviour is compatible with Prp45p being a component of the Ntc-protein complex, which is known to be involved in this step of spliceosome assembly. Moreover, co-immunoprecipitation experiments with a tagged allele of the splicing factor Prp46p, confirmed the interaction between these two proteins suggested by two-hybrid screens. Using random PCR mutagenesis, there were identified two mutants with growth defects at 37°C. The mutants, named prp45-57 and prp45-113 contained mutations in two regions, designated A and B and located respectively upstream and, downstream of the absolutely conserved SNWKN motif. To understand which of the mutations were responsible for the temperature-sensitive phenotype, the substitutions in region A and/or region B were recreated by site-directed mutagenesis. It was proved that the phenotype requires mutations in both segments, which strongly suggests that regions A and B together play roles in protein function, perhaps through intra- or inter-protein interactions. However, co-immunoprecipitation experiments revealed that these substitutions in Prp45p do not alter the interaction with Prp46p in vitro. In order to investigate the role of these mutations within the cell, two strains were created that carry these mutations in a c-Myc tagged PRP45 ORF. When growing these strains at non-permissive temperature and employing a β-galactosidase assay, it was found that they had a mild effect on pre-mRNA splicing and do not affect the transcription/translation of the reporter genes.
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Wills, Quintin Frank. "The genetics of miRNA and mRNA expression in human lymphoblastoid cell lines." Thesis, University of Oxford, 2012. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.572601.
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Human clinical genome-wide association studies (GWAS) have helped identify disease trait and pharmacogenomic loci without the need for biological under- standing. Molecular GWAS - associating genetic variation with traits such as gene expression - have been slow to fill the mechanistic gaps. While tissue specificity, lack of DNA resolution, and the need for better data integration are no dou bt important bottlenecks in molecular GWAS, there is also a very poor general understanding of which molecular phenotypes are important and how best to model them. Added to this is the clear need for a greater understanding of the strengths and weaknesses facing in vitro (and ex vivo) models as hypoth- esis generating and GWAS validation tools. The studies in this work focus on RNA expression in a popular human model: lymphoblastoid cell lines (LCLs). Chapters 2 and 3 examine microRNA (miRNA) and messenger RNA (mRNA) expression in a total of 300 genotyped human LCLs. The expression of only one miRNA could be associated with a nearby genetic variant. This result was observed in both the African and European samples studied, in a separate val- idation data set, and was technically validated with quantitative PCR. While limited genotype resolution and small sample sizes are likely to be important contributors to this low hit rate, the results strongly suggest experimental con- founders. Highly expressed miRNAs reflected the transformed nature of the cells, highly correlated miRNAs enriched for EBV and malaria associated tar- get mRNA genes, and several miRNAs that were differentially expressed be- tween the European and African samples suggested differential EBV transfer- mation. Chapter 4 presents a study on single cells from some of the same samples, to test the hypothesis that the lack of tissue spatial resolution is an important limiting factor in human genetic epidemiology. Experimental con- founders were also considered: sample growth was found to associate with the expression of several genes. Cell-to-cell gene correlations and distributions made it possible to propose how genes change their expression, functionally differ from each other, and are able to alter their behaviours without altered whole-tissue expression. The results suggest which type of genes are more likely to be susceptible to genetic effects, and propose promoter behaviours altered by genetic variants located near to 13 genes. From these whole-tissue and single cell results the broad conclusion is that, while LCLs are likely to be inappropriate for the study of miRNA genetics, their functional genomics at higher spatial resolution shows promise as a more mechanistic approach for the study of germline genetics.
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Samsonova, Anastasiia. "Structural and functional insights into YB-1 and Lin28 interplay in mRNA regulation." Thesis, université Paris-Saclay, 2020. http://www.theses.fr/2020UPASL037.
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La régulation de l'ARNm dans les cellules humaines est l'un des mécanismes essentiels permettant aux cellules de s'adapter à un nouvel environnement et de répondre aux signaux entrants. En effet, il est bien plus efficace pour une cellule de réguler l'homéostasie protéique au niveau de la traduction des ARNm plutôt qu'en jouant sur la transcription ou les mécanismes de dégradation des protéines. Dans les mécanismes de régulation de la traduction de l'ARNm, les protéines de liaison à l'ARN (RBP) jouent un rôle clé.Au cours de ce travail de recherche, nous avons démontré que Lin28, une RBP humaine avec un domaine de choc froid peut interagir avec l'ARNm en coopération avec YB1, une protéine très abondante contenant aussi un domaine de choc froid, essentielle dans la constitution des mRNPs. L'interaction entre ces deux protéines est basée sur leur haute similitude de structure, et permet potentiellement à Lin28a de réguler la traduction nombreux mRNPs en utilisant YB-1 comme «badge d'entrée» au mRNP.Pour étudier l'interaction entre Lin28 et YB-1, différentes méthodes de biologie structurale et cellulaire ont été utilisées. D'abord, l'oligomérisation des domaines de choc froid de Lin28 et YB-1 en présence d'ARNm a été démontrée in vitro, et les résidus d'acides aminés impliqués dans ce processus ont été mis en évidence par spectroscopie RMN. Ensuite, la colocalisation dans le cytoplasme de Lin28 et YB-1 a été démontrée dans un contexte cellulaire. Finalement, nous avons révélé les conséquences fonctionnelles de cette interaction, par exemple dans le cadre de la prolifération et de la différenciation cellulaires. Ces résultats éclairent un nouveau rôle de Lin28 dans le développement et l'adaptation des cellules cancéreuses à leur environnementThe mRNA regulation in human cells is one of the key mechanisms allowing the cells to adapt to a new environment and to respond to incoming signals. In terms of protein synthesis, the regulation of mRNA translation is a preferable process for cells compared to a more rigid mechanism of transcription or degradation. The RNA-binding proteins (RBPs) play a key role in the mRNA translation regulation.In the present work, we made an effort to demonstrate that a human RBP containing a cold shock domain, Lin28a, can act in cooperation with another cold shock protein YB-1, a core protein of mRNPs. The interplay between two cold shock proteins is based on their high structure similarity, that potentially gives Lin28 an opportunity to regulate the mRNA target translation in a general way using YB 1 as an “entry badge” to the mRNP.To demonstrate the interplay between Lin28 and YB 1, several methods of structural and cellular biology were used in the present study. The oligomerization of Lin28-CSD and YB-1-CSD upon RNA binding was shown in vitro, and the amino acid residues responsible for that were highlighted by NMR spectroscopy. Then, the colocalization of Lin28 and YB-1 was demonstrated in cell cytoplasm. Also, the protein interplay was shown to have functional consequences, e.g. for cell proliferation and differentiation
Books on the topic "Functional mRNA"
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Oeffinger, Marlene, and Daniel Zenklusen, eds. The Biology of mRNA: Structure and Function. Cham: Springer International Publishing, 2019. http://dx.doi.org/10.1007/978-3-030-31434-7.
