No description available.

Maternal 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.

E. faecalis est une bactérie à gram positif, responsable d’infections nosocomiales. Dans cette étude, nous avons identifié par RNA-seq 65 ARN régulateurs potentiels induits en conditions de stress et/ou d’infection chez ce pathogène opportuniste. Parmi ceux-ci, trois ARN surexprimés in vivo, en présence de sels biliaires et à pH acide ont fait l’objet d’une étude approfondie.Le premier, SRC65, s’est avéré être un petit ARN (sARN) agissant probablement en trans. Il présenterait des fonctions redondantes avec son homologue SRC90. Différentes cibles potentielles ont été identifiées et des expériences de physiologie ont révélé un rôle de SRC65 dans le déclenchement de la phase exponentielle de croissance.Le 2ème ARN étudié est une région 5’ régulatrice non traduite (5’UTR), appelée 5’1515. Elle formerait un atténuateur et agirait de manière répressive sur le gène ef1515. EF1515 est un antiterminateur de la famille BglG/SacY capable de se fixer sur 5’1515 pour réguler son expression et celle du gène ef1516 localisé en aval et codant un système de transport des sucres de type PTS. L’antiterminateur EF1515 contrôle aussi l’expression du gène ef3023 codant une protéine impliquée dans la virulence d’E. faecalis.Le dernier ARN régulateur étudié est également une 5’UTR. Celle-ci participerait à la régulation d’une hélicase à motif DEAD (CshA) codée en aval de la 5’UTR. Sa caractérisation s’inscrit dans une étude plus large concernant les éléments du métabolisme des ARN, impliquant les ribonucléases et les autres hélicases à motif DEAD d’E. faecalis. CshA aurait un rôle prépondérant pour la bactérie, en étant impliquée dans la réponse au stress, le fitness et la virulence. L’identification de l’interactome de CshA a notamment permis d’identifier l’énolase comme partenaire privilégié. / E. faecalis is a gram-positive bacterium responsible for nosocomial infections. Using RNA-seq, we identified 65 putative regulatory RNA induced under stress and/or during infection. Of these, three RNA overexpressed in vivo, in the presence of bile salts and at acidic pH have were more deeply studied.The first, SRC65, was found to be a small RNA (sRNA) probably acting in trans. It would present redundant functions with its homologous sRNA SRC90. Different potential targets were identified and physiology experiments revealed a role for SRC65 in triggering the exponential growth phase.The 2nd studied RNA is a 5’ untranslated region (5'UTR), called 5'1515 which would form an attenuator and act repressively on the ef1515 gene. EF1515 is an antiterminator of the BglG/SacY family capable of binding at 5'1515 to regulate its expression and that of the downstream gene ef1516 encoding a PTS-type sugar transporter. The EF1515 antiterminator also controls the expression of the ef3023 gene encoding a protein involved in E. faecalis virulence.The last regulatory RNA studied is also a 5'UTR. It would participate in the regulation of a DEAD-box helicase (CshA) encoded downstream of the 5'UTR. Its characterization is part of a broader study of the elements of RNA metabolism, involving ribonucleases and other DEAD-box helicases of E. faecalis. CshA would have a prominent role for the bacteria, being involved in stress response, fitness and virulence. The identification of the CshA interactome made it possible to identify enolase as a preferred partner.

Translation control is crucial during virus-host interaction, in which the host relies on the translation machinery to mount an antiviral response or induce the inflammation response to reduce virus spread, while the virus aims to take control of this system to thwart the host defense while producing viral progeny. The field of oncolytic virus (OV) therapy relies on replicating, engineered viruses that preferentially infect tumor cells to induce direct oncolysis or promote an antitumor immune response. Despite the importance of translation control in virus-host interaction, not much has been described on the interaction at the translation level between OV and cancer cells. I propose that this knowledge gap could reveal significant improvements in OV efficacy in treating cancer. In my first study, I set out to characterize the translatome of an infection-resistant breast cancer cell line infected by three clinically advanced OVs to identify residual antiviral activity in cancer cells regulated by translation control. I found the inositol phosphatase Inpp5e to be a novel antiviral gene that is translationally induced during infection via a transcript variant shift. Mechanistically, I showed that the majority of Inpp5e transcripts in uninfected cells contain a long 5’ UTR that harbor four translationally inhibitory upstream reading frames (uORF). Yet, OV infection induced the expression of a shorter 5’ UTR with a spliced intron that removes three uORFs, derepressing the translation of Inpp5e mRNA. CRISPR-Cas9 knockout of Inpp5e also enhanced the infectivity of oncolytic HSV1 and VSV. My study suggests the existence of a class of translationally regulated antiviral genes in cancer cells. In my second study, I sought to adapt the translation of transgenes to the unique translation condition imposed by the infecting virus via the incorporation of a viral 5’UTR. I identified HSV1 5’UTRs by locating the transcription start site of most HSV1 genes using RNA-seq data, then determined the 5’UTR of US11 as a potent translation enhancer during HSV1 infection. Incorporation of this 5’UTR into the transgene expression cassette inserted into the HSV1 genome enhanced transgene expression significantly at the translation level. In my third study, I set out to explore the mechanism of miR-223 mediated inflammation inhibition. miR-223 is a protective miRNA in the context of atherogenesis via suppressing inflammatory signaling. Using transcriptome and translatome profiling (RNA-seq and Ribo-seq), I found that the inhibitory effect of miR-223 on inflammation occurs primarily at the translation level. Overall, my work highlights the importance of translation control in OV-cancer cells interaction, as well as in inflammation-related diseases.

