Role of DDX RNA helicases in cancer cells

Ester Cannizzaro ROLE OF DDX RNA HELICASES IN CANCER CELLS Abstract DEAD-BOX (DDX) RNA helicases are a large family of proteins characterized by the presence of a DEAD/H (Asp-Glu-Ala-Asp/His) motif. Their main function is to unwind double stranded RNA to promote downstream molecular events. They are involved in virtually all steps of RNA metabolism such as transcription, translation, RNA export and degradation, ribosome biogenesis and pre-mRNA splicing. The aim of my work was to investigate the functions of human DDX3X and DDX54 RNA helicases, particularly in in vitro cancer models. To provide insight into their molecular functions, I identified RNAs bound by DDX3X and DDX54 in breast cancer (MCF7) cells by performing iCLIP experiments. This generated two very distinct RNA binding profiles: DDX3X preferentially bound exonic regions of mRNAs encoding translational factors, whilst DDX54 preferentially bound non-coding RNAs and intronic regions of mRNAs encoding nuclear proteins. Further bioinformatic analysis identified a few discrete binding motifs within DDX3X target RNAs. One of these, within the human JUND transcript, was validated as a DDX3X binding site using electrophoretic mobility shift assays. Notably, the levels of proteins encoded by mRNAs bound by DDX3X were altered following knockdown of the helicase. These data highlight the importance of DDX3X in maintaining appropriate levels of certain proteins, which in turn may explain at least some of the changes in phenotype observed upon DDX3X knockdown. In this regard, I showed that knocking down DDX3X or DDX54 in MCF7 cells slowed cell proliferation by inducing a G1/S phase arrest. Furthermore, a CRISPR/Cas9 dropout screen in a leukaemia cell line (MLL-AF9) showed that both helicases are essential for growth of these cells. However, loss of DDX3X or DDX54 had little effect on proliferation of immortalized NIH3T3 cells indicating that their loss is not generally lethal. In subsequent CRISPR/Cas9 dropout screens in various other cancer cell lines, including some derived from solid tumours, DDX54 was found to be essential for growth of all cell lines. In contrast, DDX3X was required for proliferation of only a subset of these. Focusing on DDX3X, I identified that the integrity of the helicase's RNA binding domains is essential for growth and cell cycle progression of an acute myeloid leukaemia cell line (OCI-AML3). Overall, my findings shed mechanistic insight upon the role of DDX RNA helicases in cancer and identify DDX3X and DDX54 as potential targets for therapeutic intervention in certain cancers

Alternative 5\u27 exons either provide or deny an initiator methionine codon to the same alpha-tubulin coding region

The primary structures of two overlapping novel alpha-tubulin cDNA clones isolated from a Macaca fascicularis testis cDNA library and the corresponding human gene are presented. Although the general structure of the human gene conforms to that of previously described mammalian alpha-tubulin genes, there is a surprising difference: the ATG initiator codon is conspicuously absent. The macaque testis cDNA similarly lacks the initiator methionine, but otherwise encodes a variant alpha-tubulin isotype precisely conserved in the human gene. RNA blot analysis in the macaque, using a 3\u27 untranslated region probe, revealed the existence of two additional related transcripts expressed in every tissue examined except the adult testis. Sequence comparisons indicate that the 2.0 kb testis transcript and one of the additional transcripts result from differential transcription of the same gene. The two transcripts differ only at the 5\u27 end as a result of the recruitment of different 5\u27 exons. Curiously, the 5\u27 exon utilized outside the testis encodes an initiator methionine in the expected location

Molecular cloning and characterization of a glucose transporter-related gene from Trypanosoma brucei

Available from British Library Document Supply Centre- DSC:DX97425 / BLDSC - British Library Document Supply CentreSIGLEGBUnited Kingdo

