+publisher:"University of Manchester" +contributor:("O\'Keefe, Raymond")

University of Manchester

1. Nancollis, Verity. Investigation into the Saccharomyces cerevisiae U5 snRNP, a core spliceosome component.

Degree: 2011, University of Manchester

URL: http://www.manchester.ac.uk/escholar/uk-ac-man-scw:106068

► The U5 snRNP is a major component of the yeast spliceosome, being part of the U4/U6.U5 tri-snRNP, the precatalytic spliceosome and the catalytically activated spliceosome.… (more)

▼ The U5 snRNP is a major component of the yeast spliceosome, being part of the U4/U6.U5 tri-snRNP, the precatalytic spliceosome and the catalytically activated spliceosome. The U5 snRNP includes, at its heart, the U5 snRNA which contains the invariant Loop 1 that functions in tethering and aligning exons during splicing. The major protein components of the U5 snRNP are the highly conserved Prp8p, the GTPase Snu114p and the helicase Brr2p. These proteins and the U5 snRNA are integral in forming the active site of the spliceosome and regulating the dynamic changes of the spliceosome. The first part of this study aimed to express and purify specific domains of Snu114p to define the structure and function of Snu114p. The N-terminal region of Snu114p was successfully expressed and purified from bacteria. Addition of the Snu114p N-terminal fragment to in vitro splicing assays resulted in a first step splicing defect, indicating a role for the N-terminus in pre-mRNA splicing. NMR studies revealed that the N-terminus of Snu114p exists as an unstructured protein domain. Mutagenesis indicated that the N-terminus of Snu114p is tolerant to mutation. A novel genetic interaction between amino acids in the N-terminus of Snu114p and the 3’ side of the U5 snRNA IL1 was identified. It is proposed here that the N-terminus of Snu114p functions to stabilise interactions of Snu114p with other proteins or snRNAs, possibly the U5 snRNA. Alternatively, the N-terminus of Snu114p may form intramolecular interactions with other regions of Snu114p to regulate Snu114p function in pre-mRNA splicing.Prp8p, Snu114p and Brr2p are known to form a stable complex but their interactions with the specific domains of the U5 snRNA are not known. The second part of this study aimed to investigate the association of Brr2p, Snu114p and Prp8p with the U5 snRNA. Mutants of the U5 snRNA were constructed in the conserved Loop 1 and the Internal Loop 1 (IL1). The influences of the U5 snRNA mutations on interactions of Prp8p, Snu114p or Brr2p with the snRNA were investigated. It was revealed that Loop 1 and both sides of IL1 of the U5 snRNA are important in association of Brr2p, Snu114p and Prp8p. Mutations in the 3’ side of IL1 drastically reduce association of Brr2p, Snu114p and Prp8p with the U5 snRNA, highlighting this region as a potential ‘protein docking’ site within the U5 snRNP. Differences seen in the associations of Brr2p, Snu114p and Prp8p with U5 snRNA mutations demonstrate that although there are intimate interactions between Brr2p, Snu114p and Prp8p, they do not associate with the U5 snRNA as a tri-protein complex. Genetic screening of BRR2 and U5 snRNA mutants reveals an interaction between the N-terminal half of Brr2p and the 3’ side of U5 snRNA IL1. This supports the proposed ‘protein docking’ site at the 3’ side of the U5 snRNA IL1.Data presented in this study increases our understanding of the regions in the U5 snRNA required for association of the essential U5 snRNP proteins, Brr2p, Snu114p and Prp8p, and goes some way to elucidating the… Advisors/Committee Members: O'Keefe, Raymond.

Subjects/Keywords: pre-mRNA splicing; U5 snRNP

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APA · Chicago · MLA · Vancouver · CSE | Export to Zotero / EndNote / Reference Manager

APA (6^th Edition):

Nancollis, V. (2011). Investigation into the Saccharomyces cerevisiae U5 snRNP, a core spliceosome component. (Doctoral Dissertation). University of Manchester. Retrieved from http://www.manchester.ac.uk/escholar/uk-ac-man-scw:106068

Chicago Manual of Style (16^th Edition):

Nancollis, Verity. “Investigation into the Saccharomyces cerevisiae U5 snRNP, a core spliceosome component.” 2011. Doctoral Dissertation, University of Manchester. Accessed January 28, 2021. http://www.manchester.ac.uk/escholar/uk-ac-man-scw:106068.

MLA Handbook (7^th Edition):

Nancollis, Verity. “Investigation into the Saccharomyces cerevisiae U5 snRNP, a core spliceosome component.” 2011. Web. 28 Jan 2021.

Vancouver:

Nancollis V. Investigation into the Saccharomyces cerevisiae U5 snRNP, a core spliceosome component. [Internet] [Doctoral dissertation]. University of Manchester; 2011. [cited 2021 Jan 28]. Available from: http://www.manchester.ac.uk/escholar/uk-ac-man-scw:106068.

Council of Science Editors:

Nancollis V. Investigation into the Saccharomyces cerevisiae U5 snRNP, a core spliceosome component. [Doctoral Dissertation]. University of Manchester; 2011. Available from: http://www.manchester.ac.uk/escholar/uk-ac-man-scw:106068

University of Manchester

2. Demain, Leigh Ann Mary. IDENTIFYING GENETIC VARIANTS IMPLICATED IN PERRAULT SYNDROME FOR IMPROVED HEARING LOSS DIAGNOSIS AND THERAPEUTICS.