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RNA-binding of LC3 to the AU-rich element of fibronectin mRNA: A structural and functional study. Ottawa: National Library of Canada, 1999.
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Bradbury, Elizabeth J., and Nicholas D. James. Mapping of neurotrophin receptors on adult sensory neurons. Edited by Paul Farquhar-Smith, Pierre Beaulieu, and Sian Jagger. Oxford University Press, 2018. http://dx.doi.org/10.1093/med/9780198834359.003.0022.
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The paper discussed in this chapter describes the first mapping of neurotrophin receptors in adult sensory neurons. Neurotrophins and their receptors were a particularly hot topic at the time, but the primary focus of interest had been in their role in development. In this paper, McMahon and colleagues characterized both mRNA and protein expression of the recently discovered trk receptors on defined populations of adult sensory neurons, correlating trk expression with other primary afferent projection neuron properties such as cell size and neuronal function. Furthermore, by showing clear correlations between the expression of different trk receptors and the physical and functional properties of defined primary afferent projections, the authors provided key evidence suggesting that nerve growth factor and neurotrophin-3 acted on functionally distinct populations of adult sensory neurons. This paper provided the basis for subsequent research on neurotrophin signalling and function in both the healthy and the diseased nervous system.
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Yue, Bai-Gong. Regulation of Adenovirus Alternative Pre-Mrna Splicing: Functional Characterization of Exotic and Intronic Splicing Enhancer Elements (Comprehensive Summaries ... from the Faculty of Medicine, 926). Uppsala Universitet, 2000.
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D, Richter Joel, ed. mRNA formation and function. San Diego: Academic Press, 1997.
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mRNA Formation and Function. Elsevier, 1997. http://dx.doi.org/10.1016/b978-0-12-587545-5.x5000-9.
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Richter, Joel D. MRNA Formation and Function. Elsevier Science & Technology Books, 1997.
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Jackson, Lynn, and Irma Fernandez. MRNA: Molecular Biology, Processing and Function. Nova Science Publishers, Incorporated, 2018.
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Oeffinger, Marlene, and Daniel Zenklusen. Biology of MRNA: Structure and Function. Springer International Publishing AG, 2021.
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Oeffinger, Marlene, and Daniel Zenklusen. The Biology of mRNA: Structure and Function. Springer, 2019.
Find full textBook chapters on the topic "Functional mRNA"
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Ladomery, Michael R., and Sebastian Oltean. "Pre-mRNA Splicing and Disease." In The Functional Nucleus, 51–69. Cham: Springer International Publishing, 2016. http://dx.doi.org/10.1007/978-3-319-38882-3_3.
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Wang, Zixing, and Yin Liu. "Predicting Functional MicroRNA-mRNA Interactions." In Methods in Molecular Biology, 117–26. New York, NY: Springer New York, 2017. http://dx.doi.org/10.1007/978-1-4939-6866-4_10.
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Singh, Ravindra N., and Natalia N. Singh. "Functional Analysis of Large Exonic Sequences Through IterativeIn VivoSelection." In Alternative pre-mRNA Splicing, 200–209. Weinheim, Germany: Wiley-VCH Verlag GmbH & Co. KGaA, 2012. http://dx.doi.org/10.1002/9783527636778.ch18.
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Ravelonandro, Michel, and Pascal Briard. "Biogenesis and functional RNAi in fruit trees." In RNAi for plant improvement and protection, 40–46. Wallingford: CABI, 2021. http://dx.doi.org/10.1079/9781789248890.0005.
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Abstract In plants, genome expression is linked to the transcribed mRNAs that are synthesized by RNA polymerase. Following its move to the cytoplasm, the generated mRNA is briefly translated to the encoded protein. If transcription and translation are dependent on the family of RNA polymerase, these two phenomena could be interfered with through the process designated as gene regulation. Thus, large molecules of RNA (single-stranded or double-stranded) consequently sliced into small molecules produce nascent small interfering RNA ranging from 21 to 27 nucleotides. This chapter revisits the biogenesis of these two types of RNAi, miRNA and siRNA, and notably their involvement in plant gene regulation. Following their sequential transcription and their specific involvement, we will consider the sources and roles of RNA interference in plants and we will look at their detection in fruit crops. We discuss their applications and the risk assessment studies in fruit crops.
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Ravelonandro, Michel, and Pascal Briard. "Biogenesis and functional RNAi in fruit trees." In RNAi for plant improvement and protection, 40–46. Wallingford: CABI, 2021. http://dx.doi.org/10.1079/9781789248890.0040.
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Abstract In plants, genome expression is linked to the transcribed mRNAs that are synthesized by RNA polymerase. Following its move to the cytoplasm, the generated mRNA is briefly translated to the encoded protein. If transcription and translation are dependent on the family of RNA polymerase, these two phenomena could be interfered with through the process designated as gene regulation. Thus, large molecules of RNA (single-stranded or double-stranded) consequently sliced into small molecules produce nascent small interfering RNA ranging from 21 to 27 nucleotides. This chapter revisits the biogenesis of these two types of RNAi, miRNA and siRNA, and notably their involvement in plant gene regulation. Following their sequential transcription and their specific involvement, we will consider the sources and roles of RNA interference in plants and we will look at their detection in fruit crops. We discuss their applications and the risk assessment studies in fruit crops.
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Saçar Demirci, Müşerref Duygu, Malik Yousef, and Jens Allmer. "Computational Prediction of Functional MicroRNA–mRNA Interactions." In Computational Biology of Non-Coding RNA, 175–96. New York, NY: Springer New York, 2019. http://dx.doi.org/10.1007/978-1-4939-8982-9_7.