Huntington’s disease is a neurodegenerative disorder caused by expansion of the CAG repeat in huntingtin exon 1. Early studies demonstrated the huntingtin gene is transcribed into two 3′UTR isoforms in normal human tissue. Decades later, researchers identified a truncated huntingtin mRNA isoform in disease but not control human brain. We speculated the amount of huntingtin 3′UTR isoforms might also vary between control and Huntington’s disease brains. We provide evidence that the abundance of huntingtin 3′UTR isoforms, including a novel mid-3′UTR isoform, differs between patient and control neural stem cells, fibroblasts, motor cortex, and cerebellum. Both alleles of huntingtin contribute to isoform changes. We show huntingtin 3′UTR isoforms are metabolized differently. The long and mid isoforms have shorter half-lives, shorter polyA tails, and more microRNA and RNA binding protein sites than the short isoform. 3′UTR Isoform changes are not limited to huntingtin. Isoforms from 11% of genes change abundance in Huntington’s motor cortex. Only 17% of genes with isoform alterations are differentially expressed in disease tissue. However, gene ontology analysis suggests they share common pathways with differentially expressed genes. We demonstrate knockdown of the RNA binding protein CNOT6 in control fibroblasts results in huntingtin isoform changes similar to those in disease fibroblasts. This study further characterizes Huntington’s disease molecular pathology and suggests RNA binding protein expression may influence mRNA isoform expression in the Huntington’s disease brain.

Bovine viral diarrhea virus (BVDV) is an important viral pathogen affecting all ages of cattle, resulting in significant economic losses worldwide. BVDV infection is associated with a diverse array of symptoms including gastrointestinal disorder, respiratory distress, fetal malformation, stillbirth, abortions, and mucosal disease (MD). Transplacental infections of fetuses between 42 and 125 days of gestation can result in immune-tolerance and the surviving fetuses become persistently infected (PI). PI animals are major reservoir of BVDV and it becomes problematic to control the disease. The objectives of this dissertation were to: 1) develop a cost-effective testing scheme to detect BVDV PI animals from exposed herds, 2) characterize two virulent BVDV-2 Mississippi isolates associated with severe hemorrhagic diseases, and 3) perform phylogenetic analysis based on sequences of 5'UTR, E2, and NS5B regions. First, we developed a BVDV testing scheme by combining pooled real-time RT-PCR with antigen capture enzyme-linked immunosorbent assay (ACE) to screen cattle herds. From positive pools individual positives were identified using ACE. Data from a three year period indicated that 92.94% PI animals were infected with BVDV-1, 3.53% with BVDV-2, and 3.53% with both BVDV-1 and BVDV-2. Analysis of the 5'UTR of 22 isolates revealed the predominance of BVDV-1b followed by BVDV-2a. Second, two virulent BVDV isolates, M10-3432 and M10-5347, were successfully recovered from an adult beef breeding cow and feedlot calf respectively. When compared to the reference strain BVDV-2 125c, five and three unique amino acids in E2 regions were different from M10-5347 and M10-3432 respectively. Phylogenetic analysis of E2 region grouped both Mississippi isolates in BVDV-2a, a subtype containing high virulent strains. M10-3432 was clustered with high virulent strain 890 while M10-5347 was clustered with high virulent strain CD87. Third, we compared the phylogenetic analyses of BVDV based on the sequences of 5'UTR, E2, and NS5B at either nucleotides or amino acids level. Although slight differences were observed, the virulent BVDV isolates were consistently classified into BVDV-2a cluster regardless of region of sequences used. Furthermore, phylogenetic tree constructed using combined two or more regions had higher posterior probability and bootstrap value than phylogenetic trees constructed using a single region

