766 research outputs found
Structural Determination of the 5\u27 Untranslated Regions of IRE-containing mRNAs
The expression of ferritin and amyloid precursor protein (APP) is post-transcriptionally regulated by iron-regulating proteins via binding to a stem-loop structure known as an iron-responsive element in the 5’-untranslated region (5’UTR) of ferritin and APP mRNAs. In this study, we used atomic force microscopy (AFM) to visualize the conformation of the 5’UTRs of ferritin heavy chain (Ferritin-H), ferritin light chain (Ferritin-L), and APP mRNA transcripts from human and mouse, and determined the secondary RNA structures using selective 2’-hydroxyl acylation analyzed by primer extension (SHAPE). The AFM imaging did not provide high resolution structural information about these RNAs, whereas the SHAPE procedure successfully interrogated the secondary RNA structures at single nucleotide resolution. To our knowledge, this is the first time that the secondary structures of the entire 5’UTRs of these RNA molecules have been experimentally mapped. This study paves the way for the further investigation of RNA-ligand interactions in these RNA molecules
Cell Death Mechanisms in Drosophila Differentiated Photoreceptor Neurons
Apoptosis, or programmed cell death, is a form of physiological cell death that is essential for normal development and homeostasis. At the end of pupal development of the Drosophila retina, cell death terminates and photoreceptor neurons complete their differentiation process. We use these terminally differentiated photoreceptor neurons as a system to study neurodegeneration. We first adapt and develop fluorescent tools for photoreceptor visualization in vivo. These tools enable a recessive genetic screen to search for genes required for the survival of differentiated photoreceptors. Many redox and mitochondrial genes were found to protect photoreceptors from late cell death. Here, we focus on the iron-storage complex, Ferritin. ferritin mutations lead to caspase activation and photoreceptor neuronal death during development and sensitize adult photoreceptor neurons to cell death stimuli. ferritin mutations provide a robust model to study the role of iron and oxidative stress in neurodegeneration. To further investigate the role of Ferritin in photoreceptor survival, we generate genetically-encoded in vivo iron and redox sensors. In summary, by developing novel tools for photoreceptor cell visualization, we explore the neuro-specific mechanisms required for lifelong photoreceptor neuron survival. We perform a photoreceptor-specific genetic screen and characterize Ferritin’s role in shielding photoreceptor cells from iron and oxidative stress-induced cell death
Studies on genetic variants of human plasma transferrin.
PhDThe work presented in this thesis is concerned with the characterisation of human
plasma transferrins showing abnormal electrophoretic mobilities on polyacrylamide
gels. The work is divided into three studies: (i) a study of transferrin variants detected by
nondenaturing polyacrylamide gel electrophoresis; (ii) a study of the plasma
concentrations of individuals showing transferrin phenotypes associated with the three
most common Tf C alleles, Tf Cl, Tf C2 and Tf C3; and (iii) a study of a reported
nondegenerate nucleotide difference in the sequence of the cloned human transferrin
gene.
In the first study, six transferrin variants (3 Tf Br,,.,,, s and 3 Tf D. 5) showing
abnormal electrophoretic mobilities on nondenaturing polyacrylamide gels, and the two
Tf C variants, Tf Cl and Tf C2 which occur at polymorphic levels (> 1%) in human
populations, were isolated and purified from human plasma. Transferrins were purified
by a combination of DEAE Sephacel anion-exchange chromatography, SP. Sephadex
cation-exchange chromatography and G-200 gel-filtration chromatography. A series of
comparative studies were then carried out on the isolated transferrins to determine
whether the six transferrin variants detected in this thesis and the Tf C2 variant, showed
similar characteristics to the wild-type Tf Cl. Transferrins were studied for sialic acid
content of the two glycan chains, and for molecular weights and isoelectric points of the
iron-free (apo) and iron-saturated (holo) transferrin forms. Metal-binding properties
were examined by studying the binding of Fe", Cu", Al" and Ga"'. Iron-binding was
studied at physiological and endosomal pH (7.5 and 5.5 respectively) using FENTA as
the iron donor. Binding of Cue*, Al"' and Ga'* were examined at physiological pH using
CUNTA, ALNTA and GANTA respectively. The ability of transferrins to retain bound
iron was examined by studying pH-induced iron release over a pH range of 6.0-4.0.
Conformational stabilities were determined by studying the iron-binding abilities of
apotransferrins following exposure to urea or thermal denaturation, and by studying iron
loss from holo transferrins following exposure to urea or thermal denaturation. Other
than expected differences in isoelectric points, and slightly faster rates of iron loss from
variant holo transferrins titrated at physiological pH, all variant transferrins were found
to show similar characteristics to Tf Cl, with identical molecular weights and sialic acid
content, similar metal-binding properties, and similar stabilities to urea or thermal
denaturation. The results indicate that the structure and function of the variant
transferrins are not adversely affected by their differences in primary structure.