Degree: 2017, University of Manchester

URL: http://www.manchester.ac.uk/escholar/uk-ac-man-scw:312267

► Perrault syndrome is a rare autosomal recessive condition characterised by sensorineural hearing loss affecting both sexes and premature ovarian insufficiency in 46, XX females. Some… (more)

▼ Perrault syndrome is a rare autosomal recessive condition characterised by sensorineural hearing loss affecting both sexes and premature ovarian insufficiency in 46, XX females. Some affected individuals present with neurological features such as ataxia, neuropathies and intellectual disability. To date six genes which cause Perrault syndrome have been identified; HSD17B4, HARS2, LARS2, CLPP, C10orf2 and ERAL1. In many cases the genetic cause of Perrault syndrome is unknown. We used whole exome sequencing to identify the genetic basis of Perrault syndrome in nine affected families. In six families we identified variants in known Perrault syndrome genes and highlighted a genotype-phenotype link between the variant LARS2 c.1565C>A p.(T522N) and low frequency sensorineural hearing loss. We also found marfanoid habitus in Perrault syndrome is not genotype specific. In three families we identified putative pathogenic variants in three novel Perrault syndrome genes; PRORP, NOP14 and DAP3. Investigation of novel Perrault genes revealed a defect of mitochondrial translation is the likely pathogenic mechanism in the case of Perrault syndrome caused by variants in PRORP, but data from a patient with Perrault syndrome caused by DAP3 showed that this is unlikely to be the mechanism in all cases. PRORP is a subunit of mitochondrial RNase P, a mitochondrial tRNA processing complex. The variant in the affected family, PRORP p.A485V, reduced mitochondrial tRNA processing by 40% resulting in accumulation of unprocessed RNA transcripts and a defect of mitochondrial translation in patient fibroblasts. DAP3 is an essential subunit of the mitochondrial ribosome. In the proband with the variant DAP3 p.C395Y, which is in trans to a large deletion, there was no defect of mitochondrial translation seen in fibroblasts. The variant DAP3 p.C395Y likely affects a non-ribosomal role of DAP3 indicating a different Perrault Syndrome pathology to that of Perrault syndrome caused by defects of PRORP. NOP14 localises to the nucleolus and functions in ribosome biogenesis but our data suggests NOP14 may have a mitochondrial function. Haploinsufficiency of NOP14 in yeast causes the slow loss of mitochondria and we saw a distinct non-nucleolar localisation of Nop14 in the mouse organ of Corti. Perrault syndrome shows large genetic and phenotypic heterogeneity. We have identified three novel Perrault syndrome genes and shed some light on the molecular pathology of Perrault syndrome. Advisors/Committee Members: O'KEEFE, RAYMOND RT, Newman, William, O'Keefe, Raymond.

Subjects/Keywords: Perrault syndrome; Primary ovarian insufficiency; Sensorineural hearing loss

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APA · Chicago · MLA · Vancouver · CSE | Export to Zotero / EndNote / Reference Manager

APA (6^th Edition):

Demain, L. A. M. (2017). IDENTIFYING GENETIC VARIANTS IMPLICATED IN PERRAULT SYNDROME FOR IMPROVED HEARING LOSS DIAGNOSIS AND THERAPEUTICS. (Doctoral Dissertation). University of Manchester. Retrieved from http://www.manchester.ac.uk/escholar/uk-ac-man-scw:312267

Chicago Manual of Style (16^th Edition):

Demain, Leigh Ann Mary. “IDENTIFYING GENETIC VARIANTS IMPLICATED IN PERRAULT SYNDROME FOR IMPROVED HEARING LOSS DIAGNOSIS AND THERAPEUTICS.” 2017. Doctoral Dissertation, University of Manchester. Accessed January 28, 2021. http://www.manchester.ac.uk/escholar/uk-ac-man-scw:312267.

MLA Handbook (7^th Edition):

Demain, Leigh Ann Mary. “IDENTIFYING GENETIC VARIANTS IMPLICATED IN PERRAULT SYNDROME FOR IMPROVED HEARING LOSS DIAGNOSIS AND THERAPEUTICS.” 2017. Web. 28 Jan 2021.

Vancouver:

Demain LAM. IDENTIFYING GENETIC VARIANTS IMPLICATED IN PERRAULT SYNDROME FOR IMPROVED HEARING LOSS DIAGNOSIS AND THERAPEUTICS. [Internet] [Doctoral dissertation]. University of Manchester; 2017. [cited 2021 Jan 28]. Available from: http://www.manchester.ac.uk/escholar/uk-ac-man-scw:312267.

Council of Science Editors:

Demain LAM. IDENTIFYING GENETIC VARIANTS IMPLICATED IN PERRAULT SYNDROME FOR IMPROVED HEARING LOSS DIAGNOSIS AND THERAPEUTICS. [Doctoral Dissertation]. University of Manchester; 2017. Available from: http://www.manchester.ac.uk/escholar/uk-ac-man-scw:312267

University of Manchester

3. Almutawa, Qamar E B a a. Impact of Chromosomal Translocations (CTs) on reproductive isolation and fitness in natural yeast isolates.

Degree: 2017, University of Manchester

URL: http://www.manchester.ac.uk/escholar/uk-ac-man-scw:307192

► Identifying the molecular mechanisms behind reproductive isolation between closely related yeast species provides avaluable understanding of their evolution. Sequence divergence and chromosomal rearrangements are the… (more)

▼ Identifying the molecular mechanisms behind reproductive isolation between closely related yeast species provides avaluable understanding of their evolution. Sequence divergence and chromosomal rearrangements are the main post-zygotic barriers behind reproductive isolation within Saccharomyces „sensu strico’ species, where hybrids are readily formed but sterile upon meiosis. Saccharomyces paradoxus and Saccharomyces cariocanus have an almost identical genome in terms of sequence, and therefore provide good model systems to explore the impact of karyotypic rearrangements on reproductive isolation. According to the biological species concept they are considered two different species despite having low sequence divergence. Since the karyotypic analysis revealed that the genomic differences are restricted to four chromosomal translocations, we hypothesized that such rearrangements may be the cause of low spore viability between them. To test this expectation, we engineered two chromosomal translocations in S. paradoxus YPS138, via Cre-loxP mediated recombination event, to render those parts of genome collinear to S.cariocanus UFRJ50816. Our analysis revealed that hybrids between S. cariocanus and engineered S. paradoxus harbouring two translocations showed a significant increase in spore viability (12.7%) compared to control hybrids harbouring five translocations (3.4%) (P=0.0031and P=0.0125, respectively, Two-sample t-test). Consequently, fitness in meiosis was improved four fold by undoing two translocations. Given this result, the prediction for spore viability in complete collinear crossing would be around 50.8 %, which is still far from the value of ca. 100%, which would be expected for strains with very low sequence divergence and belonging to the same species. This indicates that other factors may contribute to meiotic fitness in these hybrids. Further investigation was carried to determine the genome structures by using the PacBio sequencing approach. Our DNA sequencing data revealed other, previously undetected, rearrangements in S. cariocanus strain: one new reciprocal translocation between chromosomes XIII and XIV and 11 inversions distributed in 6 chromosomes. The variations in meiotic viability observed in the engineered hybrids could be because of these 5 chromosomal translocations. Further experiments were also carried out to evaluate the impact of translocations on mitotic fitness and gene expression; we observed a significant drop in the mitotic fitness of engineered translocant strains under different nutritional and temperature stresses. These changes were also accompanied with alteration in genes expression throughout the genome. Our RNA- seq data revealed that many genes were up- or down- regulated because of the translocation. Several genes with altered expression in translocant strains are correlated with morphology changes when they are up- or down- regulated. Therefore, the cell morphology was evaluated under light microscopy and different abnormal cells were detected compared to the wild type.… Advisors/Committee Members: O'KEEFE, RAYMOND RT, O'Keefe, Raymond, Delneri, Daniela.