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Grawenhoff, Julia, Sebastian Baumann, and Sebastian P. Maurer. "In Vitro Reconstitution of Kinesin-Based, Axonal mRNA Transport." In Methods in Molecular Biology, 547–68. New York, NY: Springer US, 2022. http://dx.doi.org/10.1007/978-1-0716-1990-2_29.
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AbstractMotor protein-driven transport of mRNAs on microtubules and their local translation underlie important neuronal functions such as development, growth cone steering, and synaptic plasticity. While there is abundant data on how membrane-bound cargoes such as vesicles, endosomes, or mitochondria are coupled to motor proteins, surprisingly little is known on the direct interactions of RNA–protein complexes and kinesins or dynein. Provided the potential building blocks are identified, in vitro reconstitutions coupled to Total Internal Reflection Microscopy (TIRF-M) are a powerful and highly sensitive tool to understand how single molecules dynamically interact to assemble into functional complexes. Here we describe how we assemble TIRF-M imaging chambers suitable for the imaging of single protein–RNA complexes. We give advice on optimal sample preparation procedures and explain how a minimal axonal mRNA transport complex can be assembled in vitro. As these assays work at picomolar-range concentrations of proteins and RNAs, they allow the investigation of molecules that cannot be obtained at high concentrations, such as many large or disordered proteins. This now opens the possibility to study how RNA-binding proteins (RBPs), RNAs, and microtubule-associated proteins act together in real-time at single-molecule sensitivity to create cytoplasmic mRNA distributions.
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Roy, Bijoyita. "Effects of mRNA Modifications on Translation: An Overview." In Methods in Molecular Biology, 327–56. New York, NY: Springer US, 2021. http://dx.doi.org/10.1007/978-1-0716-1374-0_20.
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AbstractThe mRNA epitranscriptome imparts diversity to gene expression by installing chemical modifications. Advances in detection methods have identified chemical modifications in eukaryotic, bacterial, and viral messenger RNAs (mRNAs). The biological functions of modifications in mRNAs still remain to be understood. Chemical modifications are introduced in synthetic mRNAs meant for therapeutic applications to maximize expression from the synthetic mRNAs and to evade the host immune response. This overview provides a background of chemical modifications found in mRNAs, with an emphasis on pseudouridine and its known effects on the mRNA life cycle, its potential applications in synthetic mRNA, and the methods used to assess its effects on mRNA translation.
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Liu, Bing, Lin Liu, Anna Tsykin, Gregory J. Goodall, Murray J. Cairns, and Jiuyong Li. "Discovering Functional microRNA-mRNA Regulatory Modules in Heterogeneous Data." In Advances in Experimental Medicine and Biology, 267–90. Dordrecht: Springer Netherlands, 2012. http://dx.doi.org/10.1007/978-94-007-5590-1_14.
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Domashevskiy, Artem V., David J. Rodriguez, Dilantha Gunawardana, and Dixie J. Goss. "Preparation of Functional, Fluorescently Labeled mRNA Capped with Anthraniloyl-m7GpppG." In Methods in Molecular Biology, 61–75. New York, NY: Springer New York, 2016. http://dx.doi.org/10.1007/978-1-4939-3625-0_4.
Full textConference papers on the topic "Functional mRNA"
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Tamim, Saleh, and Jianhua Ruan. "Functional evaluation and analysis of predicted miRNA-mRNA regulatory network." In the 2012 ACM Research in Applied Computation Symposium. New York, New York, USA: ACM Press, 2012. http://dx.doi.org/10.1145/2401603.2401618.
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Neidhart, M., J. Rethage, O. Distler, MT Peyer, ME Billingham, BA Michel, RE Gay, and S. Gay. "THU0092 The l1 mrna in rheumatoid arthritis encodes a functional retrotransposable element." In Annual European Congress of Rheumatology, Annals of the rheumatic diseases ARD July 2001. BMJ Publishing Group Ltd and European League Against Rheumatism, 2001. http://dx.doi.org/10.1136/annrheumdis-2001.969.
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Goldenkova-Pavlova, I. V., O. Mustafayev, I. V. Deineko, and A. A. Tyurin. "Fine translational control of mRNA: a complex web of mechanisms and its relevance for functional genomics and plant biotechnology." In 2nd International Scientific Conference "Plants and Microbes: the Future of Biotechnology". PLAMIC2020 Organizing committee, 2020. http://dx.doi.org/10.28983/plamic2020.089.
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Based on the results of our own research and literature data, we will present the main theoretical and experimental approaches to studying the translation efficiency of plant mRNA and highlight their contribution to functional plant genomics and biotechnology.
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Rhee, Sungmin, Sangsoo Lim, and Sun Kim. "Iterative segmented least square method for functional microRNA-mRNA module discovery in breast cancer." In 2016 IEEE International Conference on Bioinformatics and Biomedicine (BIBM). IEEE, 2016. http://dx.doi.org/10.1109/bibm.2016.7822618.
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Goldenkova-Pavlova, I. V., O. Mustafayev, I. V. Deineko, and A. A. Tyurin. "Fine translational control of mRNA: a complex net of mechanisms and its relevance for functional genomics and plant biotechnology." In IX Congress of society physiologists of plants of Russia "Plant physiology is the basis for creating plants of the future". Kazan University Press, 2019. http://dx.doi.org/10.26907/978-5-00130-204-9-2019-123.
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Sriram, Krishna, Kevin Moyung, Ross Corriden, and Paul Insel. "Abstract 3293: Solid tumors have frequent mutation, copy number variation and differential mRNA expression of GPCRs: Are such GPCRs functional oncogenes." In Proceedings: AACR Annual Meeting 2018; April 14-18, 2018; Chicago, IL. American Association for Cancer Research, 2018. http://dx.doi.org/10.1158/1538-7445.am2018-3293.
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Marquerie, G., A. Duperray, G. Uzan, and R. Berthier. "BIOSYNTHETIC PATHWAYS OF THE PLATELET FIBRINOGEN RECEPTOR IN HUMAN MEGAKARYOCYTES." In XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1642954.
