1,721,015 research outputs found
Functions and Specificities of Tristetraprolin (TTP) Family Members
No full textMembers of the tristetraprolin (TTP) family of RNA-binding proteins bind to mRNAs that contain specific AU-rich element (ARE) binding sites and promote the decay of target mRNAs. The defining feature of all TTP family members is the presence of a tandem zinc finger (TZF) domain that binds to AREs in the 3’-untranslated regions (3’-UTR) of target mRNAs. Many family members also contain a CNOT1 binding domain that has been shown to bind to CNOT1, a large scaffolding protein of the CCR4-NOT complex. Mice expressing TTP protein with the CNOT1 binding domain deleted (CNBD mice), developed only a mild inflammatory phenotype, in stark contrast to the severe phenotype of TTP KO mice, or mice expressing TTP with a C116R point mutation in the tandem zinc finger domain. These data suggest that the CNOT1 binding domain is important for some of TTP’s physiological functions, but not as critical as the TZF domain for TTP’s function. Yet, it remains unclear whether the CNOT1 binding domain of TTP is important to regulate specific targets in specific tissues.Three TTP family proteins are conserved in mammals (TTP, ZFP36L1, and ZFP36L2), encoded by the mouse genes Zfp36, Zfp36l1, and Zfp36l2, respectively. TTP, ZFP36L1, and ZFP36L2 behave similarly biochemically in assays of RNA-binding, mRNA deadenylation, and decay. Yet, knock-out (KO) mice for each gene have very different phenotypes, suggesting that each TTP family member has specific physiological functions. ZFP36 (TTP) is known for regulating cytokine expression in myeloid cells, and its deficiency leads to a severe, spontaneous, inflammatory phenotype; however, ZFP36L1 and ZFP36L2 have not been viewed as important in controlling inflammation. It is unclear whether the biochemical activities of these proteins are interchangeable or independent, and/or whether effects on target transcripts are solely dependent on the cell-specific expression of each protein. It is also unknown whether synergistic interactions exist among TTP family members and whether they can compensate for one another when the expression levels are altered.
In the major project described in this thesis, I studied potential functional overlaps of these proteins in myeloid cells, by developing myeloid-specific knock-out (M-KO) mice of these genes, singly and together. M-Zfp36-KO mice exhibited a mild inflammatory syndrome late in life, while M-Zfp36l1-KO and M-Zfp36l2-KO mice had no apparent spontaneous phenotypes. Mice with simultaneous deficiency of all three TTP family members in myeloid cells, referred to as M-triple KO mice, developed a severe spontaneous inflammatory phenotype, with a median survival of 8 weeks. Histopathological evaluation showed severe arthritis of peripheral joints and dramatic myeloid hyperplasia in tissues and bone marrow, as well as soft tissue inflammatory cell invasion. MicroCT analysis of the front and hind paws indicated severe bone loss and joint destruction and ankylosis. RNA-Seq analysis of mRNA from triple KO macrophages treated with LPS, followed by actinomycin D to inhibit transcription and allow for measurement of mRNA decay rates, demonstrated abnormal stabilization of many more cytokine and chemokine mRNAs than were seen in similar studies of cells from myeloid-specific TTP KO mice. Cytokine immunoassays also demonstrated increased levels of pro-inflammatory cytokines in serum from triple KO mice and in medium from LPS-stimulated M-triple KO macrophages. These findings suggest that simultaneous deficiency of Zfp36, Zfp36l1, and Zfp36l2 in myeloid cells leads to the synergistic development of a lethal inflammatory syndrome due to excess accumulation of pro-inflammatory cytokines. Our findings emphasize the importance of all three family members, acting in concert, in myeloid cell function.
As noted above, TTP has been shown to regulate cytokine mRNA stability, and loss of TTP leads to chronic excess levels of many pro-inflammatory cytokines. Many autoimmune diseases are characterized by chronic excess levels of the same cytokines that are increased in Zfp36-KO mice. Therefore, we speculated that increased expression of TTP could have a beneficial effect on inflammatory diseases. Mice with regulated overexpression of TTP are protected from many models of inflammatory diseases in mice. In a separate project, we and collaborators demonstrated that mice overexpressing TTP were protected from a two-stage carcinogenesis model. I used RNA-Seq to identify transcriptome changes, and found that many pro-inflammatory genes were down-regulated in the skin from mice overexpressing TTP, compared to WT, after exposure to 12-0 tetradeccanoylphorbol-13-accetate (TPA) and dimethylbenz[a]anthracene (DMBA) in an established two-stage model of skin carcinogenesis.