Subjects/Keywords: Chromosomal Translocations

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APA · Chicago · MLA · Vancouver · CSE | Export to Zotero / EndNote / Reference Manager

APA (6^th Edition):

Almutawa, Q. E. B. a. a. (2017). Impact of Chromosomal Translocations (CTs) on reproductive isolation and fitness in natural yeast isolates. (Doctoral Dissertation). University of Manchester. Retrieved from http://www.manchester.ac.uk/escholar/uk-ac-man-scw:307192

Chicago Manual of Style (16^th Edition):

Almutawa, Qamar E B a a. “Impact of Chromosomal Translocations (CTs) on reproductive isolation and fitness in natural yeast isolates.” 2017. Doctoral Dissertation, University of Manchester. Accessed January 28, 2021. http://www.manchester.ac.uk/escholar/uk-ac-man-scw:307192.

MLA Handbook (7^th Edition):

Almutawa, Qamar E B a a. “Impact of Chromosomal Translocations (CTs) on reproductive isolation and fitness in natural yeast isolates.” 2017. Web. 28 Jan 2021.

Vancouver:

Almutawa QEBaa. Impact of Chromosomal Translocations (CTs) on reproductive isolation and fitness in natural yeast isolates. [Internet] [Doctoral dissertation]. University of Manchester; 2017. [cited 2021 Jan 28]. Available from: http://www.manchester.ac.uk/escholar/uk-ac-man-scw:307192.

Council of Science Editors:

Almutawa QEBaa. Impact of Chromosomal Translocations (CTs) on reproductive isolation and fitness in natural yeast isolates. [Doctoral Dissertation]. University of Manchester; 2017. Available from: http://www.manchester.ac.uk/escholar/uk-ac-man-scw:307192

University of Manchester

4. Kirchhoffer, Olivier Auguste. Synthesis and evaluation of small molecule inhibitors of pre-mRNA splicing to effectively modify gene expression.

Degree: 2019, University of Manchester

URL: http://www.manchester.ac.uk/escholar/uk-ac-man-scw:322579

►

The spliceosome is a highly complex molecular machine that still holds a lot of secrets for contemporary biology. It plays a crucial role as one… (more)

▼

The spliceosome is a highly complex molecular machine that still holds a lot of secrets for contemporary biology. It plays a crucial role as one of the various mechanisms involved in generating a plethora of different proteins, from a limited amount of genetic information, sealed in the form of DNA in the nucleus of living cells. Previous studies have tried to dissect this complex mechanism by inhibiting the spliceosome, in order to study its biological properties. Interestingly a significant number of inhibitors found, which target the specific U2 snRNP unit of the spliceosome, have been reported to have anti-cancer activity. Some of the most promising compounds, which resulted from this drug development approach, have almost made it to the drug market at the time of writing, for their antitumor activity. A vast amount of research has emerged concerning the biological processes that ultimately led to this activity. Previous work within the Whitehead group, which focused on developing inhibitors of the U5 snRNP unit of the spliceosome, has led to the discovery of a compound derived from the steroidal antibiotic fusidic acid that displayed significant splicing inhibitory activity. Lithocholic acid later emerged as another potential starting point for developing small molecule inhibitors of the U5 snRNP, as it was found to have similar levels of inhibition as the hit compound derived from fusidic acid. Lithocholic acid was a good starting point for further SAR studies, as it isn't heavily functionalised, making it a robust compound that can easily be diversified. A variety of compounds were generated from it, with new structural features and additional functional groups that unravelled new information about the target protein. Splicing assays also revealed that several of these compounds have superseded the level of inhibition achieved by the previously outlined hit compound.

A CD with attach supplementary information has been submitted alongside the first version of the thesis

Advisors/Committee Members: O'KEEFE, RAYMOND RT, Whitehead, Roger, O'Keefe, Raymond.

Subjects/Keywords: RNA Splicing; Lithocholic acid; Splicing inhibitors; Steroid

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APA · Chicago · MLA · Vancouver · CSE | Export to Zotero / EndNote / Reference Manager

APA (6^th Edition):

Kirchhoffer, O. A. (2019). Synthesis and evaluation of small molecule inhibitors of pre-mRNA splicing to effectively modify gene expression. (Doctoral Dissertation). University of Manchester. Retrieved from http://www.manchester.ac.uk/escholar/uk-ac-man-scw:322579

Chicago Manual of Style (16^th Edition):

Kirchhoffer, Olivier Auguste. “Synthesis and evaluation of small molecule inhibitors of pre-mRNA splicing to effectively modify gene expression.” 2019. Doctoral Dissertation, University of Manchester. Accessed January 28, 2021. http://www.manchester.ac.uk/escholar/uk-ac-man-scw:322579.

MLA Handbook (7^th Edition):

Kirchhoffer, Olivier Auguste. “Synthesis and evaluation of small molecule inhibitors of pre-mRNA splicing to effectively modify gene expression.” 2019. Web. 28 Jan 2021.