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Interaction between cells and between cells and extracellular matrices are critical for a number of biological processes, including organ development, cell differenciation, cell motility, and the inimune' response. These interactions are mediated by a family of adhesion receptors that recognize short sequences such as Arg-Gly-Asp (RGD). These receptors share similar structural properties. They are heterodimers composed of a and B subunits and sometime express common epitopes. This suggests that the structural and functional relationship of these receptors may result from the transcription of related genes or may arise from cell specific post-transcriptional events. Thus, analysis of the biosynthesis and processing of these receptors would provide valuable insights into the molecular mechanism which control their expression at the surface,of different cells. Platelet membrane glycoprotein (GP) IIb-IIIa is a member of this receptor family. This protein is a non covalent heterodimer composed of two distinct polypeptides, Glib which consists of two subunits Ilba and IlbB (Mr = 116 kD, Mr = 25 kD) and GPIIIa (Mr = 100 kD, reduced). GPIIb-IIIa functions at site of platelet aggregation and serves as receptor for RGD containing factors including fibrinogen, fibronectin and von Willebrand factor. We report here on the investigation of the biosynthetic pathways of this RGD receptor in human megakaryocytes. High number of megakaryocytic cells from the megakaryoblastic stage to the polyploid mature megakaryocyte were obtained from liquid culture of cryopreserved leukocyte stem cell concentrates from patients with chronic myelogenous leukemia (CML). After sorting, using a FACS IV and indirect immunofluores-cent labeling with monoclonal antibodies anti-GPIIb-IIIa, 95 % of the cells in culture were of the megakaryocytic lineage. These megakarocytes represented an excellent tool to delineate at the molecular level events associated with the biosynthesis of GPIIb-IIIa.Metabolic labeling and pulse-chase experiments indicated that GPIIb and GPIIIa are synthesized from separate mRNA and that the two subunits of GPIIb derive from a common precursor. This was further confirmed by cell-free translation of megakaryocyte mRNA and the identification of separate cDNA containing sequences coding for the pro-GPIIb and for GPIIIa. These cDNA were isolated from a Xgt11 expression library constructed with purified megakaryocyte RNA, and were used to size the messengers coding for the two polypeptides. A single mRNA species of 3.9 kB was found to encode the pro-GPIIb, whereas two different mRNA species of 2.9 kB and 4. 1 kB were identified with the GPIIIa cDNA.The newly synthesized GPIIIa associates early with the pro-GPIIb in the rough endoplasmic reticulum. Examination of the glycosylation pathways with endoglycosidase H, tunicamycin and monensin indicated that high mannose oligosaccharides are added to the GPIIIa and pro-GPIIb polypeptide backbone. The pro-GPIIb is then processed with conversion of high mannose to the complex type carbohydrate, whereas GPIIIa remains endoH sensitive. Glycosylation of pro-GPIIb-IIIa and processing of oligosaccharides are prerequisite for proteolytic maturation of pro-GPIIb and the expression of the mature complex at the surface of the cell. Thus post-translational processing of GPIIb-IIIa requires an early assembly of the complex. This may have important implications in the maturation of megakaryocyte granules and in the molecular mechanism underlying the Glanzmann thrombastenic disease.
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He, Ming-Xiao, Courtney Anderson, Na Li, Xiao-Jun Ma, and Emily Park. "Abstract 537: Evaluation of immune function in the tumor microenvironment by RNAin situhybridization to reveal spatial information on immune cell infiltration and the expression of functional mRNA biomarkers." In Proceedings: AACR 107th Annual Meeting 2016; April 16-20, 2016; New Orleans, LA. American Association for Cancer Research, 2016. http://dx.doi.org/10.1158/1538-7445.am2016-537.
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Hubert, Christopher G., Yu Ding, Chad Toledo, Patrick J. Paddison, James M. Olson, and Jason Berndt. "Abstract 5118: A functional genetic approach in patient-derived glioblastoma stem cells reveals pre-mRNA splicing components to be cancer-lethal gene targets." In Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL. American Association for Cancer Research, 2012. http://dx.doi.org/10.1158/1538-7445.am2012-5118.
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den Biezen, Eveline, Saskia Vermeer, Diederick Keizer, Martijn Akse, Martijn van Zelst, Dianne van Strijp, Hannah Park, and Anthony Magliocco. "Abstract 740: A robust rapid mRNA based test to profile simultaneously ER, AR, PI3K and MAPK functional signaling pathway activity for precision oncology." In Proceedings: AACR Annual Meeting 2021; April 10-15, 2021 and May 17-21, 2021; Philadelphia, PA. American Association for Cancer Research, 2021. http://dx.doi.org/10.1158/1538-7445.am2021-740.
Full textReports on the topic "Functional mRNA"
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Reddy, A. S. N. Functional analysis of U1-70K interacting SR proteins in pre-mRNA splicing in Arabidopsis. Office of Scientific and Technical Information (OSTI), November 2008. http://dx.doi.org/10.2172/941683.
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Stern, David, and Gadi Schuster. Manipulation of Gene Expression in the Chloroplast. United States Department of Agriculture, September 2000. http://dx.doi.org/10.32747/2000.7575289.bard.
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The steady-state level of a given mRNA is determined by its rates of transcription and degradation. The stabilities of chloroplast mRNAs vary during plant development, in part regulating gene expression. Furthermore, the fitness of the organelle depends on its ability to destroy non-functional transcripts. In addition, there is a resurgent interest by the biotechnology community in chloroplast transformation due to the public concerns over pollen transmission of introduced traits or foreign proteins. Therefore, studies into basic gene expression mechanisms in the chloroplast will open the door to take advantage of these opportunities. This project was aimed at gaining mechanistic insights into mRNA processing and degradation in the chloroplast and to engineer transcripts of varying stability in Chlamydomonas reinhardtii cells. This research uncovered new and important information on chloroplast mRNA stability, processing, degradation and translation. In particular, the processing of the 3' untranslated regions of chloroplast mRNAs was shown to be important determinants in translation. The endonucleolytic site in the 3' untranslated region was characterized by site directed mutagensis. RNA polyadenylation has been characterized in the chloroplast of Chlamydomonas reinhardtii and chloroplast transformants carrying polyadenylated sequences were constructed and analyzed. Data obtained to date suggest that chloroplasts have gene regulatory mechanisms which are uniquely adapted to their post-endosymbiotic environment, including those that regulate RNA stability. An exciting point has been reached, because molecular genetic studies have defined critical RNA-protein interactions that participate in these processes. However, much remains to be learned about these multiple pathways, how they interact with each other, and how many nuclear genes are consecrated to overseeing them. Chlamydomonas is an ideal model system to extend our understanding of these areas, given its ease of manipulation and the existing knowledge base, some of which we have generated.
