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199 results on '"O'Keefe, Raymond T."'

1. Combining a prioritization strategy and functional studies nominates 5’UTR variants underlying inherited retinal disease

Author

Dueñas Rey, Alfredo, del Pozo Valero, Marta, Bouckaert, Manon, Wood, Katherine A, Van den Broeck, Filip, Daich Varela, Malena, Thomas, Huw B, Van Heetvelde, Mattias, De Bruyne, Marieke, Van de Sompele, Stijn, Bauwens, Miriam, Lenaerts, Hanne, Mahieu, Quinten, Josifova, Dragana, Rivolta, Carlo, O’Keefe, Raymond T, Ellingford, Jamie, Webster, Andrew R, Arno, Gavin, Ayuso, Carmen, De Zaeytijd, Julie, Leroy, Bart P, De Baere, Elfride, and Coppieters, Frauke

Published
2024
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2. Novel homozygous variants in PRORP expand the genotypic spectrum of combined oxidative phosphorylation deficiency 54

Author

Smith, Thomas B., Rea, Alessandro, Thomas, Huw B., Thompson, Kyle, Oláhová, Monika, Maroofian, Reza, Zamani, Mina, He, Langping, Sadeghian, Saeid, Galehdari, Hamid, Lotan, Nava Shaul, Gilboa, Tal, Herman, Kristin C., McCorvie, Thomas J., Yue, Wyatt W., Houlden, Henry, Taylor, Robert W., Newman, William G., and O’Keefe, Raymond T.

Published
2023
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5. Structural determination of oleanane-28,13β-olide and taraxerane-28,14β-olide fluoro­lactonization products from the reaction of oleanolic acid with SelectfluorTM.

Author

Eadsforth, Megan A., Kong, Linghan, Whitehead, George, Vitórica-Yrezábal, Iñigo J., O'Keefe, Raymond T., Bryce, Richard A., and Whiteheada, Roger C.

Subjects
CRYSTAL structure, TRITERPENOIDS, METHANOL, X-rays, ACIDS
Abstract

The X-ray crystal structure data of 12-α-fluoro-3β-hy­droxy­olean-28,13β-olide methanol hemisolvate, 2C30H47FO3·CH3OH, (1), and 12-α-fluoro-3β-hy­droxy­taraxer-28,14β-olide methanol hemisolvate, 2C30H47FO3·CH3OH, (2), are described. The fluoro­lactonization of oleanolic acid using SelectfluorTM yielded a mixture of the six-membered δ-lactone (1) and the unusual seven-membered γ-lactone (2) following a 1,2-shift of methyl C-27 from C-14 to C-13. [ABSTRACT FROM AUTHOR]

Published
2024
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6. Uncovering genetic mechanisms of hypertension through multi-omic analysis of the kidney

Author

Eales, James M., Jiang, Xiao, Xu, Xiaoguang, Saluja, Sushant, Akbarov, Artur, Cano-Gamez, Eddie, McNulty, Michelle T., Finan, Christopher, Guo, Hui, Wystrychowski, Wojciech, Szulinska, Monika, Thomas, Huw B., Pramanik, Sanjeev, Chopade, Sandesh, Prestes, Priscilla R., Wise, Ingrid, Evangelou, Evangelos, Salehi, Mahan, Shakanti, Yusif, Ekholm, Mikael, Denniff, Matthew, Nazgiewicz, Alicja, Eichinger, Felix, Godfrey, Bradley, Antczak, Andrzej, Glyda, Maciej, Król, Robert, Eyre, Stephen, Brown, Jason, Berzuini, Carlo, Bowes, John, Caulfield, Mark, Zukowska-Szczechowska, Ewa, Zywiec, Joanna, Bogdanski, Pawel, Kretzler, Matthias, Woolf, Adrian S., Talavera, David, Keavney, Bernard, Maffia, Pasquale, Guzik, Tomasz J., O’Keefe, Raymond T., Trynka, Gosia, Samani, Nilesh J., Hingorani, Aroon, Sampson, Matthew G., Morris, Andrew P., Charchar, Fadi J., and Tomaszewski, Maciej

Published
2021
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7. Structural determination of oleanane-28,13β-olide and taraxerane-28,14β-olide fluorolactonization products from the reaction of oleanolic acid with Selectfluor™.