Vancouver:

Kirchhoffer OA. Synthesis and evaluation of small molecule inhibitors of pre-mRNA splicing to effectively modify gene expression. [Internet] [Doctoral dissertation]. University of Manchester; 2019. [cited 2021 Jan 28]. Available from: http://www.manchester.ac.uk/escholar/uk-ac-man-scw:322579.

Council of Science Editors:

Kirchhoffer OA. Synthesis and evaluation of small molecule inhibitors of pre-mRNA splicing to effectively modify gene expression. [Doctoral Dissertation]. University of Manchester; 2019. Available from: http://www.manchester.ac.uk/escholar/uk-ac-man-scw:322579

5. Coyne, Victoria Lee. Characterization of long non-coding RNAs in the Hox Complex of Drosophila.

Degree: 2016, University of Manchester

URL: http://www.manchester.ac.uk/escholar/uk-ac-man-scw:305935

► Long non-coding RNAs (lncRNAs) are often defined as transcripts >200nts that have no discernable protein-coding ability (Quinn and Chang, 2016). Although relatively little is understood… (more)

▼ Long non-coding RNAs (lncRNAs) are often defined as transcripts >200nts that have no discernable protein-coding ability (Quinn and Chang, 2016). Although relatively little is understood about the molecular mechanisms of lncRNA function, they have established roles in regulation of gene expression during development, cell differentiation and pluripotency (Fatica and Bozzoni, 2014; Luo et al., 2016; Quinn and Chang, 2016; Rinn and Chang, 2012) across vastly diverse organisms ranging from plants to humans (Ulitsky and Bartel, 2013). LncRNAs have also been associated with numerous pathological conditions, such as cancers (Brunner et al., 2012), cardiovascular disease and neurodegeneration (Chen et al., 2013). Investigations into lncRNAs in wide ranging organisms, have revealed that many influence gene activity by forming ribonucleoprotein complexes that affect the conformational state of chromatin (Rinn and Chang, 2012). A genomic region that has revealed several functional lncRNAs in diverse organisms is the Hox complex (Pauli et al., 2011; Pettini, 2012; Rinn et al., 2007). The Hox complex encodes a set of transcription factors (TFs), physically clustered in the genome, which provide morphological identity along the anterior to posterior axis of developing embryos (Mallo and Alonso, 2013), throughout the majority of bilatarian animals (Moreno et al., 2011). Misexpression or mutation of Hox genes causes morphological and pathophysiological defects (Quinonez and Innis, 2014). We investigated clustering of lncRNAs throughout the D. melanogaster genome using available annotations and carried out RNA-seq in D. virilis to expand the repertoire of lncRNAs and identify clusters of lncRNAs. We found the Hox complex to be heavily enriched with lncRNAs in both organisms, and syntenic transcripts from D. melanogaster could be identified in D. pseudoobscura and D. virilis. Several lncRNAs aligned with polycomb response elements (PREs); transcription of PREs has previously been linked to a switch in their activity (Herzog et al., 2014). However, we found that transcribed PREs in D. melanogaster move positions relative to the protein-coding genes in other drosophilids, whilst the transcriptional units remain in the same syntenic region. Conservation of syntenic transcripts without evidence of remaining a PRE suggest that the transcription is not linked to PRE function, agreeing with recent findings that transcription of PREs does not affect their function (Kassis and Muller, 2015). We investigated functions of a novel lncRNA and adjacent PRE in the Hox complex by ectopic expression and utilization of other genetic manipulation tools. Overexpression of either the lncRNA or PRE and partial duplication of the lncRNA caused phenotypes such as missing halteres and/or T3 legs, misshaped T3 legs or malformed abdominal segments. The observations that ectopic expression of this lncRNA and an adjacent regulatory element from the Hox complex causes phenotypes that can be linked to adjacent Hox gene misregulation, Antp and Ubx, suggest that they… Advisors/Committee Members: O'KEEFE, RAYMOND RT, GRIFFITHS-JONES, SAMUEL SR, O'Keefe, Raymond, Griffiths-Jones, Samuel, Ronshaugen, Matthew.

Subjects/Keywords: long non-coding RNA evolution development Hox

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APA · Chicago · MLA · Vancouver · CSE | Export to Zotero / EndNote / Reference Manager

APA (6^th Edition):

Coyne, V. L. (2016). Characterization of long non-coding RNAs in the Hox Complex of Drosophila. (Doctoral Dissertation). University of Manchester. Retrieved from http://www.manchester.ac.uk/escholar/uk-ac-man-scw:305935

Chicago Manual of Style (16^th Edition):

Coyne, Victoria Lee. “Characterization of long non-coding RNAs in the Hox Complex of Drosophila.” 2016. Doctoral Dissertation, University of Manchester. Accessed January 28, 2021. http://www.manchester.ac.uk/escholar/uk-ac-man-scw:305935.

MLA Handbook (7^th Edition):

Coyne, Victoria Lee. “Characterization of long non-coding RNAs in the Hox Complex of Drosophila.” 2016. Web. 28 Jan 2021.

Vancouver:

Coyne VL. Characterization of long non-coding RNAs in the Hox Complex of Drosophila. [Internet] [Doctoral dissertation]. University of Manchester; 2016. [cited 2021 Jan 28]. Available from: http://www.manchester.ac.uk/escholar/uk-ac-man-scw:305935.

Council of Science Editors:

Coyne VL. Characterization of long non-coding RNAs in the Hox Complex of Drosophila. [Doctoral Dissertation]. University of Manchester; 2016. Available from: http://www.manchester.ac.uk/escholar/uk-ac-man-scw:305935

University of Manchester

6. Estrada-Rivadeneyra, Diego. Use of the ncRNA deletion and overexpression collections in Saccharomyces cerevisiae for the study of ncRNAs and mode of action of orphan drugs.

