1,721,007 research outputs found
INSIGHTS INTO HOW THE 3´UTR MEDIATES EXPRESSION OF A CONSERVED RNA-BINDING PROTEIN AND CONTRIBUTES TO GERMLINE DEVELOPMENT IN C. ELEGANS
Get PDFMaternal mRNA regulation is essential to germline and embryo development in metazoans. Over the past few decades, it has become clear that many RNA-binding proteins (RBPs) containing highly conserved RNA-binding domains orchestrate spatiotemporal expression pattern of germline and embryonic genes to control gametogenesis and embryogenesis in the nematode Caenorhabditis elegans. These RBPs bind regulatory elements situated primarily in the UTRs of their target mRNAs to regulate expression by influencing transcript stability or translational efficiency. The 3´UTR is the main determinant of patterned expression in the germline of C. elegans. MEX-3 is a KH-domain RBP that is required for anterior cell fate specification and maintenance of germ cell totipotency. MEX-3 is expressed in mitotic germ cells, maturing oocytes, and early embryos. MEX-3 is absent in the meiotic pachytene region as well as the diplotene loop region. The 3´UTR of mex-3 is sufficient to confer MEX-3’s expression to a transgenic reporter. Here, I assessed the importance of the endogenous 3´UTR of mex-3 to MEX-3’s expression pattern and function using CRISPR/Cas9 mutagenesis followed by molecular and phenotypic analysis. 3´UTR deletion allelic series demonstrated that the endogenous 3´UTR of mex-3 is indeed required for MEX-3’s pattern in the germline in vivo. I identified regions of the 3´UTR that contribute to repression of MEX-3 in different regions of the germline. Surprisingly, the 3´UTR was dispensable for viability. However, several 3´UTR deletions exhibited reduced fertility. Analysis of the transcriptome of these mutants revealed that the 3´UTR deletions altered expression of soma-specific genes, consistent with MEX-3’s role in repressing somatic gene programs. These data sets also showed that mex-3 mRNA levels do not correlate with MEX-3 protein levels. In order to determine which germline RBPs regulate expression of mex-3 through its 3´UTR, I used RNAi to knock down several candidate RBPs including three that were previously shown to regulate expression of MEX-3. My RNAi studies showed that GLD-1, LIN-41, and OMA-1/2 repress expression of mex-3 through its 3´UTR in the meiotic pachytene region, diplotene loop region, and oocytes in the proximal end, respectively. Furthermore, I have identified DAZ-1, an RRM-containing RBP, as a novel repressor of MEX-3 expression in the distal mitotic germ cells. Using RNAi, I demonstrated that poly(A) tail length control and the translation initiation factor IFE-3 contribute to MEX-3’s expression in the germline. Poly(A) polyadenylation and deadenylation cycles govern expression of mex-3 in the distal mitotic germ cells, while IFE-3 contributes to repression of mex-3 in the meiotic pachytene region, presumably by control of translation initiation. Using high throughput sequencing-based poly(A) tail assay, I have shown that the poly(A) tail length distribution of mex-3 mRNA shifts towards shorter tails in the mex-3 3´UTR deletion mutants with reduced fertility phenotypes. Our study is the first as far as we know to address the importance of an endogenous 3´UTR to in vivo expression and function in C. elegans germline. It will be interesting to determine how different RBPs and cis-regulatory elements orchestrate the spatiotemporal expression pattern of a single germline gene. It will also be interesting to assess whether other germline 3´UTRs are similarly dispensable for viability, and if so, what role do 3´UTRs play in enhancing reproductive success.Interdisciplinary Graduate Progra
The Post-Transcriptional Regulation Mechanism and Functional Importance of a Key Maternal mRNA, GLP-1
Get PDFTranslational control of maternal mRNAs is a major form of gene regulation during germline development and embryogenesis. C. elegans Notch homolog glp-1 maternal gene is necessary for germ cell proliferation, and embryonic fate determination. The RNA binding proteins POS-1 and GLD-1 directly regulate the translation of GLP-1 protein by binding to the specific elements within the glp-1 3’ UTR. When POS-1 or GLD-1 binding is disrupted by mutation of their respective elements, the expression pattern of a glp-1 3’ UTR reporter transgene changes in both the germline and in embryos. The mechanism by which POS-1 and GLD-1 mediate translation repression is not well understood. Previous work showed that loss of pos-1 increases the average polyA tail length of endogenous glp-1 transcripts in embryos. In this dissertation, we show that mutation of either the GLD-1 or POS-1 binding motifs in transgenic reporters does not change polyA site selection. This result rules out alternative polyA site selection as a mechanism of regulation. We also show that wild-type glp-1 