Author

Eadsforth, Megan A., Kong, Linghan, Whitehead, George, Vitórica-Yrezábal, Iñigo J., O'Keefe, Raymond T., Bryce, Richard A., and Whitehead, Roger C.

Subjects
CRYSTAL structure, TRITERPENOIDS, METHANOL, X-rays, ACIDS
Abstract

The X-ray crystal structure data of 12-α-fluoro-3β-hy­droxy­olean-28,13β-olide methanol hemisolvate, 2C30H47FO3·CH3OH, (1), and 12-α-fluoro-3β-hy­droxy­taraxer-28,14β-olide methanol hemisolvate, 2C30H47FO3·CH3OH, (2), are described. The fluoro­lactonization of oleanolic acid using SelectfluorTM yielded a mixture of the six-membered δ-lactone (1) and the unusual seven-membered γ-lactone (2) following a 1,2-shift of methyl C-27 from C-14 to C-13. [ABSTRACT FROM AUTHOR]

Published
2024
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15. Novel homozygous variants in PRORPexpand the genotypic spectrum of combined oxidative phosphorylation deficiency 54

Author

Smith, Thomas B., Rea, Alessandro, Thomas, Huw B., Thompson, Kyle, Oláhová, Monika, Maroofian, Reza, Zamani, Mina, He, Langping, Sadeghian, Saeid, Galehdari, Hamid, Lotan, Nava Shaul, Gilboa, Tal, Herman, Kristin C., McCorvie, Thomas J., Yue, Wyatt W., Houlden, Henry, Taylor, Robert W., Newman, William G., and O’Keefe, Raymond T.

Abstract

Biallelic hypomorphic variants in PRORPhave been recently described as causing the autosomal recessive disorder combined oxidative phosphorylation deficiency type 54 (COXPD54). COXPD54 encompasses a phenotypic spectrum of sensorineural hearing loss and ovarian insufficiency (Perrault syndrome) to leukodystrophy. Here, we report three additional families with homozygous missense PRORPvariants with pleiotropic phenotypes. Each missense variant altered a highly conserved residue within the metallonuclease domain. In vitro mitochondrial tRNA processing assays with recombinant TRMT10C, SDR5C1 and PRORP indicated two COXPD54-associated PRORPvariants, c.1159A>G (p.Thr387Ala) and c.1241C>T (p.Ala414Val), decreased pre-tRNAIlecleavage, consistent with both variants impacting tRNA processing. No significant decrease in tRNA processing was observed with PRORPc.1093T>C (p.Tyr365His), which was identified in an individual with leukodystrophy. These data provide independent evidence that PRORPvariants are associated with COXPD54 and that the assessment of 5′ leader mitochondrial tRNA processing is a valuable assay for the functional analysis and clinical interpretation of novel PRORPvariants.

Published
2023
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16. Bi-allelic FRA10AC1 variants in a multisystem human syndrome

Author

Banka, Siddharth, primary, Shalev, Stavit, additional, Park, Soo-Mi, additional, Wood, Katherine A, additional, Thomas, Huw B, additional, Wright, Helen L, additional, Alyahya, Mohammed, additional, Bankier, Sean, additional, Alimi, Ola, additional, Chervinsky, Elena, additional, Zeef, Leo A H, additional, and O’Keefe, Raymond T, additional

Published
2022
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17. MRSD: a quantitative approach for assessing suitability of RNA-seq in the investigation of mis-splicing in Mendelian disease

Author

Rowlands, Charlie F., Taylor, Algy, Rice, Gillian I, Whiffin, Nicola, Hall, Hildegard Nikki, Newman, William G., Black, Graeme C.M., Investigators, kConFab, O'Keefe, Raymond T., Hubbard, Simon, Douglas, Andrew, Baralle, Diana, Briggs, Tracy A., and Ellingford, Jamie M.