Degree: 2019, University of Manchester

URL: http://www.manchester.ac.uk/escholar/uk-ac-man-scw:322447

► Analysis of eukaryotic transcriptomes has revealed the existence of thousands of previously unannotated noncoding RNAs (ncRNAs), most of them with unknown functions. Recent evidence suggests… (more)

▼ Analysis of eukaryotic transcriptomes has revealed the existence of thousands of previously unannotated noncoding RNAs (ncRNAs), most of them with unknown functions. Recent evidence suggests that these novel transcripts play important roles in gene regulation, particularly under stress conditions. The main objective of this study was to use the recently developed ncRNA deletion and overexpression collections in Saccharomyces cerevisiae to study the functions of ncRNAs in eukaryotes and obtain information on the drug mode of action and possible ncRNA targets of orphan drugs. A high-throughput assay on solid media was used to systematically test the haploid (MATa) and diploid heterozygous ncRNA deletion collections in the presence of two orphan drugs: lithium citrate and riluzole. A total of seventeen ncRNAs were identified from this initial screening and subjected to liquid growth assays to confirm the results observed on solid media, quantify the growth differences, and detect the growth stages being affected. Moreover, by cloning and transforming the previously deleted ncRNAs back into the corresponding deletion strains we discovered evidence that suggests that four of the ncRNAs identified in this study might act in trans. Furthermore, high-throughput screening of the ncRNA overexpression collection in yeast revealed the existence of ncRNAs that conferred increased or decreased resistance to lithium when overexpressed. Analysis of gene expression by qPCR revealed that overexpression of the ncRNA SUT378 leads to downregulation of the overlapping gene TUM1, which confers increased resistance to lithium. Additionally, we present in this study results and methods from experiments performed with the objective of providing further evidence and resources that will serve as a base for the future study of ncRNAs. In conclusion, the yeast ncRNA deletion and overexpression collections have proven to be effective and inexpensive resources to obtain information on the functions of ncRNAs and discover possible ncRNA targets. Given that most basic biological processes are conserved within eukaryotes, data obtained from these assays could help us better understand the regulatory functions of ncRNAs in other eukaryotes and reveal the drug mode of action and targets of orphan drugs in humans. Advisors/Committee Members: DELNERI, DANIELA DEA, O'Keefe, Raymond, Delneri, Daniela.

Subjects/Keywords: ncRNA; yeast genetics; orphan drugs; chemogenomics; riluzole; lithium; HDAC inhibitors; drug screening

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APA · Chicago · MLA · Vancouver · CSE | Export to Zotero / EndNote / Reference Manager

APA (6^th Edition):

Estrada-Rivadeneyra, D. (2019). Use of the ncRNA deletion and overexpression collections in Saccharomyces cerevisiae for the study of ncRNAs and mode of action of orphan drugs. (Doctoral Dissertation). University of Manchester. Retrieved from http://www.manchester.ac.uk/escholar/uk-ac-man-scw:322447

Chicago Manual of Style (16^th Edition):

Estrada-Rivadeneyra, Diego. “Use of the ncRNA deletion and overexpression collections in Saccharomyces cerevisiae for the study of ncRNAs and mode of action of orphan drugs.” 2019. Doctoral Dissertation, University of Manchester. Accessed January 28, 2021. http://www.manchester.ac.uk/escholar/uk-ac-man-scw:322447.

MLA Handbook (7^th Edition):

Estrada-Rivadeneyra, Diego. “Use of the ncRNA deletion and overexpression collections in Saccharomyces cerevisiae for the study of ncRNAs and mode of action of orphan drugs.” 2019. Web. 28 Jan 2021.

Vancouver:

Estrada-Rivadeneyra D. Use of the ncRNA deletion and overexpression collections in Saccharomyces cerevisiae for the study of ncRNAs and mode of action of orphan drugs. [Internet] [Doctoral dissertation]. University of Manchester; 2019. [cited 2021 Jan 28]. Available from: http://www.manchester.ac.uk/escholar/uk-ac-man-scw:322447.

Council of Science Editors:

Estrada-Rivadeneyra D. Use of the ncRNA deletion and overexpression collections in Saccharomyces cerevisiae for the study of ncRNAs and mode of action of orphan drugs. [Doctoral Dissertation]. University of Manchester; 2019. Available from: http://www.manchester.ac.uk/escholar/uk-ac-man-scw:322447

University of Manchester

7. Tandiono, Moses. Yeast Models for the Craniofacial Disorders Mandibulofacial Dysostosis Guion Almeida Type and Burn McKeown Syndrome.

Degree: 2016, University of Manchester

URL: http://www.manchester.ac.uk/escholar/uk-ac-man-scw:300458

► Proper formation of the cranial and facial features in vertebrates is a crucial event that requires the tight regulation of differentiation and proliferation of cranial… (more)

▼ Proper formation of the cranial and facial features in vertebrates is a crucial event that requires the tight regulation of differentiation and proliferation of cranial neural crest cells (CNCC). Similar to Treacher Collins syndrome, both mandibulofacial dysostoses Guion-Almeida (MFDGA) and Burn McKeown Syndrome (BMKS) patients share some malformed craniofacial features. Genetic characterisation of MFDGA and BMKS has identified spontaneous heterozygous mutations and deletions in EFTUD2 (hSnu114) and in the promoter regions of TXNL4A (hDib1) combined with one null allele, respectively. Both genes encode for highly conserved U5 small nuclear ribonucleoproteins (snRNP), and the mutations results in reduced expression of hSnu114 and hDib1, both of which are essential components of the spliceosome required for pre-mRNA splicing. In this work, yeast models of MFDGA and BMKS were used in growth assays to identify growth sensitivities to ER stress factors, providing evidence that ER stress may affect CNCC survival, proliferation and differentiation during craniofacial development. The profiling of GAL1-regulated SNU114 and DIB1 strains displayed different proliferation rates when SNU114 and DIB1 expression was switched on or off, indicating that expression of both SNU114 and DIB1 needs to be tightly regulated to prevent mis-splicing of pre-mRNAs. The inhibition of SNU114 and DIB1 expression induced a markedly reduced and a slightly reduced tri-snRNP assembly in primer extension assay, respectively, revealing their functional link in tri-snRNP assembly and in pre-mRNA splicing. Additionally, an accumulation of larger U2 snRNA complexes were observed in both SNU114 and DIB1 depleted extracts. This study suggest a link in reduced tri-snRNP assembly to the mis-splicing of a subset of pre-mRNAs that are crucial not only for proliferation and differentiation, but for restoring ER homeostasis during NCC development. Finally, the elimination of antibiotic activity of a fusidic acid derivative through targeted chemical modifications indicates the generation of a splicing inhibitor that potentially targets Snu114p. It is suggested that the reduced expression of EFTUD2 and TXNL4A results in neural crest cell apoptosis due to aberrant mis-splicing of crucial genes involved in differentiation and proliferation during embryogenesis, leading to MFDGA and BMKS. Advisors/Committee Members: CROSTHWAITE, SUSAN SK, O'Keefe, Raymond, Crosthwaite, Susan.