The second study examined the relationship between plasma transferrin concentration
and transferrin phenotypes representing five of the six most common Tf C phenotypes
(i. e. Tf Cl, Tf C2, Tf C2-1, Tf C3-1 and Tf C3-2), to determine whether plasma
concentrations were dependent on transferrin phenotype as suggested in literature.
Transferrin phenotypes of 931 unrelated individuals were determined by electrophoresis
of plasma on polyacrylamide isoelectric focusing gels. Plasma transferrin concentrations
were determined by single radial immunodiffusion using rabbit anti-human transferrin
IgG. A significant difference was found between the plasma concentrations of the five
transferrin phenotypes (p < 0.01) indicating that transferrin concentration was dependent
on phenotype. The results suggest that the two Tf C alleles, Tf C2 and Tf C3 are
associated with low plasma concentrations.
The third study was initiated to investigate a report in literature that a nondegenerate
nucleotide difference of adenine for guanine at base 1086 in exon 8 of the human
transferrin gene, may indicate the presence of a hitherto unrecognised transferrin variant
with Asn rather than a wild-type Asp at position 310 of the amino acid sequence. The
nucleotide sequenceo f exon 8 from 220 unrelatedi ndividuals showing transferrin
phenotypes associated with the three most common Tf C alleles, Tf Cl, Tf C2 and Tf
C3, and from 5 individuals showing abnormal transferrin phenotypes in the first study,
were amplified by polymerase chain reaction. Amplified products were digested with
the restriction endonuclease, Fok I which has a single recognition site in exon 8
containing the proposed wild-type guanine at base 1086, or with Bsm I which also
shows a single recognition site containing the proposed adenine nucleotide. The study
failed to detect the presence of the proposed transferrin variant, confirming that the
wild-type guanine was present in all 225 individuals.Biotechnology and Biological Sciences Research Counci
MECHANISMS OF REGULATION OF TAU IRES MEDIATED TRANSLATION
The translation of most eukaryotic mRNAs occurs in a cap-dependent manner. However, a subset of mRNAs are capable of initiating translation in a cap-independent manner by utilizing sequences in their 5’ UTR called IRES. It was previously shown that the 5’ UTR of the tau mRNA contains an IRES. In this study I show that IRES dependent translation of tau IRES is regulated at multiple levels in order to regulate the expression of the tau protein.
Tau protein is ubiquitously expressed but is concentrated in the brain. In this study, I utilized neural and non-neural cell lines to show that tau IRES is utilized differently (in some cases, up to 50% of total tau translation) depending on the cell type. For many IRES containing mRNAs, IRES activity is enhanced in conditions when cap-dependent translation is shut down, such as during cellular stress and mitosis. In this study, I show that tau IRES activity is upregulated during increased iron, poly (I:C), and extracellular Aβ exposure, which are stress conditions commonly observed in neurodegenerative diseases. Further, I show that tau IRES is differentially regulated by various upstream stimuli through their downstream signaling kinases. However, a comparison of the effect of various signaling pathways on tau and APP IRES suggested that the specific regulation of these IRESes occur downstream of mTOR signaling.
Most IRESes require binding by certain non-canonical factors called IRES trans acting factors (ITAFs) for internal initiation. ITAFs can be positive or negative, thus enhancing or inhibiting IRES function. Examination of sequences in the tau 5’ UTR led us to analyze four different RNA binding proteins as putative ITAFs for tau. Out of these, I identified two proteins – polypyrimidine tract binding protein (PTB) and neural PTB (nPTB) as inhibitory ITAFs of tau IRES. Altering the expression of PTB and nPTB in vitro and in cells negatively influenced tau IRES activity and protein expression.