In a third project described in this thesis, we hypothesized that the C-terminal portion of TTP, which contains the CNOT1 binding domain, is vital to recruit exonucleases and promote deadenylation of the target mRNA. To determine if deletion of the CNOT1 binding domain of TTP in mice has effects on transcript turnover in mice, I chose four tissues in which TTP is expressed (liver, spleen, colon, and adipose tissue), and performed transcriptome analysis and differential gene expression analysis in these tissues from WT, TTP KO, and CNBD mice. We found that potential TTP target transcripts were differentially regulated in tissues from mice expressing TTP protein lacking the CNOT1 binding domain. Some transcripts were up-regulated to similar levels in tissues from both TTP KO and TTP CNBD mice, while other transcripts were up-regulated at higher levels in tissues from TTP KO mice than in tissues from TTP CNBD mice. These data suggest that the CNOT1 binding domain is important, but not the only factor necessary, for the ability of TTP to regulate mRNA stability in tissues, such as liver, spleen, colon, and adipose tissue.
The work described in this dissertation increases our understanding of the functions and specificity of TTP family members, and the therapeutic potential of TTP and its family members in the treatment of inflammatory diseases.
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Characterization of a Full-Length TTP Family Member Association with RNA Sequence Elements
No full textPost-transcriptional regulation of cytoplasmic mRNAs is an efficient mechanism of regulating the amounts of active protein within a eukaryotic cell. RNA sequence elements located in the untranslated regions of mRNAs can influence transcript degradation or translation through associations with RNA-binding proteins. Tristetraprolin (TTP) is the best known member of a family of CCCH zinc finger proteins that targets adenosine-uridine rich element (ARE) binding sites in the 3’ untranslated regions (UTRs) of mRNAs, promoting transcript deadenylation through the recruitment of deadenylases. More specifically, TTP has been shown to bind AREs located in the 3’-UTRs of transcripts with known roles in the inflammatory response. The mRNA-binding region of the protein is the highly conserved CCCH tandem zinc finger (TZF) domain. The synthetic TTP TZF domain has been shown to bind with high affinity to the 13-mer sequence of UUUUAUUUAUUUU. However, the binding affinities of full-length TTP family members to the same sequence and its variants are unknown. Furthermore, the distance needed between two overlapping or neighboring UUAUUUAUU 9-mers for tandem binding events of a full-length TTP family member to a target transcript has not been explored. To address these questions, we recombinantly expressed and purified the full-length C. albicans TTP family member Zfs1. Using full-length Zfs1, tagged at the N-terminus with maltose binding protein (MBP), we determined the binding affinities of the protein to the optimal TTP binding sequence, UUAUUUAUU. Fluorescence anisotropy experiments determined that the binding affinities of MBP-Zfs1 to non-canonical AREs were influenced by ionic buffer strength, suggesting that transcript selectivity may be affected by intracellular conditions. Furthermore, electrophoretic mobility shift assays (EMSAs) revealed that separation of two core AUUUA sequences by two uridines is sufficient for tandem binding of MBP-Zfs1. Finally, we found evidence for tandem binding of MBP-Zfs1 to a 27-base RNA oligonucleotide containing only a single ARE-binding site, and showed that this was concentration and RNA length dependent; this phenomenon had not been seen previously. These data suggest that the association of the TTP TZF domain and the TZF domains of other species, to ARE-binding sites is highly conserved. Domains outside of the TZF domain may mediate transcript selectivity in changing cellular conditions, and promote protein-RNA interactions not associated with the ARE-binding TZF domain. In summary, the evidence presented here suggests that Zfs1-mediated decay of mRNA targets may require additional interactions, in addition to ARE-TZF domain associations, to promote transcript destabilization and degradation. These studies further our understanding of post-transcriptional steps in gene regulation.</p
ZFP36L3: a Unique Member of the Tristetraprolin Family of RNA-Binding Tandem Zinc Finger Proteins
No full textMembers of the tristetraprolin (TTP) family of CCCH tandem zinc finger proteins bind to AU-rich elements in the 3' untranslated regions of certain cellular mRNAs, leading to their deadenylation and destabilization. Studies in knockout mice have demonstrated roles for three of the family members, TTP, ZFP36L1 (L1), and ZFP36L2 (L2), in inflammation, chorioallantoic fusion, and hematopoiesis, respectively. However, little is known about a recently-discovered TTP family member, ZFP36L3 (L3). Although L3 exhibits similar general biochemical functions to other members of the TTP family, initial studies of this family member revealed a number of unique characteristics.First, L3 does not shuttle between the nucleus and cytoplasm like TTP, L1, and L2. Through studies of L3 deletion mutants, we determined that a nuclear localization signal that resides within the conserved tandem zinc finger domain was functional, although the C-terminal nuclear export sequence was non-functional. We then demonstrated that the unique repeat domain of L3 was responsible for the "full-time" cytoplasmic localization of the protein and was able to override the ability of the nuclear localization signal to direct transport into the nucleus.In addition, L3 