The effect of oestradiol-17beta on RNA synthesis in the uterus of the immature rat

1) The action of oestradiol at a molecular level, together with the current concepts of INA synthesis, maturation and transport are reviewed, IINA synthesis is investigated and characterized in the uterus of 18-21 day old rats responding to oestradiol-17beta. 2) The observed stimulation of RNA synthesis, in the uterus of rats treated with oestradiol-17beta is markedly dependent on the route of injection of precursor. After intraperitoneal administration of radioactive precursors, response is low and variable. Conversely, intravenous injection of precursor gives rise to a marked stimulation of RNA synthesis which is far in excess of the stimulated uptake into the acid-soluble pools and indicates that the increased uptake into RNA represents a real stimulation in RNA synthesis. Subcutaneous injection of precursor gives an intermediate response. 3) 18-21 day old rats respond maximally to a dose of 0.3?g/rat or more of oestradiol-17beta, the degree of response being dependent on the weight of the animals within the age range, 4) In vitro synthesis of ISIA, in uteri excised from oestrogen-treated rats, is only slightly stimulated and the increase can be accounted for by hormone-activated uptake of precursor. 5) The purification and separation of uterine RNA on polyacrylamide gels, agarose gels and sucrose density gradients is described. 6) The earliest detected effect of oestradiol is the stimulated synthesis of a very-high molecular weight RNA from approximately 30min after the administration of oestrndiol-17beta. The rapid synthesis and decay of this species, together with its nuclear location, absence of methylation and its base composition, permits its identification as heterogeneous nuclear RNA. The HnRNA made in response to oestradiol varies considerably in size. 7) Since evidence is accummulating that HnRNA contains polynucleotide sequences which ultimately become messengers, it is suggested that the stimulated production of this species in the uterus of oestrogen-treated rats may reflect hormone-induced rRNA synthesis and the translation of the messengers into protein may be a necessary prerequisite for stimulated rRNA synthesis and subsequent hormone augmented differentiation. 8) A striking change in the uteri of rats exposed to oestradiol is the stimulated synthesis of ribosomal RNA. When purified RNA is separated on 2.7% polyacrylamide gels, synthesis can he followed from the 45S precursor, through the 32S precursor and into the ribosomal submit species. Synthesis is first stimulated at, or shortly after, 1h of oestrogen treatment and is greatly increased 2 and 4h after hormone administration. Some evidence is presented that, in addition to increasing the rate of transcription of rRNA, oestradiol may also stimulate the rate of rRNA maturation together with its transport into the cytoplasm. 9) The fate of newly-synthesized ribosomes in oestrogen-treated uterine cells is investigated. As a consequence of hormone administration, pre-existing and newly-synthesized ribosomes appear to aggregate into polysomes but there are few membrane-bound ribosomes either before or after hormone treatment. The features of the induced production of ribosomes in immature rat uteri are discussed in relation to the current concepts of their involvement in hormone action. 10) Oestrogen-induced synthesis of tRNA may precede slightly the increase in rRNA synthesis, since the labelling of 45 RNA is clearly elevated 1h after hormone administration. Synthesis of both 45 and 55 RNA is strongly stimulated after 2 and 4h of oestrogen treatment. 11) The increased synthesis of rRNA in response to oestrodiol-17beta is acre strongly inhibited by actinomycin D than the synthesis of other RNA species. Cycloheximide, depending on the time of administration and dosage, inhibits either RNA synthesis or the maturation of rRNA