Molecular functional analysis of the tumor suppressor gene PDCD4

Das Pdcd4-Gen (Programmed Cell Death 4) ist ein neues Tumorsupressorgen, das ursprünglich als ein während der Apoptose aktiviertes Gen identifiziert wurde. Um die molekularen Funktionen des Pdcd4 Tumorsuppressorgens zu charakterisieren, wurde ein Knock-Down-System mittels RNAi Technologie und ein Knock Out System mittels homologer Rekombination entwickelt. Mittels der 1D SDS PAGE- und der 2D-SDS-PAGE-Analyse konnten einige Transkriptionsfaktoren (ATF 2, c Myc, c Jun, CEBPß) sowie folgende Proteine als Pdcd4-Zielproteine identifiziert werden: Cytokeratin 17 (CK 17), Cytokeratin 8 (CK 8) und C2 und C3, die zur Familie der Aldo-Keton-Reduktase 1 (AKR1) gehören, sowie die Glutamyl Prolyl Bifunctional tRNA synthetase (GluProRS). Diese beiden hier entwickelten Systeme sowie die hier identifizierten Zielproteine könnten zur Klärung der molekularen Funktionen des Tumorsuppressor Pdcd4 beitragen. The Programmed Cell Death 4 gene (Pdcd4) is a novel tumor suppressor gene originally identified as a gene upregulated during apoptosis. To study the molecular functions of the Pdcd4 tumor suppressor gene by a reverse genetic approach, a knock down system and a knock-out system were developed in the HeLa cells using siRNA mediated RNA interference (RNAi) and in the DT40 cells using homologous recombination, respectively. The 1D and 2D SDS PAGE analysis in HeLa cells identified several transcription factors (ATF-2, c-Myc, c-Jun, CEBPß) and proteins viz., cytokeratin 17 (CK 17), Aldo Keto Reductase 1 (AKR1) family members C2 and C3, cytokeratin 8 (CK 8) and Glutamyl Prolyl Bifunctional tRNA synthetase (GluProRS) as novel molecular targets of Pdcd4. The two systems developed in the present work will be useful in further elucidating the molecular functions of tumor suppressor Pdcd4 by utilizing the molecular targets identified here

Genes: Multigene Families, Control of Gene Expression, Genetic contributions to Human Diseases, including Chromosomal Fragile Sites and ‘Dynamic’ and ‘Non-self’ Mutations

The early work in this thesis utilizes the general approach of comparative analysis. In order to find out the relationship between entities (either functional or genetic) my colleagues and I have attempted to identify the important elements by detecting similarity between those entities that act in a similar manner. The philosophy behind this approach is simply that when two distinct objects perform a similar process then the requirements essential for that process will be revealed as similarities between those objects above a noise of difference between them. The use of comparative analysis in biological systems is an attempt to identify natural order from apparent chaos. This work includes but is not limited to :- 1. discovery of the family of kallikrein genes and exploration of their roles in biology, 2. identification of the DNA sequence elements required for hormonal and heavy metal control of metallothionein gene expression 3. discovery of at least some of the necessary and sufficient conditions for the appearance of fragile sites on chromosomes, and their consequent contributions to disease, 4. the molecular properties of repeat DNA sequence expansion that lead to dynamic mutation and consequent fragile site expression and / or disease pathogenesis. In a sense the use of genetic animal models in order to study gene function and pathogenesis follows similar logic of comparative analysis – the mutation of a single endogenous gene or the expression of a single introduced mutated gene in a (presumed) constant genetic background to enable the biological consequences of the genetic mutation or aberrant gene expression by comparing animals from the ‘wild-type’ or parent line with those that now carry the mutation or altered gene. This approach has been utilized in the most recent work contained herein as a means to determine gene function and / or to model human genetic disease pathogenesis, specifically pathogenic mechanisms of the protein WWOX in cancer and expanded repeat RNAs in neurodegenerative diseases. The culmination of this recent work is the development of an hypothesis – 4. that expanded repeat double-stranded RNA leads to neurodegeneration through its recognition by the RNA-binding pattern recognition receptors as a ‘non-self’ or foreign nucleic acid due to a paucity of RNA modification. The resultant pathogenic mechanism is therefore autoinflammatory disease. Given the wide range and variety of evidence of inflammatory activation in neurodegenerative diseases in general, this mechanism is therefore hypothesized to be the general causal mechanism for most (or all) of these diseases. A specific Introduction - highlighting the nature and significance of the work, and a Conclusion – of how this work has contributed to knowledge, are given at the start of each chapter, while the impact of the various components of this work is indicated by the number of citations for each of the included publications. Authorship contributions to each of the included publications in this work are also indicated with each specific reference.Thesis (DSc) -- University of Adelaide, School of Biological Sciences, 202

Microbiology for Allied Health Students

This open textbook is a remix of Openstax Microbiology, CC-BY 4.0, and created through an Affordable Learning Georgia Round Six Textbook Transformation Grant. The textbook has the following supplemental materials within this repository: This is a collection of instructional materials for the following open textbook and lab manual: Microbiology for Allied Health Students Lab Manual Microbiology for Allied Health Students Instructional Materials Authors\u27 Description: Microbiology for Allied Health Students is designed to cover the scope and sequence requirements for the single semester Microbiology course for non-majors and allied health students. The book presents the core concepts of microbiology with a focus on applications for careers in allied health. The pedagogical features of Microbiology for Allied Health Students make the material interesting and accessible to students while maintaining the career-application focus and scientific rigor inherent in the subject matter. The scope and sequence of Microbiology for Allied Health Students has been developed and vetted with input from numerous instructors at institutions across the U.S. It is designed to meet the needs of most microbiology courses allied health students. With these objectives in mind, the content of this textbook has been arranged in a logical progression from fundamental to more advanced concepts. The opening chapters present an overview of the discipline, with individual chapters focusing on cellular biology as well as each of the different types of microorganisms and the various means by which we can control and combat microbial growth. The focus turns to microbial pathogenicity, emphasizing how interactions between microbes and the human immune system contribute to human health and disease. The last several chapters of the text provide a survey of medical microbiology, presenting the characteristics of microbial diseases organized by body system. Accessible files with optical character recognition (OCR) and auto-tagging provided by the Center for Inclusive Design and Innovation.https://oer.galileo.usg.edu/biology-textbooks/1015/thumbnail.jp