Along with sequences in the 5’ UTR, the sequences in the 3’ UTR of an mRNA may also affect its translation, either through direct interaction between the RNA sequences, or through interaction by RNA binding proteins. In this study, I show that the tau 3’ UTR enhances IRES-dependent translation of tau, and this interaction requires the entire tau 3’UTR. Overall, I show that the tau IRES is a unique tool utilized by the mRNA to regulate tau protein expression
Analysis of RNA-Protein interactions involved in calicivirus translation and replication
The interaction of host-cell nucleic acid-binding proteins with the genomes of
positive-stranded RNA viruses is known to play a role in the translation and
replication of many viruses. To date, however, the characterisation of similar
interactions with the genomes of members of the Caliciviridae family has been
limited to in vitro binding analysis. In this study, feline calicivirus (FCV) and murine
norovirus (MNV) have been used as model systems to identify and characterise the
role of host-cell factors that interact with the viral RNA and RNA structures that
regulate virus replication. It was demonstrated that RNA-binding proteins such as
polypyrimidine tract-binding protein (PTB), poly(C)-binding proteins (PCBPs) and
La protein interact with the extremities of MNV and FCV genomic and subgenomic
RNAs. PTB acted as a negative-regulator in FCV translation and is possibly involved
in the switch between translation and replication during the late stages of the
infection, as PTB is exported from the nucleus to the cytoplasm, where calicivirus
replication takes place. Furthermore, using the MNV reverse-genetics system,
disruption of 5' end stem-loops reduced infectivity ~15-20 fold, while disruption of an
RNA structure that is suspected to be part of the subgenomic RNA synthesis promoter
and an RNA structure at the 3' end completely inhibited virus replication. Restoration
of infectivity by repair mutations in the subgenomic promoter region and the recovery
of viruses that contained repressor mutations within the disrupted structures, in both
the subgenomic promoter region and the 3’ end, confirmed a functional role for these
RNA secondary structures. Overall this study has yielded new insights into the role of
RNA structures and RNA-protein interactions in the calicivirus life cycle
Strukturelle Untersuchung an Häm-bindenden Proteinen
Heme is a widely known cofactor molecule in many enzymes like hemoglobin, cytochromes, and myoglobins. It is attached to these proteins covalently and classifies them as hemoproteins. In the last decade, a growing number of reports on the regulatory role of heme in various molecular and cellular processes uncovered the signaling role of heme. Here, heme binds transiently or with a low affinity to a variety of proteins and regulates their function. Inspite of the number of reports on its regulatory role, they are poorly defined at the structural level. Heme binds to these proteins through special amino acids called heme regulatory motifs (HRMs) such as cysteine-proline (CP), histidine and tyrosine. Studies conducted on CBS and the IL-36 cytokine family members during this project validated the heme-peptide knowledge at the protein level
Iron as Therapeutic Targets in Human Diseases Volume 2
Iron is an essential element for almost all organisms, a cofactor playing a crucial role in a number of vital functions, including oxygen transport, DNA synthesis, and respiration. However, its ability to exchange electrons renders excess iron potentially toxic, since it is capable of catalyzing the formation of highly poisonous free radicals. As a consequence, iron homeostasis is tightly controlled by sophisticated mechanisms that have been partially elucidated. Because of its biological importance, numerous disorders have been recently linked to the deregulation of iron homeostasis, which include not only the typical disorders of iron overload and deficiency but also cancer and neurodegenerative diseases. This leads iron metabolism to become an interesting therapeutic target for novel pharmacological treatments against these diseases. Several therapies are currently under development for hematological disorders, while other are being considered for different pathologies. The therapeutic targeting under study includes the hepcidin/ferroportin axis for the regulation of systemic iron homeostasis, complex cytosolic machineries for the regulation of the intracellular iron status and its association with oxidative damage, and reagents exploiting proteins of iron metabolism such as ferritin and transferrin receptor. A promising potential target is a recently described form of programmed cell death named ferroptosis, in which the role of iron is essential but not completely clarified. This Special Issue has the aim to summarize the state-of-the-art, and the latest findings published in the iron field, as well as to elucidate future directions
Understanding the role of eIF4A in gene regulation in health and disease
Eukaryotic initiation factor 4A (eIF4A) is an ATP-dependent RNA helicase responsible for unwinding the secondary structure of mRNAs. In humans, eIF4A exists as three separate paralogs: eIF4AI and eIF4AII possess a high degree of homology while eIF4AIII is distinct. Knockdown of eIF4AII had no effect on the expression of a reporter construct containing a structured RNA hairpin. Knockdown of eIF4AI and treatment with hippuristanol (an eIF4A inhibitor) caused a dramatic reduction in the hairpin-mediated gene. This reporter system was developed as part of this project to act as a screen for eIF4A activity along with an in vitro screening approach.
The activity of eIF4A is suppressed in vivo by the tumour suppressor PDCD4. The fact that loss of PDCD4 function increases the severity of DNA damage is probably attributable its eIF4A-suppressive activity.
Based on previous microarray data, it was supposed that eIF4A inhibition may be therapeutically beneficial in the treatment of Alzheimer's disease. As part of this project, it was demonstrated that eIF4A suppression significantly reduced the expression of reporter genes preceded by the 5’ UTRs of genes predicted to play harmful roles in Alzheimer’s disease. The expression of reporter genes preceded by the 5’ UTR sequences of genes predicted to be beneficial in Alzheimer's were not affected by this suppression.
Reporter plasmids containing the 5’ UTR sequences of the oncogenes ODC1, EGFR and VEGFA have high requirements for eIF4A as estimated using hippuristanol. eIF4A inhibition did not significantly affect the reporters containing the 5’ UTRs of non-pathogenic genes. The EGFR 5’ UTR was found to contain an IRES which explains why EGFR is upregulated in response to hypoxia
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