Abstract

Background: RNA-sequencing of patient biosamples is a promising approach to delineate the impact of genomic variants on splicing, but variable gene expression between tissues complicates selection of appropriate tissues. Relative expression level is often used as a metric to predict RNA-sequencing utility. Here, we describe a gene- and tissue-specific metric to inform the feasibility of RNA-sequencing, overcoming some issues with using expression values alone.Results: We derive a novel metric, Minimum Required Sequencing Depth (MRSD), for all genes across three human biosamples (whole blood, lymphoblastoid cell lines (LCLs) and skeletal muscle). MRSD estimates the depth of sequencing required from RNA-sequencing to achieve user-specified sequencing coverage of a gene, transcript or group of genes of interest. MRSD predicts levels of splice junction coverage with high precision (90.1-98.2%) and overcomes transcript region-specific sequencing biases. Applying MRSD scoring to established disease gene panels shows that LCLs are the optimum source of RNA, of the three investigated biosamples, for 69.3% of gene panels. Our approach demonstrates that up to 59.4% of variants of uncertain significance in ClinVar predicted to impact splicing could be functionally assayed by RNA-sequencing in at least one of the investigated biosamples.Conclusions: We demonstrate the power of MRSD as a metric to inform choice of appropriate biosamples for the functional assessment of splicing aberrations. We apply MRSD in the context of Mendelian genetic disorders and illustrate its benefits over expression-based approaches. We anticipate that the integration of MRSD into clinical pipelines will improve variant interpretation and, ultimately, diagnostic yield.

Published
2022

20. MRSD: A quantitative approach for assessing suitability of RNA-seq in the investigation of mis-splicing in Mendelian disease

Author

Rowlands, Charlie F., primary, Taylor, Algy, additional, Rice, Gillian, additional, Whiffin, Nicola, additional, Hall, Hildegard Nikki, additional, Newman, William G., additional, Black, Graeme C.M., additional, O’Keefe, Raymond T., additional, Hubbard, Simon, additional, Douglas, Andrew G.L., additional, Baralle, Diana, additional, Briggs, Tracy A., additional, and Ellingford, Jamie M., additional

Published
2022
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22. Bi-allelic variants in the mitochondrial RNase P subunit PRORP cause mitochondrial tRNA processing defects and pleiotropic multisystem presentations

Author

Hochberg, Irit, primary, Demain, Leigh A.M., additional, Richer, Julie, additional, Thompson, Kyle, additional, Urquhart, Jill E., additional, Rea, Alessandro, additional, Pagarkar, Waheeda, additional, Rodríguez-Palmero, Agustí, additional, Schlüter, Agatha, additional, Verdura, Edgard, additional, Pujol, Aurora, additional, Quijada-Fraile, Pilar, additional, Amberger, Albert, additional, Deutschmann, Andrea J., additional, Demetz, Sandra, additional, Gillespie, Meredith, additional, Belyantseva, Inna A., additional, McMillan, Hugh J., additional, Barzik, Melanie, additional, Beaman, Glenda M., additional, Motha, Reeya, additional, Ng, Kah Ying, additional, O’Sullivan, James, additional, Williams, Simon G., additional, Bhaskar, Sanjeev S., additional, Lawrence, Isabella R., additional, Jenkinson, Emma M., additional, Zambonin, Jessica L., additional, Blumenfeld, Zeev, additional, Yalonetsky, Sergey, additional, Oerum, Stephanie, additional, Rossmanith, Walter, additional, Yue, Wyatt W., additional, Zschocke, Johannes, additional, Munro, Kevin J., additional, Battersby, Brendan J., additional, Friedman, Thomas B., additional, Taylor, Robert W., additional, O’Keefe, Raymond T., additional, and Newman, William G., additional

Published
2021
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23. New insights into Perrault syndrome, a clinically and genetically heterogeneous disorder

Author

Faridi, Rabia, primary, Rea, Alessandro, additional, Fenollar-Ferrer, Cristina, additional, O’Keefe, Raymond T., additional, Gu, Shoujun, additional, Munir, Zunaira, additional, Khan, Asma Ali, additional, Riazuddin, Sheikh, additional, Hoa, Michael, additional, Naz, Sadaf, additional, Newman, William G., additional, and Friedman, Thomas B., additional