Drug development progress in duchenne muscular dystrophy

Duchenne muscular dystrophy (DMD) is a severe, progressive, and incurable X-linked disorder caused by mutations in the dystrophin gene. Patients with DMD have an absence of functional dystrophin protein, which results in chronic damage of muscle fibers during contraction, thus leading to deterioration of muscle quality and loss of muscle mass over time. Although there is currently no cure for DMD, improvements in treatment care and management could delay disease progression and improve quality of life, thereby prolonging life expectancy for these patients. Furthermore, active research efforts are ongoing to develop therapeutic strategies that target dystrophin deficiency, such as gene replacement therapies, exon skipping, and readthrough therapy, as well as strategies that target secondary pathology of DMD, such as novel anti-inflammatory compounds, myostatin inhibitors, and cardioprotective compounds. Furthermore, longitudinal modeling approaches have been used to characterize the progression of MRI and functional endpoints for predictive purposes to inform Go/No Go decisions in drug development. This review showcases approved drugs or drug candidates along their development paths and also provides information on primary endpoints and enrollment size of Ph2/3 and Ph3 trials in the DMD space

Hematology

Hematology encompasses the physiology and pathology of blood and of the blood-forming organs. In common with other areas of medicine, the pace of change in hematology has been breathtaking over recent years. There are now many treatment options available to the modern hematologist and, happily, a greatly improved outlook for the vast majority of patients with blood disorders and malignancies. Improvements in the clinic reflect, and in many respects are driven by, advances in our scientific understanding of hematological processes under both normal and disease conditions. Hematology - Science and Practice consists of a selection of essays which aim to inform both specialist and non-specialist readers about some of the latest advances in hematology, in both laboratory and clinic

Comparative genomic analyses of Entamoeba species

Amoebiasis is the third-most common cause of mortality worldwide from a disease borne of a parasitic infection. It affects up to 50 million people annually, of which 40,000 to 100,000 cases are fatal. Entamoeba histolytica is an obligate protozoon parasite of humans and is the aetiological agent of the disease. Recent suggestions that other members of the Entamoeba genus are human-infective, and potentially pathogenic, have been investigated here. A draft assembly and annotation of the 25 Mb genome of E. moshkovskii strain Laredo is presented, to which multiple E. moshkovskii strains were mapped. The E. moshkovskii genome was found to be approximately 200 times more variable than that of E. histolytica. Performance of the four-haplotype test revealed that genetic recombination does not seem to occur in E. moshkovskii. As such, it is suggested that it be referred to as a ‘species complex’, rather than an individual species. A comparative genomic analysis of E. histolytica HM-1:IMSS, E. moshkovskii Laredo, E. invadens IP-1 and the avirulent E. dispar SAW760 was performed. Subsequent comparative analyses against members of genera representative of the diversity in the Unikonts clade enabled the identification of orthologous gene families unique to the Entamoeba genus. Analysis of virulence factors within this set revealed that gene families involved in adhesion of amoebic trophozoites to host cells play a key role in the development of invasive amoebiasis. The Gal/GalNAc lectins and members of the BspA family are of particular interest, being present in all analysed species, except for E. dispar. The presence of these key families, plus cysteine proteases, in the E. moshkovskii genome suggests that some sequence types within this species complex may be pathogenic. E. invadens was found to possess larger numbers of more variable genes within many virulence factor families, including the BspA family and the Gal/GalNAc lectins. This suggests that sequence diversity facilitates E. invadens’ polyxenous lifestyle. Finally, a novel species recently isolated from a human faecal sample - E. bangladeshi, strain 8237 – was sequenced. Its genome was assembled using multiple de novo genome assemblers and coding sequences were assembled individually. A combination of all methods tested was found to be beneficial in maximising the number of gene sequences assembled, which is advised as good practice in future similar assemblies. The phylogeny of E. bangladeshi, achieved using the combined assemblies’ outputs suggested that the novel species is human-infective. The work presented here utilised modern comparative genomic techniques to improve understanding of Entamoeba species, their capacity for causing disease and their potential impact upon the epidemiology of amoebiasis