Indiana University-Purdue University Indianapolis (IUPUI) / Cyclin Dependent Kinase regulation is often manipulated by cancer cells to promote unlimited proliferation. P27 is an important regulator of Cyclin E/CDK 2, which has been found in low amounts in many types of malignant cancers. Lovastatin has been shown to cause cell cycle arrest in the G1 phase of the cell cycle by increasing the P27 protein. There has been some question, however, if lovastatin regulates P27 at the transcriptional or translational level. Although it has been claimed that P27 expression regulation is due to an IRES located in its 5’UTR, other studies suggested that P27 expression is regulated at the level of transcription. To further investigate the regulation mechanism of P27 expression, the 5’-UTR of P27 and its deletion mutants were examined using a luciferase reporter gene in HeLa cells following exposure to lovastatin. It was found that lovastatin stimulated a significant 1.4 fold increase in its promoter activity of the full length 5’UTR (575). Deletion of 35 nucleotides from the 5’ end of the UTR eliminated the lovastatin-induced increase in promoter activity. Further mapping analyses of the first 35 bases showed that two regions, M1 (575-559) and M3 (543-527), were less sensitive to lovastatin than the other mutated constructs. Since M1 and M3 still showed some activity, a construct was created with deletions in both the M1 and M3 regions. This showed no increase in luciferase activity when exposed to lovastatin. Looking at RNA levels, there was a 1.5 fold increase in RNA when the full length 5’UTR was inserted into HeLa cells and exposed to 81 µM of lovastatin. In contrast, there was no increase in RNA when M1/M3 (575-559; 543-527) was inserted into HeLa cells and exposed to 81 µM of lovastatin. In addition, there was a 1.6 fold increase in endogenous P27 RNA levels after HeLa cells were exposed to 81 µM of lovastatin. In all of these experiments, there seems to be two promoters that work cooperatively: M1 (575-559) and M3 (543-527).

No description available.

京都大学 / 新制・課程博士 / 博士(理学) / 甲第24082号 / 理博第4849号 / 新制||理||1694(附属図書館) / 京都大学大学院理学研究科生物科学専攻 / (主査)准教授 柘植 知彦, 教授 森 和俊, 教授 川口 真也 / 学位規則第4条第1項該当 / Doctor of Science / Kyoto University / DGAM

Nescient Helix Loop Helix 2 (Nhlh2) is a member of the basic helix-loop-helix transcription factor family. Mice with a targeted deletion of Nhlh2, called N2KO mice, show adult onset obesity in both males and females. Nhlh2 regulates other genes by binding to the E-box in the promoter region of these genes. This transcription factor regulates many other transcription factors including MC4R and PC1/3 which are associated with human obesity. The Nhlh2 promoter has been analyzed for putative transcription factors binding sites. These putative binding sites have been tested to be the regulators of Nhlh2 by transactivation assays with mutant promoters, Electrophoretic Shift Assay (EMSA), and Chromatin Immunoprecipitation Assay (ChIP) as methods to investigate the DNA-protein binding. The results of these experiments showed that the Nhlh2 promoter has five Signal Transducer and Activator of Transcription 3 (Stat3) binding site motifs at -47, -65, -80, -281, -294 and two Nuclear Factor Kappa-Light-Chain-Enhancer of Activated B Cells (NFκB) binding site motifs at -67 and -135. While NFκB acts as a negative regulator of Nhlh2, this research showed that Stat3 acts as a regulator for the Nhlh2 basal expression and leptin stimulation. The ChIP assay using chromatin from mouse hypothalamus and antibodies against Stat3 and the NFκB subunits P50, P65, and c-Rel demonstrated that all of these antibodies were able to pull down the part of the Nhlh2 promoter containing the binding sites of Stat3 and NFκB. The EMSA results not only demonstrated that NFκB and Stat3 binding site motifs are real binding sites, but also exists the possibility of a relationship between these transcription factors to regulate Nhlh2 expression with leptin stimulation. An effort in analyzing the human NHLH2 3'UTR showed that one of the SNPs located at position 1568 in the NHLH2 mRNA (NHLH2A^1568G) which converts adenosine to guanine might have the potential to decrease the mRNA stability. For more investigation about this SNP, the mouse Nhlh2 tail was cloned into 2 different vectors and these vectors were subjected to site directed mutagenesis to create the 3'UTR SNP that convert A to G. One of these vectors used luciferase as a reporter gene for expression while the other one was used to measure Nhlh2 mRNA stability. These vectors were transfected into hypothalamic cell line N29/2 to test the effect of this SNP on Nhlh2 expression. This study demonstrated that this SNP down regulated luciferase expression and also decreased Nhlh2 mRNA stability. Taken together, this study demonstrated that Nhlh2 could be regulated transcriptionally by both NFκB and Stat3 transcription factors and post-transcripitionally by the 3'UTR SNP that converts adenosine to guanine. / Ph. D.