Some studies on the low molecular weight RNA components of mammalian cells

Until recently, RNA was considered to belong to one of three categories; transfer RNA(tRNA), ribosomal RNA or messenger RNA. In addition however, the development of more sophisticated analytical techniques such as polyacrylamide gel electrophoresis, coupled with the improved cell fractionation procedures, has enabled the detection of hitherto unrecognised low molecular weight RNA species in both the nuclear and cytoplasmic fractions of eukaryete cells. The aim of this work was to investigate the identity and molecular characteristics of these various low molecular weight RNA components in the nuclei and cytoplasm of normal, malignant and virus transformed animal cells and to define the molecular processes involved in their biogenesis. (A) Low Molecular Weight RNAs of the Nucleus. Polyacrylamide gel electrophoresis of extracts of isolated mammalian nuclei indicate the existence of approximately fourteen distinct low molecular weight RNA species with a size range of 80-350 nucleotides in length. These RNA components are stable and appear each to be present to the extent of approximately 105 molecules per cell. Many of these RNA molecules are methylated either in the base or ribose moeities and they are distributed between the nuclealar and nucleoplasmic fractions of the nucleus. Analysis of their patterns of synthesis in synchronised cell populations indicate two distinct patterns of synthesis, for some appear to be synthesised only at, or just after, the time of maximum DNA synthesis during the S phase of the cell cycle whilst others appear to be synthesised continually. In addition these low molecular weight methylated RNA components appear to be confined to the nucleus for no methylated RNA components of similar size and base composition can be found in the cytoplasm, (B) Low Molecular Weight RNAs of the Cytoplasm, Electrophoresis of cytoplasmic preparations from mammalian tissue culture cell lines reveals the presence of approximately thirteen low molecular weight RNA components with, a size range of 80-350 nucleotides in length. The bulk of these RNA components possess lower electrophoretic mobilities than the 5s RNA component from the cytoplasmic ribosomes. Kinetics of labelling studies indicate them to be synthesised in a fairly linear fashion and to show no precursor-product relationship one to another. They appear to be fairly stable, being less metabolically stable than tRNA or the RNA of the cytoplasmic ribosomes, but possessing total life spans of the order of approximately 24-30 hours. Unlike the low molecular weight RNA components of the nucleus they appear to be devoid of methylated nucleosides, the only methylated low molecular weight cytoplasmic RNA component being transfer RNA. Studies on the intracellular location of these RNA spedies indicate that they are absent from the cell sap, are not associated with the mitochondrial fraction but are associated principally with the microsomal fraction and appear in fact to be found on the polyribosomes. They are released from the polyribosomes subsequent to EDTA treatment and appear to be released in the form of free RNA strands rather than as ribonucleoprotein associates. Nucleotide composition analysis indicates these RNA species to possess an average' (G+C) content of 54% and experiments employing ethidium bromide, which specifically inhibits mitochondrial RNA synthesis, indicate that they are not derived from the transcription of mitochondrial DNA. Inhibitor studies using low levels of actinomycin D suggest them to be of nucleoplasmic origin whilst experiments employing puromycin and cordycepin suggest that their function is not that of mRNA. Exporiments utilising the adenosine analogue, toyocamycin, raise the possibility that they could represent the cleavage products of some larger precursor molecule(s). Analyses of the synthesis of these low molecular weight RNA components of the cytoplasm in synchronised cell populations indicate that they are not synthesised coordinately with the DNA during the S phase of the cell cycle but are synthesised throughout the S and phases with a rate which apparently increases through these periods of the cell cycle. Additional experiments investigating the effects of the drug amanitin on RNA synthesis in mammalian cells indicate that the in vitro effects of the drug are different from the in vivo effects and suggest, that an extranucleolar control of nucleolar function exists in mammalian cells

The maturation of low molecular weight RNA in mammalian cells

Abstract Not Provided

Activating senescence in p16-positive Basal-like breast cancer.

PhDBreast cancer is the most common cancer in the UK and Basal-like breast cancer (a highly aggressive subtype) accounts for approximately 8-22% of all cases depending on ethnicity. Unlike most human malignancies and indeed other PAM50 breast cancer subtypes, the vast majority of Basal-like tumours are positive for wild type p16. This p16 signature is associated with a particularly poor prognosis and p16-positive Basal-like breast cancer remains the most clinically challenging subtype and is the focus of this project. Pro-senescence therapies are gaining momentum as attractive strategies for the treatment of those breast cancers with current unmet clinical need. To identify targets for pro-senescence therapy in p16-positive Basal-like breast cancer, a genome‐wide siRNA screen and two subsequent validation screens using two p16-positive cancer cell lines were performed. Screening revealed 20 siRNAs that induced senescence within both cancer cell lines. Strikingly, 11 of these 20 siRNAs targeted ribosomal proteins, implicating disrupted ribosomal biosynthesis in senescence activation in p16-positive Basal-like breast cancer. Importantly, subsequent experiments in normal human mammary epithelial cells established that specific ribosomal protein knockdown is well tolerated by normal cells. Analysis of the METABRIC data set showed a high degree of ribosomal dysregulation in Basal-like tumours and revealed that all 11 ribosomal hits identified were frequently overexpressed in p16-positive Basal-like breast cancers. Kaplan Meier analysis confirmed that elevated expression of six of the 11 ribosomal proteins correlates with a reduced overall survival in these women, further supporting a role for these proteins as drivers of disease. These six ribosomal hits, associated with the poorest patient survival, were prioritised for further validation. Senescence induction was found to be highly stable, and associated with dramatic changes to nucleolar morphology, reminiscent of the nucleolar signature observed upon premature senescence induction in normal human mammary epithelial cells. In addition, siRNA rescue experiments indicated that senescence initiation is dependent on p16 and p21 expression and is accompanied by p16 nuclear translocation and p21 degradation. Further, ribosomal protein silencing in MDA-MB-231 cells (p16-null Basal-like breast cancer cell line) resulted in a ‘death-like’ phenotype, partially dependent on p21 expression suggesting that, within a cancer context, ribosomal protein silencing may induce a differential response depending on the status of p16. In conclusion, it is proposed that these six ribosomal candidates may form the basis of a novel pro-senescence therapy for p16-positive Basal-like breast cancer. They may also represent novel prognostic biomarkers for this disease subset and may help to improve disease stratification and future directed personalised therapies.Medical Research Council PhD Studentship (Grant number: RGJZZY4)