Subjects/Keywords: MFDGA; BMKS; Mandibulofacial dysostosis; SNU114; DIB1; Fusidic acid; Splicing inhibitor

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APA · Chicago · MLA · Vancouver · CSE | Export to Zotero / EndNote / Reference Manager

APA (6^th Edition):

Tandiono, M. (2016). Yeast Models for the Craniofacial Disorders Mandibulofacial Dysostosis Guion Almeida Type and Burn McKeown Syndrome. (Doctoral Dissertation). University of Manchester. Retrieved from http://www.manchester.ac.uk/escholar/uk-ac-man-scw:300458

Chicago Manual of Style (16^th Edition):

Tandiono, Moses. “Yeast Models for the Craniofacial Disorders Mandibulofacial Dysostosis Guion Almeida Type and Burn McKeown Syndrome.” 2016. Doctoral Dissertation, University of Manchester. Accessed January 28, 2021. http://www.manchester.ac.uk/escholar/uk-ac-man-scw:300458.

MLA Handbook (7^th Edition):

Tandiono, Moses. “Yeast Models for the Craniofacial Disorders Mandibulofacial Dysostosis Guion Almeida Type and Burn McKeown Syndrome.” 2016. Web. 28 Jan 2021.

Vancouver:

Tandiono M. Yeast Models for the Craniofacial Disorders Mandibulofacial Dysostosis Guion Almeida Type and Burn McKeown Syndrome. [Internet] [Doctoral dissertation]. University of Manchester; 2016. [cited 2021 Jan 28]. Available from: http://www.manchester.ac.uk/escholar/uk-ac-man-scw:300458.

Council of Science Editors:

Tandiono M. Yeast Models for the Craniofacial Disorders Mandibulofacial Dysostosis Guion Almeida Type and Burn McKeown Syndrome. [Doctoral Dissertation]. University of Manchester; 2016. Available from: http://www.manchester.ac.uk/escholar/uk-ac-man-scw:300458

University of Manchester

8. Godinjak, Lamija. Investigating Burn-McKeown Syndrome and Mandibulofacial Dysostosis, Guion-Almeida type through genetic interaction studies of Dib1 and Snu114 in S. cerevisiae.

Degree: 2018, University of Manchester

URL: http://www.manchester.ac.uk/escholar/uk-ac-man-scw:314241

► Removal, or splicing, of intervening sequences from the pre-messenger RiboNucleic Acid (pre-mRNA) to form mature mRNA in eukaryotes is carried out by the spliceosome. During… (more)

▼ Removal, or splicing, of intervening sequences from the pre-messenger RiboNucleic Acid (pre-mRNA) to form mature mRNA in eukaryotes is carried out by the spliceosome. During the stepwise assembly of the spliceosome, the catalytic core of the spliceosome is created as the B complex is activated. Proteins of U5 small nuclear RiboNucleo Protein particles (snRNPs), Prp8, Brr2, Snu114 and Dib1, are core splicing factors which play crucial roles in activation of the B complex. As Prp8 releases its inhibition on the helicase Brr2, the U4/U6 duplex unwinds and completes the formation of the catalytic core of the spliceosome. GTP has to bind Snu114, a GTPase, to support U4/U6 duplex unwinding while Dib1 leaves B complex during the activation of B complex. Compound heterozygosity of core splicing factor DIB1 (TXLN4A) and haploinsufficiency of core splicing factor SNU114 (EFTUD2) cause the craniofacial disorders Burn McKeown Syndrome (BMKS) and Mandibulofacial Dysostosis, Guion-Almeida type (MFDGA) respectively. Patients with BMKS and MFDGA share similar disease phenotypes. As BMKS and MFDGA have overlapping phenotypes and Snu114, in its GTP state, activates the B complex while Dib1 has to leave to allow B complex activation, we hypothesize that Snu114 and Dib1 are functionally linked. A synthetic genetic screen was performed to identify if genetic interactions exist between Snu114 and Dib1. For the synthetic genetic screen, a haploid yeast strain, having SNU114-DIB1 double knock out and the complementing wild type genes on a URA3 plasmid, was successfully constructed. The constructed strain was then transformed with different combinations of snu114 and dib1 mutants. Two snu114 mutations, V266P and K146I, were in the GTP domain while P860K was found in the IV domain of Snu114. Mutants of dib1 were dib1 (K34A+D36R) mutant 1, dib1 (E72A+E75A) mutant 3 and dib1 (E66A+D69A) mutant 4. Using a plasmid shuffle assay, where the URA3 plasmid with the complementing wild type genes was counter-selected with 5-Fluoroorotic acid, a positive genetic interaction between the dib1 (K34A+D36R) mutant 1 and snu114 mutant V266P was observed. A putative positive genetic interaction suggests that Snu114, in its GTP state, acts antagonistically to Dib1 in the activation of complex B and suggests that Snu114 is responsible for the removal of Dib1 from the B complex to form the activated B complex. Although further investigation is needed to explain how Snu114, in its GTP state, could remove Dib1, the positive interaction between Snu114 and Dib1 provides evidence on why BMKS and MFDGA share similar phenotypes. Advisors/Committee Members: PAVITT, GRAHAM GD, O'Keefe, Raymond, Pavitt, Graham.