Published
2021
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27. Bi-allelic variants in the mitochondrial RNase P subunit PRORP cause mitochondrial tRNA processing defects and pleiotropic multisystem presentations

Author

Hochberg, Irit, Demain, Leigh A.M., Richer, Julie, Thompson, Kyle, Urquhart, Jill E., Rea, Alessandro, Pagarkar, Waheeda, Rodríguez-Palmero, Agustí, Schlüter, Agatha, Verdura, Edgard, Pujol, Aurora, Quijada-Fraile, Pilar, Amberger, Albert, Deutschmann, Andrea J., Demetz, Sandra, Gillespie, Meredith, Belyantseva, Inna A., McMillan, Hugh J., Barzik, Melanie, Beaman, Glenda M., Motha, Reeya, Ng, Kah Ying, O'Sullivan, James, Williams, Simon G., Bhaskar, Sanjeev S., Lawrence, Isabella R., Jenkinson, Emma M., Zambonin, Jessica L., Blumenfeld, Zeev, Yalonetsky, Sergey, Oerum, Stephanie, Rossmanith, Walter, Yue, Wyatt W., Zschocke, Johannes, Munro, Kevin J., Battersby, Brendan J., Friedman, Thomas B., Taylor, Robert W., O'Keefe, Raymond T., Newman, William G., Hochberg, Irit, Demain, Leigh A.M., Richer, Julie, Thompson, Kyle, Urquhart, Jill E., Rea, Alessandro, Pagarkar, Waheeda, Rodríguez-Palmero, Agustí, Schlüter, Agatha, Verdura, Edgard, Pujol, Aurora, Quijada-Fraile, Pilar, Amberger, Albert, Deutschmann, Andrea J., Demetz, Sandra, Gillespie, Meredith, Belyantseva, Inna A., McMillan, Hugh J., Barzik, Melanie, Beaman, Glenda M., Motha, Reeya, Ng, Kah Ying, O'Sullivan, James, Williams, Simon G., Bhaskar, Sanjeev S., Lawrence, Isabella R., Jenkinson, Emma M., Zambonin, Jessica L., Blumenfeld, Zeev, Yalonetsky, Sergey, Oerum, Stephanie, Rossmanith, Walter, Yue, Wyatt W., Zschocke, Johannes, Munro, Kevin J., Battersby, Brendan J., Friedman, Thomas B., Taylor, Robert W., O'Keefe, Raymond T., and Newman, William G.

Abstract

Human mitochondrial RNase P (mt-RNase P) is responsible for 5' end processing of mitochondrial precursor tRNAs, a vital step in mitochondrial RNA maturation, and is comprised of three protein subunits: TRMT10C, SDR5C1 (HSD10), and PRORP. Pathogenic variants in TRMT10C and SDR5C1 are associated with distinct recessive or x-linked infantile onset disorders, resulting from defects in mitochondrial RNA processing. We report four unrelated families with multisystem disease associated with bi-allelic variants in PRORP, the metallonuclease subunit of mt-RNase P. Affected individuals presented with variable phenotypes comprising sensorineural hearing loss, primary ovarian insufficiency, developmental delay, and brain white matter changes. Fibroblasts from affected individuals in two families demonstrated decreased steady state levels of PRORP, an accumulation of unprocessed mitochondrial transcripts, and decreased steady state levels of mitochondrial-encoded proteins, which were rescued by introduction of the wild-type PRORP cDNA. In mt-tRNA processing assays performed with recombinant mt-RNase P proteins, the disease-associated variants resulted in diminished mitochondrial tRNA processing. Identification of disease-causing variants in PRORP indicates that pathogenic variants in all three subunits of mt-RNase P can cause mitochondrial dysfunction, each with distinct pleiotropic clinical presentations.