transgenic reporter embryos have a shorter average polyA tail length compared to transgenic reporters with mutated GLD-1 or POS-1 binding motifs. We have studied the effect of cytoplasmic polymerases, deadenylases and translation initiation factors on our transgenic reporters. Our RNAi experiments show that two cytoplasmic polyA polymerases, GLD-2 and GLD-4, have strikingly different effects on the expression of reporter transgenes harboring GLD-1 or POS-1 binding motif mutations. By contrast, none of the deadenylases affect the transgenic reporter expression. We also observed strong derepression of all reporters upon reduction of the translation initiation factor ife-3. The results reveal that POS-1 and GLD-1 exert their repressive effects in different ways through cytoplasmic polyA polymerase activity, while IFE-3 mediated translation repression is independent of both POS-1 and GLD-1. Lastly, we have examined the biological significance of glp-1 3’UTR to the worm reproduction by using CRISPR/Cas9 mutagenesis to generate glp-1 3’UTR mutations in the endogenous locus. Characterization of a 71 base pair mutation that deletes the GLD-1 and POS-1 binding sites in the glp-1 3’UTR reveals a 2-fold reduction in the number of embryos produced and a 4-fold reduction in the hatch rate. Imaging results show that the mutant embryos appear to have patterning defects. Together, our results show that the glp-1 3’UTR contributes to reproductive health but is not essential to fertility.Interdisciplinary Graduate Program2025-08-1
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
Sequence and Target Specificity of the C. elegans Cell Fate Specification Factor POS-1: A Dissertation
Get PDFIn most metazoans, early embryogenesis is controlled by the translational regulation of maternally supplied mRNA. Sequence-specific RNA-binding proteins play an important role in regulating early embryogenesis, yet their specificities and regulatory targets are largely unknown. To understand how these RNA-binding proteins select their targets, my research focused on the C. elegans CCCH-type tandem zinc finger protein POS-1. Embryos lacking maternally supplied POS-1 die prior to gastrulation, and exhibit defects in the specification of pharyngeal, intestinal, and germline precursor cells. To identify the regulatory targets that contribute to the POS-1 mutant phenotype, we set out to determine the sequence specificity of POS-1 in vitro, and then use this information to identify regulatory targets in vivo. Using a candidate-based search, we identified a twelve-nucleotide fragment of the mex-3 3' untranslated region (3' UTR) to which POS-1 binds with high affinity. Using quantitative fluorescent electrophoretic mobility shift assays, I determined the affinity of the RNA-binding domain of POS-1 for a panel of single nucleotide mutations of this sequence, and then defined a consensus binding element based on this dataset. POS-1 recognizes the degenerate element UAU 2-3 RDN 1-3 G, where R is any purine (adenosine or guanine), and D is any base except cytosine. A bioinformatics analysis revealed the presence of this element in approximately 40% of C. elegans 3' UTRs, suggesting that POS-1 is capable of binding to and perhaps regulating many transcripts in vivo. POS-1 binding sites alone are not sufficient to pattern the expression of a reporter, suggesting that other factors may contribute to POS-1 specificity. To address the mechanism of POS-1-mediated translational regulation, I investigated the translational regulation of the C. elegans Notch homolog glp-1. Previous work demonstrated that glp-1 translation is repressed in the early embryo in a POS-1-dependent fashion, though it was not clear if this regulation was direct. The glp-1 3' UTR contains two POS-1 binding sites within five nucleotides of each other, and these sites are within a thirty nucleotide region of the 3' UTR required for proper spatiotemporal translation of glp-1. The POS-1 sites overlap with a negative regulatory element that is recognized by GLD-1, and a positive regulatory element recognized by an unknown factor. Both POS-1 and GLD-1 bind to an RNA containing these sites in vitro, and POS-1 competes with GLD-1 for binding. Both proteins are required for translational repression of a glp-1 3' UTR reporter in embryos. Furthermore, only one of the two POS-1 binding sites is required for repression, and the required site is wholly contained within a previously characterized positive regulatory element. Based on this, we propose that POS-1 does not regulate its targets by recruiting regulatory machinery, but instead by competing with factors that do. Thus, sites of POS-1 regulation are highly context dependent, which may contribute to POS-1 specificity.Biochemistry and Molecular Pharmacolog
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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