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APA · Chicago · MLA · Vancouver · CSE | Export to Zotero / EndNote / Reference Manager

APA (6^th Edition):

Godinjak, L. (2018). Investigating Burn-McKeown Syndrome and Mandibulofacial Dysostosis, Guion-Almeida type through genetic interaction studies of Dib1 and Snu114 in S. cerevisiae. (Doctoral Dissertation). University of Manchester. Retrieved from http://www.manchester.ac.uk/escholar/uk-ac-man-scw:314241

Chicago Manual of Style (16^th Edition):

Godinjak, Lamija. “Investigating Burn-McKeown Syndrome and Mandibulofacial Dysostosis, Guion-Almeida type through genetic interaction studies of Dib1 and Snu114 in S. cerevisiae.” 2018. Doctoral Dissertation, University of Manchester. Accessed January 28, 2021. http://www.manchester.ac.uk/escholar/uk-ac-man-scw:314241.

MLA Handbook (7^th Edition):

Godinjak, Lamija. “Investigating Burn-McKeown Syndrome and Mandibulofacial Dysostosis, Guion-Almeida type through genetic interaction studies of Dib1 and Snu114 in S. cerevisiae.” 2018. Web. 28 Jan 2021.

Vancouver:

Godinjak L. Investigating Burn-McKeown Syndrome and Mandibulofacial Dysostosis, Guion-Almeida type through genetic interaction studies of Dib1 and Snu114 in S. cerevisiae. [Internet] [Doctoral dissertation]. University of Manchester; 2018. [cited 2021 Jan 28]. Available from: http://www.manchester.ac.uk/escholar/uk-ac-man-scw:314241.

Council of Science Editors:

Godinjak L. Investigating Burn-McKeown Syndrome and Mandibulofacial Dysostosis, Guion-Almeida type through genetic interaction studies of Dib1 and Snu114 in S. cerevisiae. [Doctoral Dissertation]. University of Manchester; 2018. Available from: http://www.manchester.ac.uk/escholar/uk-ac-man-scw:314241

University of Manchester

9. Shamsah, Sara. A gene deletion strategy to identify the function of a non-coding RNA in the eukaryotic genome using the model organism Saccharomyces cerevisiae.

Degree: 2015, University of Manchester

URL: http://www.manchester.ac.uk/escholar/uk-ac-man-scw:280682

► Non-coding RNA (nc-RNA) plays an important role in biological processes. To understand a non-coding RNA function, we constructed twelve molecular bar-coded deletion mutants in Saccharomyces… (more)

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APA · Chicago · MLA · Vancouver · CSE | Export to Zotero / EndNote / Reference Manager

APA (6^th Edition):

Shamsah, S. (2015). A gene deletion strategy to identify the function of a non-coding RNA in the eukaryotic genome using the model organism Saccharomyces cerevisiae. (Doctoral Dissertation). University of Manchester. Retrieved from http://www.manchester.ac.uk/escholar/uk-ac-man-scw:280682

Chicago Manual of Style (16^th Edition):

Shamsah, Sara. “A gene deletion strategy to identify the function of a non-coding RNA in the eukaryotic genome using the model organism Saccharomyces cerevisiae.” 2015. Doctoral Dissertation, University of Manchester. Accessed January 28, 2021. http://www.manchester.ac.uk/escholar/uk-ac-man-scw:280682.

MLA Handbook (7^th Edition):

Shamsah, Sara. “A gene deletion strategy to identify the function of a non-coding RNA in the eukaryotic genome using the model organism Saccharomyces cerevisiae.” 2015. Web. 28 Jan 2021.

Vancouver:

Shamsah S. A gene deletion strategy to identify the function of a non-coding RNA in the eukaryotic genome using the model organism Saccharomyces cerevisiae. [Internet] [Doctoral dissertation]. University of Manchester; 2015. [cited 2021 Jan 28]. Available from: http://www.manchester.ac.uk/escholar/uk-ac-man-scw:280682.

Council of Science Editors:

Shamsah S. A gene deletion strategy to identify the function of a non-coding RNA in the eukaryotic genome using the model organism Saccharomyces cerevisiae. [Doctoral Dissertation]. University of Manchester; 2015. Available from: http://www.manchester.ac.uk/escholar/uk-ac-man-scw:280682

University of Manchester

10. Woods, Steven. Pre-mRNA splicing in the filamentous ascomycete Neurospora crassa.

Degree: 2018, University of Manchester

URL: http://www.manchester.ac.uk/escholar/uk-ac-man-scw:314345

►

Pre-mRNA splicing is a eukaryotic phenomenon enabling multiple protein isoforms to be encoded by the same gene; the protein sequence can be altered by the… (more)

▼

Pre-mRNA splicing is a eukaryotic phenomenon enabling multiple protein isoforms to be encoded by the same gene; the protein sequence can be altered by the use of alternative splice sites. Using RNA-Seq data I explored the full complement of introns excised from pre-mRNAs in the model organism Neurospora crassa to investigate splicing of introns with noncanonical splice site sequences. I report the splice site pairs observed in my data and provide evidence that AT dinucleotides can function as splice acceptors in N. crassa. This is the first time that AT dinucleotides have been reported as splice acceptors outside of rare occurrences in the human transcriptome. I present the serendipitous discovery of the first reported N. crassa twintron and show that N. crassa introns can be at least twice as long (~3.6 kb) as the NC12 genome annotation suggests. These results highlight the limitations imposed when transcriptome analysis considers only the most widely accepted features of introns valid. Light is an important stimulus for N. crassa biology, including the onset of asexual development and the synthesis of photoprotective pigments. To investigate the contribution of alternative splicing to the N. crassa light response, I generated a measure of intron retention, the most common mode of alternative splicing in lower eukaryotes. Candidate introns with differential read coverage between dark-grown and light-pulsed cultures reveal intronic read distributions consistent with alternative transcription start sites, rather than intron retention. Thus, it is unlikely that light-regulated splicing is a phenomenon acting in N. crassa. Introns exist in a branched lariat conformation after splicing, but are rapidly debranched and degraded. I created a Neurospora crassa knockout strain lacking RNA lariat debranching activity, which enables enrichment of excised introns and will facilitate future efforts to understand pre-mRNA splicing in this organism.

An Excel spreadsheet of inferred introns is provided on a CD.

Advisors/Committee Members: PAVITT, GRAHAM GD, O'KEEFE, RAYMOND RT, GRIFFITHS-JONES, SAMUEL SR, Crosthwaite, Susan, Pavitt, Graham, O'Keefe, Raymond, Griffiths-Jones, Samuel.