Published
2021

29. Comparison of in Silico Strategies to Prioritize Rare Genomic Variants Impacting RNA Splicing for the Diagnosis of Genomic Disorders

Author

Rowlands, Charlie, primary, Thomas, Huw B, additional, Lord, Jenny, additional, Wai, Htoo A, additional, Arno, Gavin, additional, Beaman, Glenda, additional, Sergouniotis, Panagiotis, additional, Gomes-Silva, Beatriz, additional, Campbell, Christopher, additional, Gossan, Nicole, additional, Hardcastle, Claire, additional, Webb, Kevin, additional, O’Callaghan, Christopher, additional, Hirst, Robert A, additional, Ramsden, Simon, additional, Jones, Elizabeth, additional, -Smith, Jill Clayton, additional, Webster, Andrew R, additional, Douglas, Andrew GL, additional, O’Keefe, Raymond T, additional, Newman, William G, additional, Baralle, Diana, additional, Black, Graeme CM, additional, and Ellingford, Jamie M, additional

Published
2021
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30. Analysis of U8 snoRNA Variants in Zebrafish Reveals How Bi-allelic Variants Cause Leukoencephalopathy with Calcifications and Cysts

Author

Badrock, Andrew P, Uggenti, Carolina, Wacheul, Ludivine, Crilly, Siobhan, Jenkinson, Emma, Rice, Gillian G.I., Kasher, Paul R, Lafontaine, Denis, Crow, Yanick Joseph, O'Keefe, Raymond T, Badrock, Andrew P, Uggenti, Carolina, Wacheul, Ludivine, Crilly, Siobhan, Jenkinson, Emma, Rice, Gillian G.I., Kasher, Paul R, Lafontaine, Denis, Crow, Yanick Joseph, and O'Keefe, Raymond T

Abstract

How mutations in the non-coding U8 snoRNA cause the neurological disorder leukoencephalopathy with calcifications and cysts (LCC) is poorly understood. Here, we report the generation of a mutant U8 animal model for interrogating LCC-associated pathology. Mutant U8 zebrafish exhibit defective central nervous system development, a disturbance of ribosomal RNA (rRNA) biogenesis and tp53 activation, which monitors ribosome biogenesis. Further, we demonstrate that fibroblasts from individuals with LCC are defective in rRNA processing. Human precursor-U8 (pre-U8) containing a 3′ extension rescued mutant U8 zebrafish, and this result indicates conserved biological function. Analysis of LCC-associated U8 mutations in zebrafish revealed that one null and one functional allele contribute to LCC. We show that mutations in three nucleotides at the 5′ end of pre-U8 alter the processing of the 3′ extension, and we identify a previously unknown base-pairing interaction between the 5′ end and the 3′ extension of human pre-U8. Indeed, LCC-associated mutations in any one of seven nucleotides in the 5′ end and 3′ extension alter the processing of pre-U8, and these mutations are present on a single allele in almost all individuals with LCC identified to date. Given genetic data indicating that bi-allelic null U8 alleles are likely incompatible with human development, and that LCC is not caused by haploinsufficiency, the identification of hypomorphic misprocessing mutations that mediate viable embryogenesis furthers our understanding of LCC molecular pathology and cerebral vascular homeostasis., SCOPUS: ar.j, info:eu-repo/semantics/published

Published
2020

34. Expanding the genotypic spectrum of TXNL4A variants in Burn‐McKeown syndrome.

Author

Wood, Katherine A., Ellingford, Jamie M., Thomas, Huw B., Douzgou, Sofia, Beaman, Glenda M., Hobson, Emma, Prescott, Katrina, O'Keefe, Raymond T., and Newman, William G.

Subjects
RNA splicing, GENOTYPES, BINDING sites, TRANSCRIPTION factors, TELEOLOGY
Abstract