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Meidan, Rina, and Robert Milvae. Regulation of Bovine Corpus Luteum Function. United States Department of Agriculture, March 1995. http://dx.doi.org/10.32747/1995.7604935.bard.
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The main goal of this research plan was to elucidate regulatory mechanisms controlling the development, function of the bovine corpus luteum (CL). The CL contains two different sterodigenic cell types and therefore it was necessary to obtain pure cell population. A system was developed in which granulosa and theca interna cells, isolated from a preovulatory follicle, acquired characteristics typical of large (LL) and small (SL) luteal cells, respectively, as judged by several biochemical and morphological criteria. Experiments were conducted to determine the effects of granulosa cells removal on subsequent CL function, the results obtained support the concept that granulosa cells make a substaintial contribution to the output of progesterone by the cyclic CL but may have a limited role in determining the functional lifespan of the CL. This experimental model was also used to better understand the contribution of follicular granulosa cells to subsequent luteal SCC mRNA expression. The mitochondrial cytochrome side-chain cleavage enzyme (SCC), which converts cholesterol to pregnenolone, is the first and rate-limiting enzyme of the steroidogenic pathway. Experiments were conducted to characterize the gene expression of P450scc in bovine CL. Levels of P450scc mRNA were higher during mid-luteal phase than in either the early or late luteal phases. PGF 2a injection decreased luteal P450scc mRNA in a time-dependent manner; levels were significantly reduced by 2h after treatment. CLs obtained from heifers on day 8 of the estrous cycle which had granulosa cells removed had a 45% reduction in the levels of mRNA for SCC enzymes as well as a 78% reduction in the numbers of LL cells. To characterize SCC expression in each steroidogenic cell type we utilized pure cell populations. Upon luteinization, LL expressed 2-3 fold higher amounts of both SCC enzymes mRNAs than SL. Moreover, eight days after stimulant removal, LL retained their P4 production capacity, expressed P450scc mRNA and contained this protein. In our attempts to establish the in vitro luteinization model, we had to select the prevulatory and pre-gonadotropin surge follicles. The ratio of estradiol:P4 which is often used was unreliable since P4 levels are high in atretic follicles and also in preovulatory post-gonadotropin follicles. We have therefore examined whether oxytocin (OT) levels in follicular fluids could enhance our ability to correctly and easily define follicular status. Based on E2 and OT concentrations in follicular fluids we could more accurately identify follicles that are preovulatory and post gonadotropin surge. Next we studied OT biosynthesis in granulosa cells, cells which were incubated with forskolin contained stores of the precursor indicating that forskolin (which mimics gonadotropin action) is an effective stimulator of OT biosynthesis and release. While studying in vitro luteinization, we noticed that IGF-I induced effects were not identical to those induced by insulin despite the fact that megadoses of insulin were used. This was the first indication that the cells may secrete IGF binding protein(s) which regonize IGFs and not insulin. In a detailed study involving several techniques, we characterized the species of IGF binding proteins secreted by luteal cells. The effects of exogenous polyunsaturated fatty acids and arachidonic acid on the production of P4 and prostanoids by dispersed bovine luteal cells was examined. The addition of eicosapentaenoic acid and arachidonic acid resulted in a dose-dependent reduction in basal and LH-stimulated biosynthesis of P4 and PGI2 and an increase in production of PGF 2a and 5-HETE production. Indomethacin, an inhibitor of arachidonic acid metabolism via the production of 5-HETE was unaffected. Results of these experiments suggest that the inhibitory effect of arachidonic acid on the biosynthesis of luteal P4 is due to either a direct action of arachidonic acid, or its conversion to 5-HETE via the lipoxgenase pathway of metabolism. The detailed and important information gained by the two labs elucidated the mode of action of factors crucially important to the function of the bovine CL. The data indicate that follicular granulosa cells make a major contribution to numbers of large luteal cells, OT and basal P4 production, as well as the content of cytochrome P450 scc. Granulosa-derived large luteal cells have distinct features: when luteinized, the cell no longer possesses LH receptors, its cAMP response is diminished yet P4 synthesis is sustained. This may imply that maintenance of P4 (even in the absence of a Luteotropic signal) during critical periods such as pregnancy recognition, is dependent on the proper luteinization and function of the large luteal cell.
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Fields, Michael J., Mordechai Shemesh, and Anna-Riitta Fuchs. Significance of Oxytocin and Oxytocin Receptors in Bovine Pregnancy. United States Department of Agriculture, August 1994. http://dx.doi.org/10.32747/1994.7568790.bard.
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Oxytocin has multiple actions in bovine reproductive tract and it was our purpose to determine the nature of these actions and their significance for the physiology of bovine reproduction. The bovine oxytocin receptors (OTR) gene was cloned and its expression studied during the cycle and pregnancy. OTR mRNA changed in parallel with OTR with control occurring mainly at the transcriptional level. However, the endocrine regulation of OTR were found in endometrium and cervical mucosa at estrus and at parturition. In both tissues OTR were suppressed in the luteal phase and early pregnancy. Whereas cervical OTR remained suppressed throughout pregnancy, endometrial OTR began to increase soon after implantation and reached higher concentrations in midpregnancy than at estrus. OTR in caruncles did not increase until third trimester, and OTR in cervical mucosa, cotyledons and fetal membranes increased only at term. Myometrial OTR showed less variation and OTR were present throughout the cycle and pregnancy but increased significantly during mid- and late pregnancy. OTR were localized in endometrial epithelial cells and lumina epithelial cells of cervical mucosa as determined by immunohistochemistry. Endometrial OTR were functional throughout pregnancy and mediated PGF release from day 50 onwards in a receptor density related manner. OTR in cervical mucosa mediated PGE release both in vivo and in vitro, as shown in cyclic cows. The ontogeny of uterine OTR was studied from third trimester fetal stage until puberty. OTR were present in endometrium and cervical mucosa in high concentrations throughout this period; myometrial OTR began to increase somewhat later but also reached adult values by 6-mo of age. In the prepuberal heifers OT injections failed to initiate PGF2a, release. The influence of steroids on the effect of OT was examined. Ovariectomy and E2 were without effect, but P4 with or without E2 induced a massive PGF2a release in response to OT in spite of reduced OTR. Bovine cyclooxygenases (COX-1 and COX-2) were cloned and their expression studied in the endometrium of prepuberal heifers and pregnant cows. Untreated and E2 treated prepuberal heifers did not express COX-2 but P4 treated heifers did express the mRNA for COX-2, albeit weakly. During the second half of pregnancy COX-2 mRNA was strongly expressed in cotyledons and somewhat less in caruncles, whereas endometrium, myometrium and cervical mucosa showed only weak, if any, COX-2 mRNA under basal conditions. However, 2 h after OT injection significant increases in COX-2 mRNA were found in endometrial RNA. Thus OT is capable of inducing the expression of the inducible COX-2 gene, and hence the conversion of arachidonic acid to prostanoids. The results indicate that the functions of OT are numerous and probably essential for successful pregnancy and parturition.