Studies on the conformation of HeLa cell 5.8s rRNA

The low molecular weight ribosomal RNA species, 5.8S rRNA, is found in the larger ribosomal subunit of eukaryotic cells; it occurs in equimolar ratios to the 28S rRNA molecule and is hydrogen-bonded to that component. The nucleotide sequences of 5.8S rRNA from several organisms have been determined and a secondary structure proposed, based on maximised base pairing. The aim of this work has been to investigate the conformation of HeLa cell 5.8S rRNA. The approach to this investigation involved the use of the technique of chemical modification. The assumption behind such an approach is that a base residue which is not modified by a reagent specific for that base is inaccessible. Bases involved in double helical stems of the molecule are unreactive and the reaction will be diminished if a base is involved in maintaining the tertiary structure through stacking or base pairing. Only bases not involved in such interactions will be significantly modified by a reagent specific for that base. The correlation between the three dimensional structure of yeast tRNAPhe and the results obtained from chemical modification studies suggest that such studies carried out on 5.8S rRNA should provide useful data concerning the conformation. Two modifying reagents, were used in this study; sodium bisulphite, which reacts specifically with non-hydrogen bonded cytidine residues, converting them to uridine, and carbodiimide, which under the conditions used has high specificity for non-hydrogen bonded uridine residues. When these techniques are coupled with 'fingerprinting' procedures it is possible to investigate the structure of 5.8S rRNA. When these studies were carried out on isolated 5.8S rRNA at 2

Comparative analysis of RNA binding properties of Rpl22 proteins

After the whole genome duplication event, Saccharomyces cerevisiae lost 90 % of its paralogs. 59 ribosomal protein genes (RPG) retained a duplicated form. The cell has to balance expression of RPGs as a part of adaptation to changing conditions, thus ensuring the production of the right ratio of ribosomal proteins (RP). In addition, RPGs contain 1/3 of all introns found in the S. cerevisiae genome. Rpl22A/B are part of the large ribosomal subunit where they contact 25S rRNA. Deletion of these RPGs results in 2X slower growth in comparison with WT cells. Within their extraribosomal function Rpl22A/B are able to intragenically and intergenically regulate their expression. Binding of Rpl22A/B to the intronic part of pre-mRNA of RPL22A or RPL22B results in splicing inhibition, which is stronger in the case of the RPL22B intron (RPL22Bi). However, the exact mechanism is not known. We know that Rpl22s from various organisms bind a hairpin structure that can be found in 25S rRNA also. Since the predicted structure of RPL22Bi does not contain this binding motif, it rises a question of whether the RNA binding surface of Rpl22A/B is different or more extensive than the one with which Rpl22 contacts 25S rRNA. We compared the ability of Rpl22 from different organisms to complement the functions of Rpl22A/B. These...Po události celogenomové duplikace (WGD) ztratila Saccharomyces cerevisiae 90 % paralogů. 59 genů pro ribozomální proteiny (RPG) si zachovalo duplikovanou kopii. Buňka je schopna v rámci adaptace na měnící se podmínky balancovat expresi RPG, a zajišťovat tak produkci ribozomálních proteinů (RP) ve správném poměru. RPG navíc obsahují 1/3 veškerých intronů vyskytujících se v genomu S. cerevisiae. Rpl22A/B jsou součástí velké ribozomální podjednotky, kde kontaktují 25S rRNA. Delece těchto RPG vede k 2X pomalejšímu růstu v porovnání s buňkami WT. Rpl22A/B jsou v rámci své extraribozomální funkce schopny intragenově a intergenově regulovat svoji expresi. Výsledkem vazby Rpl22A/B na intron pre-mRNA RPL22A nebo RPL22B je inhibice sestřihu, která je silnější v případě intronu RPL22B (RPL22Bi). Přesný mechanismus ovšem není známý. Víme, že Rpl22 z různých organismů vážou strukturu vlásenky, která se vyskytuje mimo jiné i v 25S rRNA. Jelikož predikovaná struktura RPL22Bi vlásenku neobsahuje, vyvolává to otázku, zda je RNA vazebný povrch Rpl22A/B odlišný nebo rozsáhlejší než ten, kterým kontaktuje 25S rRNA. Porovnali jsme schopnost Rpl22 z různých organismů zastoupit funkce Rpl22A/B. Tyto Rpl22 mají substituci v několika pozicích aminokyselin (AK). Tato práce rozebírá vliv odlišného složení AK Rpl22 na funkce...Katedra buněčné biologieDepartment of Cell BiologyFaculty of SciencePřírodovědecká fakult

Investigation of Neurexin 2 as a Candidate for Parkinson's Disease

Biomedical Sciences: Molecular Biology and Human Genetic