The developmental disorder Burn‐McKeown Syndrome (BMKS) is characterised by choanal atresia and specific craniofacial features. BMKS is caused by biallelic variants in the pre‐messenger RNA splicing factor TXNL4A. Most patients have a loss‐of‐function variant in trans with a 34‐base pair (bp) deletion (type 1 Δ34) in the promoter region. Here, we identified two patients with BMKS. One individual has a TXNL4A c.93_94delCC, p.His32Argfs *21 variant combined with a type 1 Δ34 promoter deletion. The other has an intronic TXNL4A splice site variant (c.258‐3C>G) and a type 1 Δ34 promoter deletion. We show the c.258‐3C>G variant and a previously reported c.258‐2A>G variant, cause skipping of the final exon of TXNL4A in a minigene splicing assay. Furthermore, we identify putative transcription factor binding sites within the 56 bp of the TXNL4A promoter affected by the type 1 and type 2 Δ34 and use dual luciferase assays to identify a 22 bp repeated motif essential for TXNL4A expression within this promoter region. We propose that additional variants affecting critical transcription factor binding nucleotides within the 22 bp repeated motif could be relevant to BMKS aetiology. Finally, our data emphasises the need to analyse the non‐coding sequence in individuals where a single likely pathogenic coding variant is identified in an autosomal recessive disorder consistent with the clinical presentation. [ABSTRACT FROM AUTHOR]

Published
2022
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35. Leukoencephalopathy with calcifications and cysts: Genetic and phenotypic spectrum

Author

Crow, Yanick J, primary, Marshall, Heather, additional, Rice, Gillian I, additional, Seabra, Luis, additional, Jenkinson, Emma M, additional, Baranano, Kristin, additional, Battini, Roberta, additional, Berger, Andrea, additional, Blair, Edward, additional, Blauwblomme, Thomas, additional, Bolduc, Francois, additional, Boddaert, Natalie, additional, Buckard, Johannes, additional, Burnett, Heather, additional, Calvert, Sophie, additional, Caumes, Roseline, additional, Ng, Andy Cheuk‐Him, additional, Chiang, Diana, additional, Clifford, David B, additional, Cordelli, Duccio M, additional, Burca, Anna, additional, Demic, Natasha, additional, Desguerre, Isabelle, additional, De Waele, Liesbeth, additional, Di Fonzo, Alessio, additional, Dunham, S. Richard, additional, Dyack, Sarah, additional, Elmslie, Frances, additional, Ferrand, Mickaël, additional, Fisher, Gemma, additional, Karimiani, Ehsan Ghayoor, additional, Ghoumid, Jamal, additional, Gibbon, Frances, additional, Goel, Himanshu, additional, Hilmarsen, Hilde T, additional, Hughes, Imelda, additional, Jacob, Anu, additional, Jones, Elizabeth A, additional, Kumar, Ram, additional, Leventer, Richard J, additional, MacDonald, Shelley, additional, Maroofian, Reza, additional, Mehta, Sarju G, additional, Metz, Imke, additional, Monfrini, Edoardo, additional, Neumann, Daniela, additional, Noetzel, Michael, additional, O'Driscoll, Mary, additional, Õunap, Katrin, additional, Panzer, Axel, additional, Parikh, Sumit, additional, Prabhakar, Prab, additional, Ramond, Francis, additional, Sandford, Richard, additional, Saneto, Russell, additional, Soh, Calvin, additional, Stutterd, Chloe A, additional, Subramanian, Gopinath M, additional, Talbot, Kevin, additional, Thomas, Rhys H, additional, Toro, Camilo, additional, Touraine, Renaud, additional, Wakeling, Emma, additional, Wassmer, Evangeline, additional, Whitney, Andrea, additional, Livingston, John H, additional, O'Keefe, Raymond T, additional, and Badrock, Andrew P, additional

Published
2020
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38. EFTUD2 missense variants disrupt protein function and splicing in mandibulofacial dysostosis Guion‐Almeida type

Author

Thomas, Huw B., primary, Wood, Katherine A., additional, Buczek, Weronika A., additional, Gordon, Christopher T., additional, Pingault, Véronique, additional, Attié‐Bitach, Tania, additional, Hentges, Kathryn E., additional, Varghese, Vinod C., additional, Amiel, Jeanne, additional, Newman, William G., additional, and O'Keefe, Raymond T., additional

Published
2020
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39. Analysis of U8 snoRNA Variants in Zebrafish Reveals How Bi-allelic Variants Cause Leukoencephalopathy with Calcifications and Cysts