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Sessa, Guido, and Gregory Martin. A functional genomics approach to dissect resistance of tomato to bacterial spot disease. United States Department of Agriculture, January 2004. http://dx.doi.org/10.32747/2004.7695876.bard.
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The research problem. Bacterial spot disease in tomato is of great economic importance worldwide and it is particularly severe in warm and moist areas affecting yield and quality of tomato fruits. Causal agent of spot disease is the Gram-negative bacterium Xanthomonas campestris pv. vesicatoria (Xcv), which can be a contaminant on tomato seeds, or survive in plant debris and in association with certain weeds. Despite the economic significance of spot disease, plant protection against Xcvby cultural practices and chemical control have so far proven unsuccessful. In addition, breeding for resistance to bacterial spot in tomato has been undermined by the genetic complexity of the available sources of resistance and by the multiple races of the pathogen. Genetic resistance to specific Xcvraces have been identified in tomato lines that develop a hypersensitive response and additional defense responses upon bacterial challenge. Central goals of this research were: 1. To identify plant genes involved in signaling and defense responses that result in the onset of resistance. 2. To characterize molecular properties and mode of action of bacterial proteins, which function as avirulence or virulence factors during the interaction between Xcvand resistant or susceptible tomato plants, respectively. Our main achievements during this research program are in three major areas: 1. Identification of differentially expressed genes during the resistance response of tomato to Xcvrace T3. A combination of suppression subtractive hybridization and microarray analysis identified a large set of tomato genes that are induced or repressed during the response of resistant plants to avirulent XcvT3 bacteria. These genes were grouped in clusters based on coordinate expression kinetics, and classified into over 20 functional classes. Among them we identified genes that are directly modulated by expression of the type III effector protein AvrXv3 and genes that are induced also during the tomato resistance response to Pseudomonas syringae pv. tomato. 2. Characterization of molecular and biochemical properties of the tomato LeMPK3MAP kinase. A detailed molecular and biochemical analysis was performed for LeMPK3 MAP kinase, which was among the genes induced by XcvT3 in resistant tomato plants. LeMPK3 was induced at the mRNA level by different pathogens, elicitors, and wounding, but not by defense-related plant hormones. Moreover, an induction of LeMPK3 kinase activity was observed in resistant tomato plants upon Xcvinfection. LeMPK3 was biochemically defined as a dual-specificity MAP kinase, and extensively characterized in vitro in terms of kinase activity, sites and mechanism of autophosphorylation, divalent cation preference, Kₘand Vₘₐₓ values for ATP. 3. Characteriztion of molecular properties of the Xcveffector protein AvrRxv. The avirulence gene avrRxvis involved in the genetic interaction that determines tomato resistance to Xcvrace T1. We found that AvrRxv functions inside the plant cell, localizes to the cytoplasm, and is sufficient to confer avirulence to virulent Xcvstrains. In addition, we showed that the AvrRxv cysteine protease catalytic core is essential for host recognition. Finally, insights into cellular processes activated by AvrRxv expression in resistant plants were obtained by microarray analysis of 8,600 tomato genes. Scientific and agricultural significance: The findings of these activities depict a comprehensive and detailed picture of cellular processes taking place during the onset of tomato resistance to Xcv. In this research, a large pool of genes, which may be involved in the control and execution of plant defense responses, was identified and the stage is set for the dissection of signaling pathways specifically triggered by Xcv.
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Lers, Amnon, and Pamela J. Green. LX Senescence-Induced Ribonuclease in Tomato: Function and Regulation. United States Department of Agriculture, September 2003. http://dx.doi.org/10.32747/2003.7586455.bard.
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Natural leaf senescence, which occurs even when growth conditions are near optimal, has a negative influence on yield. Postharvest induced senescence contributes to the losses of quality in flowers, foliage, and vegetables. Strategies designed to control the senescence process in crop plants could therefore have great applied significance. However, the successful design of such strategies requires a better insight into the senescence machinery and control in higher plants. A main feature of senescence is the hydrolysis of macromolecules by hydrolases of various types such as ribonucleases (RNases) and proteases. Previously we had identified and characterized the tomato LX RNase gene demonstrating its transcript to be highly and specifically induced during senescence. This reported study was focused on LX but also had broadened our research to other senescence-associated nucleic acids degrading enzymes to learn about their function and the regulation of their encoding genes. Beside tomato we used parsley and Arabidopsis for the study of: the bi-functional nuclease which has a role in senescence. The study of different senescence- associated nucleases in few plant systems will allow a more general view on function and regulation of these enzymes in senescence. The specific original proposed objectives included: 1. Study the consequences of alterations in LX RNase level on tomato leaf senescence and general development; 2. Analyze stimuli which may participate in senescence-specific activation of the LX gene; 3. Clone the senescence-associated BFNI nuclease gene homologue from tomato. 4. Further characterize the sequences required for senescence-specific gene expression. Homozygous transgenic plants in which LX gene was either inhibited or over-expressed were generated. In both of these LX mutated plants no major phenotypic consequences were observed, which may suggests that LX is not essential for plant growth under optimal growth conditions. Lack of any abnormalities in the LX over-expressing lines suggests that special system exist to allow function of the RNase only when needed. Detailed analyses of growth under stress and consequences to RNA metabolism are underway. We have analyzed LX expression on the protein level demonstrating that it is involved also in petal senescing. Our results suggest that LX is responding to complex regulation involving developmental, organ dependent factors and responds differently to hormonal or environmental stimuli in the different plant organs. The cloned 1.4 kb promoter was cloned and its analysis revealed that probably not all required elements for senescence induction are included. Biochemical analysis of senescence-associated be-functional nucleases in the different plants, tomato, parsley and Arabidopsis, suggests they belong to a sub-class within the type I plant nucleases. The parsley PcNUC1/2 nuclease protein was purified from senescing leaves its and activity was studied in vitro revealing endo-, double strand, nucleolytic activity and exo-nucleolytic activity. Its encoding gene was cloned and found to be induced on the mRNA level. The promoter of the related Arabidopsis BFNI nuclease was shown in both tomato and Arabidopsis to be able and direct senescence-specific expression suggesting that, at least part, the gene is regulated on the transcriptional level and that the mechanism for this senescence-specific regulation is conserved between different plants. Few plants in which the BFNI gene is mutated were identified which are subjected now to detailed analysis. Our results suggest that the senescence-related nucleic acid degrading enzymes share similarities in both function and regulation between different plants and possibly have important functions in processes un-related to senescence. Still, the function of these enzymes, at least in some cases is not essential to plant development under optimal growth conditions. We are now at the stage which permits in depth investigation of the specific functions and mode of molecular regulation of senescence-associated nucleases with the aid of the research tools developed. The isolated senescence-specific promoter, shown to be active in heterologous plant system, could be utilized in agricultural-related biotechnological applications for retardation of senescence.