is specifically expressed in rodent yolk sac and placenta, while the other members of the TTP family exhibit relatively ubiquitous expression. We further examined the expression of L3 at both the RNA and protein level. Through northern and western blotting, we demonstrated the expression of L3 during mid-to-late gestation in mouse placenta. We also performed immunostaining of placental sections to demonstrate that this protein is exclusively expressed in the cytoplasm of the labyrinthine trophoblast cells and trophoblast giant cells of the placenta.L3 most likely binds to and promotes the decay of a certain set of mRNA transcripts. Because of its specific sites of expression, we hypothesized that L3 may regulate the decay of a set of mRNAs that are important for the development or physiology of the placenta. We employed the ribonucleoprotein immunoprecipitation-microarray analysis of mouse placenta lysates to identify possible mRNA targets of L3. Our study identified approximately 400 transcripts that were enriched in immunoprecipitates using a highly specific L3 antibody. Some of these transcripts could be bound and downregulated by L3 in a physiological setting. Our top candidate transcript, based on relative enrichment and sequence analysis, was B-type natriuretic peptide, a hormone well-known for its role in cardiac physiology. We confirmed the expression of B-type natriuretic peptide in mouse placenta through northern blotting and in situ hybridization histochemistry. We also verified the ability of L3 to directly bind to and promote the degradation of this transcript in electrophoretic mobility shift assays and co-transfection assays, respectively.Lastly, L3 demonstrates a unique migration characteristic in denaturing polyacrylamide gel electrophoresis as compared to TTP, L1, and L2. It migrates as two distinct species of Mr ~90,000 and ~100,000. We investigated the basis for this unusual migration in studies of deletion mutants and serine mutants. We found that both phosphorylation and the presence of the conserved C-terminus are required for the existence of the slower-migrating species. We then focused our study on phosphorylation of the C-terminus and discovered that the phosphorylation of Ser721 may play a role in creating the slower-migrating species. We also identified four other phosphorylated residues with mass spectrometry. Finally, we examined the effect of the C-terminus on the function of L3 and determined that this conserved region is not required for mRNA binding or to promote mRNA deadenylation or degradation in our assays.The work described in this dissertation increases our understanding of this unique tristetraprolin family member, L3. Additional study of this protein is required to further elucidate its role in the physiology of rodent placenta, and to determine whether this role is subsumed by one of the other TTP family members in the placentas of other mammals.</p
Going Beyond Counting First Authors in Author Co-citation Analysis
Get PDFThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Variations on the Author
Get PDF“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
Appropriate Similarity Measures for Author Cocitation Analysis
Get PDFWe provide a number of new insights into the methodological discussion about author cocitation analysis. We first argue that the use of the Pearson correlation for measuring the similarity between authors’ cocitation profiles is not very satisfactory. We then discuss what kind of similarity measures may be used as an alternative to the Pearson correlation. We consider three similarity measures in particular. One is the well-known cosine. The other two similarity measures have not been used before in the bibliometric literature. Finally, we show by means of an example that our findings have a high practical relevance.information science;Pearson correlation;cosine;similarity measure;author cocitation analysis
Dispelling the Myths Behind First-author Citation Counts
Get PDFWe conducted a full-scale evaluative citation analysis study of scholars in the XML research field to explore just how different from each other author rankings resulting from different citation counting methods actually are, and to demonstrate the capability of emerging data and tools on the Web in supporting more realistic citation counting methods. Our results contest some common arguments for the continued
use of first-author citation counts in the evaluation of scholars, such as high correlations between author rankings by first-author citation counts and other citation
counting methods, and high costs of using more realistic citation counting methods that are not well-supported by the ISI databases. It is argued that increasingly available digital full text research papers make it possible for citation analysis studies to go beyond what the ISI databases have directly supported and to employ more
sophisticated methods
koamabayili/VECTRON-author-checklist: VECTRON author checklist
No full textWe have done our best to complete the author checklist relating to the use of animals in the hut study. Note that the objective for the hut study was to evaluate the IRS treatment applications for residual efficacy against Anopheles mosquitoes, including the local An. coluzzii mosquito population. Cows were only used to attract mosquitoes into the huts and no tests were carried out directly on the cows. The author checklist is intended for use with studies where experiments are carried out on animals, which is why we have had such difficulty in completing this for the hut study, as many of the questions do not relate to how the cows were used
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