Author

Badrock, Andrew P., primary, Uggenti, Carolina, additional, Wacheul, Ludivine, additional, Crilly, Siobhan, additional, Jenkinson, Emma M., additional, Rice, Gillian I., additional, Kasher, Paul R., additional, Lafontaine, Denis L.J., additional, Crow, Yanick J., additional, and O’Keefe, Raymond T., additional

Published
2020
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43. Advanced Methods for the Analysis of Altered Pre-mRNA Splicing in Yeast and Disease

Author

O'Keefe, Raymond T and Thomas, Huw

Abstract

Splicing of pre-messenger RNA (pre-mRNA) transcripts is a fundamental process in all eukaryotes that provides a mechanism of increasing the proteomic diversity within a cell that can be tightly regulated in a dynamic manner. While constitutive and alternative splicing are necessary for the correct development and regulation of cells/organisms, aberrant splicing is now associated with an increasingly varied number of human diseases, such as neurological and developmental diseases, and cancer. Studies of splicing mechanisms and regulation are often achieved in nonhuman model organisms such as yeast. Yeasts possess homologs to many of the core spliceosome components of higher organisms, including humans, and as such yeast species are now a well-established model organism for understanding how differential splicing of transcripts can alter the phenotype of a cell or organism. Here we describe methods to investigate pre-mRNA splicing in yeast cells using modern RNA-Seq technology and bioinformatics software. Details of traditional validation methods are also described.

Published
2019

44. The invariant U5 snRNA loop 1 sequence is dispensable for the first catalytic step of pre-mRNA splicing in yeast

Author

O'Keefe, Raymond T., Norman, Christine, and Newman, Andrew J.

Subjects
Catalytic RNA -- Research, Yeast -- Genetic aspects, RNA splicing -- Research, Biological sciences
Abstract

An in vitro reconstitution system was developed to study the role of U5 snRNA in the two catalytic steps of pre-mRNA splicing. It is known that the invariant loop 1 sequence of U5 interacts with exon sequences at the 5' splice location before the first catalytic step. An in vitro analysis of U5 mutations shows that the first transesterification precisely occurs when the U5 loop is absent. Thus, this sequence is not a major component of the spliceosomal active site for the first catalytic step. The second catalytic step was found to tolerate mutations in the invariant sequence.

Published
1996

46. Dynamic organization of DNA replication in mammalian cell nuclei: spatially and temporally defined replication of chromosome-specific alpha-satellite DNA sequences

Author

O'Keefe, Raymond T., Henderson, Scott C., and Spector, David L.

Subjects
Chromosome replication -- Research, Biological sciences
Abstract

Temporal and spatial patterns of DNA replication in mammalian cells were investigated using fluorescence microscopy, confocal laser scanning microscopy and immunoelectron microscopy. Results indicate that there are five patterns of replication occuring at specific sites in the nucleus and at specific periods in the S-phase of the cell cycle. Further, it was shown that alpha-satellite DNA sequences replicate at the middle of the S-phase. The replication of specific DNA sequences were likewise shown to follow specific temporal and spatial patterns.

Published
1992

47. Large-scale profiling of noncoding RNA function in yeast

Author

Parker, Steven, Fraczek, Marcin G., Wu, Jian, Shamsah, Sara, Manousaki, Alkisti, Dungrattanalert, Kobchai, de Almeida, Rogerio Alves, Invernizzi, Edith, Burgis, Tim, Omara, Walid, Griffiths-Jones, Sam, Delneri, Daniela, and O’Keefe, Raymond T.

Subjects
deletion collection, Heterozygote, RNA, Untranslated, 3' Utr, lcsh:QH426-470, Yeast and Fungal Models, Saccharomyces cerevisiae, Haploidy, Research and Analysis Methods, Biochemistry, Noncoding RNA, Saccharomyces, Model Organisms, Untranslated Regions, Gene Expression Regulation, Fungal, Manchester Institute of Biotechnology, Databases, Genetic, Fitness, Genetics, Small nucleolar RNAs, Non-coding RNA, Evolutionary Biology, Biology and life sciences, Population Biology, Messenger RNA, Gene Expression Profiling, Organisms, Fungi, Eukaryota, RNA, Fungal, Functional genomics, Gene Pool, ResearchInstitutes_Networks_Beacons/manchester_institute_of_biotechnology, Yeast, Gene regulation, Nucleic acids, lcsh:Genetics, Phenotype, Experimental Organism Systems, RNA, Gene expression, Genetic Fitness, Transfer RNA, Population Genetics, Gene Deletion, Research Article
Abstract