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Schuster, Gadi, and David Stern. Integration of phosphorus and chloroplast mRNA metabolism through regulated ribonucleases. United States Department of Agriculture, August 2008. http://dx.doi.org/10.32747/2008.7695859.bard.
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New potential for engineering chloroplasts to express novel traits has stimulated research into relevant techniques and genetic processes, including plastid transformation and gene regulation. This proposal continued our long time BARD-funded collaboration research into mechanisms that influence chloroplast RNA accumulation, and thus gene expression. Previous work on cpRNA catabolism has elucidated a pathway initiated by endonucleolytic cleavage, followed by polyadenylation and exonucleolytic degradation. A major player in this process is the nucleus-encoded exoribonuclease/polymerasepolynucleotidephoshorylase (PNPase). Biochemical characterization of PNPase has revealed a modular structure that controls its RNA synthesis and degradation activities, which in turn are responsive to the phosphate (P) concentration. However, the in vivo roles and regulation of these opposing activities are poorly understood. The objectives of this project were to define how PNPase is controlled by P and nucleotides, using in vitro assays; To make use of both null and site-directed mutations in the PNPgene to study why PNPase appears to be required for photosynthesis; and to analyze plants defective in P sensing for effects on chloroplast gene expression, to address one aspect of how adaptation is integrated throughout the organism. Our new data show that P deprivation reduces cpRNA decay rates in vivo in a PNPasedependent manner, suggesting that PNPase is part of an organismal P limitation response chain that includes the chloroplast. As an essential component of macromolecules, P availability often limits plant growth, and particularly impacts photosynthesis. Although plants have evolved sophisticated scavenging mechanisms these have yet to be exploited, hence P is the most important fertilizer input for crop plants. cpRNA metabolism was found to be regulated by P concentrations through a global sensing pathway in which PNPase is a central player. In addition several additional discoveries were revealed during the course of this research program. The human mitochondria PNPase was explored and a possible role in maintaining mitochondria homeostasis was outlined. As polyadenylation was found to be a common mechanism that is present in almost all organisms, the few examples of organisms that metabolize RNA with no polyadenylation were analyzed and described. Our experiment shaded new insights into how nutrient stress signals affect yield by influencing photosynthesis and other chloroplast processes, suggesting strategies for improving agriculturally-important plants or plants with novel introduced traits. Our studies illuminated the poorly understood linkage of chloroplast gene expression to environmental influences other than light quality and quantity. Finely, our finding significantly advanced the knowledge about polyadenylation of RNA, the evolution of this process and its function in different organisms including bacteria, archaea, chloroplasts, mitochondria and the eukaryotic cell. These new insights into chloroplast gene regulation will ultimately support plant improvement for agriculture
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Raghothama, Kashchandra G., Avner Silber, and Avraham Levy. Biotechnology approaches to enhance phosphorus acquisition of tomato plants. United States Department of Agriculture, January 2006. http://dx.doi.org/10.32747/2006.7586546.bard.
Full textAbstract:
Abstract: Phosphorus is one of the least available macronutrient in the soil. The high affinity phosphate transporters are known to be associated with phosphate acquisition under natural conditions. Due to unique interactions of phosphate with soil particles, up to 80% of the applied phosphates may be fixed forcing the farmers to apply 4 to 5 times the fertilizers necessary for crop production. Efficient uptake and utilization of this essential nutrient is essential for sustainability and profitability of agriculture. Many predictions point to utilization/exhaustion of high quality phosphate rocks within this century. This calls for efforts to improve the ability of plants to acquire and utilize limiting sources of phosphate in the rhizosphere. Two important molecular and biochemical components associated with phosphate efficiency are phosphate transporters and phosphatases. This research project is aimed at defining molecular determinants of phosphate acquisition and utilization in addition to generating phosphate uptake efficient plants. The main objectives of the project were; Creation and analysis of transgenic tomato plants over-expressing phosphatases and transporters Characterization of the recently identified members (LePT3 and LePT4) of the Pi transporter family Generate molecular tools to study genetic responses of plants to Pi deficiency During the project period we have successfully identified and characterized a novel phosphate transporter associated with mycorrhizal symbiosis. The expression of this transporter increases with mycorrhizal symbiosis. A thorough characterization of mutant tomato lacking the expression of this gene revealed the biological significance of LePT3 and another novel gene LePT4. In addition we have isolated and characterized several phosphate starvation induced genes from tomato using a combination of differential and subtractive mRNA hybridization techniques. One of the genes, LePS2 belongs to the family of phospho-protein phosphatase. The functionality of the recombinant protein was determined using synthetic phosphor-peptides. Over expression of this gene in tomato resulted in significant changes in growth, delay in flowering and senescence. It is anticipated that phospho-protein phosphatase may have regulatory role in phosphate deficiency responses of plants. In addition a novel phosphate starvation induced glycerol 3-phosphate permease gene family was also characterized. Two doctoral research students are continuing the characterization and functional analysis of these genes. Over expression of high affinity phosphate transporters in tobacco showed increased phosphate content under hydroponic conditions. There is growing evidence suggesting that high affinity phosphate transporters are crucial for phosphate acquisition even under phosphate sufficiency conditions. This project has helped train several postdoctoral fellows and graduate students. Further analysis of transgenic plants expressing phosphatases and transporters will not only reveal the biological function of the targeted genes but also result in phosphate uptake and utilization efficient plants.