Subjects/Keywords: RNA; Splicing; Neurospora

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APA · Chicago · MLA · Vancouver · CSE | Export to Zotero / EndNote / Reference Manager

APA (6^th Edition):

Woods, S. (2018). Pre-mRNA splicing in the filamentous ascomycete Neurospora crassa. (Doctoral Dissertation). University of Manchester. Retrieved from http://www.manchester.ac.uk/escholar/uk-ac-man-scw:314345

Chicago Manual of Style (16^th Edition):

Woods, Steven. “Pre-mRNA splicing in the filamentous ascomycete Neurospora crassa.” 2018. Doctoral Dissertation, University of Manchester. Accessed January 28, 2021. http://www.manchester.ac.uk/escholar/uk-ac-man-scw:314345.

MLA Handbook (7^th Edition):

Woods, Steven. “Pre-mRNA splicing in the filamentous ascomycete Neurospora crassa.” 2018. Web. 28 Jan 2021.

Vancouver:

Woods S. Pre-mRNA splicing in the filamentous ascomycete Neurospora crassa. [Internet] [Doctoral dissertation]. University of Manchester; 2018. [cited 2021 Jan 28]. Available from: http://www.manchester.ac.uk/escholar/uk-ac-man-scw:314345.

Council of Science Editors:

Woods S. Pre-mRNA splicing in the filamentous ascomycete Neurospora crassa. [Doctoral Dissertation]. University of Manchester; 2018. Available from: http://www.manchester.ac.uk/escholar/uk-ac-man-scw:314345

11. Concilio, Maria Grazia. NMR characterisation of a novel neamine antibiotic 12 and its interaction with a conserved 27mer RNA motif of 16S rRNA.

Degree: 2013, University of Manchester

URL: http://www.manchester.ac.uk/escholar/uk-ac-man-scw:203926

►

In recent times, the growing challenge of antibiotic resistance has prompted intense efforts to elucidate the mechanism of action of antibiotics at the molecular level,… (more)

▼

In recent times, the growing challenge of antibiotic resistance has prompted intense efforts to elucidate the mechanism of action of antibiotics at the molecular level, using techniques such as NMR spectroscopy. Blocking protein synthesis is an effective way of combating bacterial infection and many antibiotics function in this manner. Interest in the involvement of RNA in protein biosynthesis has increased following extensive studies on the binding of antibiotic drugs to specific target sites on ribosomal RNA. A systematic NMR study of a novel antibiotic derived from neamine and its interaction with a conserved and highly stable 27mer RNA motif of the A-site 16S rRNA was carried out. The antibiotic showed well resolved and dispersed resonances including exchange retarded amide protons suggesting a stable, folded conformation of the drug. The NMR results obtained provide a structural rationale to modify selected groups on the neamine 12 antibiotic to enhance the affinity for the RNA. The empirical Gibbs energy (ΔG=-10.70 kcal/mol) and secondary structure were predicted for the 27mer RNA by the Vienna RNAfold software exhibiting a good thermodynamic stability. Similarly, the 27mer RNA exhibited well resolved and stable exchangeable imino and amino resonances in the lowfield region of the 1H-NMR spectrum. The NMR spectra of the 27mer RNA-neamine 12 complex showed small but detectable changes in chemical shift and linewidth, indicating weak interaction between the neamine 12 and the 27mer RNA. These changes can be qualitatively interpreted as changes in the local conformation of the 27mer RNA and the neamine 12, arising from the formation of their complex. The NMR results obtained have laid a solid foundation to determine the three dimensional solution state structures of neamine 12 after necessary modification and the complex with the RNA to elucidate the mechanism of action of the antibiotic.

The phenomenon of antibiotic resistance is a global health threat and efforts are being made worldwide to discover new antibiotics. In this project a technique called NMR spectroscopy has been applied to study the properties of a novel antibioc called neamine 12 and its interaction with a model RNA nucleic acid system.

None

Advisors/Committee Members: O'KEEFE, RAYMOND RT, Ramesh, Vasudevan, O'Keefe, Raymond.

Subjects/Keywords: Antibiotic resistance, Neamine antibiotic, RNA, NMR; Molecular modeling

…University of Manchester certain rights to use such Copyright, including for administrative… …antibiotic and the staff of the University of Manchester for having measured all the spectra. I…

10 more images…

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APA · Chicago · MLA · Vancouver · CSE | Export to Zotero / EndNote / Reference Manager

APA (6^th Edition):

Concilio, M. G. (2013). NMR characterisation of a novel neamine antibiotic 12 and its interaction with a conserved 27mer RNA motif of 16S rRNA. (Doctoral Dissertation). University of Manchester. Retrieved from http://www.manchester.ac.uk/escholar/uk-ac-man-scw:203926

Chicago Manual of Style (16^th Edition):

Concilio, Maria Grazia. “NMR characterisation of a novel neamine antibiotic 12 and its interaction with a conserved 27mer RNA motif of 16S rRNA.” 2013. Doctoral Dissertation, University of Manchester. Accessed January 28, 2021. http://www.manchester.ac.uk/escholar/uk-ac-man-scw:203926.

MLA Handbook (7^th Edition):

Concilio, Maria Grazia. “NMR characterisation of a novel neamine antibiotic 12 and its interaction with a conserved 27mer RNA motif of 16S rRNA.” 2013. Web. 28 Jan 2021.

Vancouver:

Concilio MG. NMR characterisation of a novel neamine antibiotic 12 and its interaction with a conserved 27mer RNA motif of 16S rRNA. [Internet] [Doctoral dissertation]. University of Manchester; 2013. [cited 2021 Jan 28]. Available from: http://www.manchester.ac.uk/escholar/uk-ac-man-scw:203926.

Council of Science Editors:

Concilio MG. NMR characterisation of a novel neamine antibiotic 12 and its interaction with a conserved 27mer RNA motif of 16S rRNA. [Doctoral Dissertation]. University of Manchester; 2013. Available from: http://www.manchester.ac.uk/escholar/uk-ac-man-scw:203926

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