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Tucker, Mark L., Shimon Meir, Amnon Lers, Sonia Philosoph-Hadas, and Cai-Zhong Jiang. Elucidation of signaling pathways that regulate ethylene-induced leaf and flower abscission of agriculturally important plants. United States Department of Agriculture, January 2012. http://dx.doi.org/10.32747/2012.7597929.bard.
Full textAbstract:
The Problem: Abscission is a highly regulated process, occurring as a natural terminal stage of development, in which various organs are separated from the parent plant. In most plant species, the process is initiated by a decrease in active auxin in the abscission zone (AZ) and an increase in ethylene, and may be accelerated by postharvest or environmental stresses. Another potential key regulator in abscission is IDA (Inflorescence Deficient in Abscission), which was identified as an essential peptide signal for floral organ abscission in Arabidopsis. However, information is still lacking regarding the molecular mechanisms integrating all these regulators. In our previous BARD funded research we made substantial progress towards understanding these molecular events in tomato, and the study is still in progress. We established a powerful platform for analysis of genes for regulatory proteins expressed in AZ. We identified changes in gene expression for several transcription factors (TFs) directly linked to ethylene and auxin signaling and several additional regulatory proteins not so obviously linked to these hormones. Moreover, we demonstrated using a virus-induced gene silencing (VIGS) assay that several play a functional role in the onset of abscission. Based on these results we have selected 14 genes for further analysis in stably transformed tomato plants. All 14 genes were suppressed by RNA interference (RNAi) using a constitutive promoter, and 5 of them were also suppressed using an abscission-specific promoter. Transformations are currently at different stages of progress including some lines that already display an abscission phenotype. Objectives: We propose here to (1) complete the functional analysis of the stably transformed tomato plants with T2 lines and perform transcriptome analysis using custom abscission-specific microarrays; (2) conduct an indepth analysis of the role of IDA signaling in tomato leaf and flower abscission; (3) perform transcriptome and proteome analyses to extend the earlier gene expression studies to identify transcripts and proteins that are highly specific to the separation layer (i.e., target cells for cell separation) prior to the onset of abscission; (4) extend and compliment the work in tomato using a winnowed set of genes in soybean. Methodology: Next Generation Sequencing (NGS) of mRNA will be used to further increase the list of abscission-associated genes, and for preparation of a custom tomato abscission microarray to test altered gene expression in transgenic plants. Tandem mass spectrometry (LC-MS/MS) of protein extracts from leaf petiole, flower pedicel and their AZ tissues will be used to identify the proteome of the AZ before and during abscission. AZ-specific gene promoters will be used in stably transformed tomato plants to reduce non-target phenotypes. The bean pod mottle virus (BPMV) plasmid vectors will be used for VIGS analysis in soybean. Expected Contribution: Our study will provide new insights into the regulation of ethylene-induced abscission by further revealing the role of key regulators in the process. This will permit development of novel techniques for manipulating leaf and flower abscission, thereby improving the postharvest performance of agriculturally important crops.
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Meidan, Rina, and Joy Pate. Roles of Endothelin 1 and Tumor Necrosis Factor-A in Determining Responsiveness of the Bovine Corpus Luteum to Prostaglandin F2a. United States Department of Agriculture, January 2004. http://dx.doi.org/10.32747/2004.7695854.bard.
Full textAbstract:
The corpus luteum (CL) is a transient endocrine gland that has a vital role in the regulation of the estrous cycle, fertility and the maintenance of pregnancy. In the absence of appropriate support, such as occurs during maternal recognition of pregnancy, the CL will regress. Prostaglandin F2a (PGF) was first suggested as the physiological luteolysin in ruminants several decades ago. Yet, the cellular mechanisms by which PGF causes luteal regression remain poorly defined. In recent years it became evident that the process of luteal regression requires a close cooperation between steroidogenic, endothelial and immune cells, all resident cells of this gland. Changes in the population of these cells within the CL closely consort with the functional changes occurring during various stages of CL life span. The proposal aimed to gain a better understanding of the intra-ovarian regulation of luteolysis and focuses especially on the possible reasons causing the early CL (before day 5) to be refractory to the luteolytic actions of PGF. The specific aims of this proposal were to: determine if the refractoriness of the early CL to PGF is due to its inability to synthesize or respond to endothelin–1 (ET-1), determine the cellular localization of ET, PGF and tumor necrosis factor a (TNF a) receptors in early and mid luteal phases, determine the functional relationships among ET-1 and cytokines, and characterize the effects of PGF and ET-1 on prostaglandin production by luteal cell types. We found that in contrast to the mature CL, administration of PGF2a before day 5 of the bovine cycle failed to elevate ET-1, ETA receptors or to induce luteolysis. In fact, PGF₂ₐ prevented the upregulation of the ET-1 gene by ET-1 or TNFa in cultured luteal cells from day 4 CL. In addition, we reported that ECE-1 expression was elevated during the transitionof the CL from early to mid luteal phase and was accompanied by a significant rise in ET-1 peptide. This coincides with the time point at which the CL gains its responsiveness to PGF2a, suggesting that ability to synthesize ET-1 may be a prerequisite for luteolysis. We have shown that while ET-1 mRNA was exclusively localized to endothelial cells both in young and mature CL, ECE-1 was present in the endothelial cells and steroidogenic cells alike. We also found that the gene for TNF receptor I is only moderately affected by the cytokines tested, but that the gene for TNF receptor II is upregulated by ET-1 and PGF₂ₐ. However, these cytokines both increase expression of MCP-1, although TNFa is even more effective in this regard. In addition, we found that proteins involved in the transport and metabolism of PGF (PGT, PGDH, COX-2) change as the estrous cycle progresses, and could contribute to the refractoriness of young CL. The data obtained in this work illustrate ET-1 synthesis throughout the bovine cycle and provide a better understanding of the mechanisms regulating luteal regression and unravel reasons causing the CL to be refractory to PGF2a.