Noncoding RNAs (ncRNAs) are emerging as key regulators of cellular function. We have exploited the recently developed barcoded ncRNA gene deletion strain collections in the yeast Saccharomyces cerevisiae to investigate the numerous ncRNAs in yeast with no known function. The ncRNA deletion collection contains deletions of tRNAs, snoRNAs, snRNAs, stable unannotated transcripts (SUTs), cryptic unstable transcripts (CUTs) and other annotated ncRNAs encompassing 532 different individual ncRNA deletions. We have profiled the fitness of the diploid heterozygous ncRNA deletion strain collection in six conditions using batch and continuous liquid culture, as well as the haploid ncRNA deletion strain collections arrayed individually onto solid rich media. These analyses revealed many novel environmental-specific haplo-insufficient and haplo-proficient phenotypes providing key information on the importance of each specific ncRNA in every condition. Co-fitness analysis using fitness data from the heterozygous ncRNA deletion strain collection identified two ncRNA groups required for growth during heat stress and nutrient deprivation. The extensive fitness data for each ncRNA deletion strain has been compiled into an easy to navigate database called Yeast ncRNA Analysis (YNCA). By expanding the original ncRNA deletion strain collection we identified four novel essential ncRNAs; SUT527, SUT075, SUT367 and SUT259/691. We defined the effects of each new essential ncRNA on adjacent gene expression in the heterozygote background identifying both repression and induction of nearby genes. Additionally, we discovered a function for SUT527 in the expression, 3’ end formation and localization of SEC4, an essential protein coding mRNA. Finally, using plasmid complementation we rescued the SUT075 lethal phenotype revealing that this ncRNA acts in trans. Overall, our findings provide important new insights into the function of ncRNAs., Author summary Genomes from different organisms produce noncoding RNAs that are not translated to make proteins and whose functions are largely unknown. There are approximately 2,000 noncoding RNAs that make up almost 25% of the yeast genome compared to the approximately 6,000 protein coding genes that make up 70% of the yeast genome. With this large number of ncRNAs there is a need for large-scale studies to determine the functional roles of ncRNAs. We take advantage of a recently developed resource of 532 yeast strains in which individual noncoding RNA genes have been deleted. We grow these yeast noncoding RNA deletion strains in different conditions and catalogue how each noncoding RNA contributes to cell growth. Improvement or inhibition of cell growth under particular conditions implicates the deleted RNA in cellular responses to those conditions. We have also investigated in more detail the function of specific noncoding RNAs, revealing examples of how a deletion influences nearby genes, and other examples of noncoding RNAs that regulate genes at distant genomic locations. We have made our extensive data on the fitness of noncoding RNA deletion mutants publically available for searching and bulk download in a new online resource, called Yeast ncRNA Analysis (YNCA). This large-scale analysis of noncoding RNA deletion mutants reveals the importance of many noncoding RNAs in cellular function.

Published
2018

48. A homozygous missense variant in CHRM3 associated with familial urinary bladder disease

Author

Beaman, Glenda M., primary, Galatà, Gabriella, additional, Teik, Keng W., additional, Urquhart, Jill E., additional, Aishah, Ali, additional, O'Sullivan, James, additional, Bhaskar, Sanjeev S., additional, Wood, Katherine A., additional, Thomas, Huw B., additional, O'Keefe, Raymond T., additional, Woolf, Adrian S., additional, Stuart, Helen M., additional, and Newman, William G., additional

Published
2019
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49. Disease modeling of core pre-mRNA splicing factor haploinsufficiency

Author

Wood, Katherine A, primary, Rowlands, Charlie F, additional, Qureshi, Wasay Mohiuddin Shaikh, additional, Thomas, Huw B, additional, Buczek, Weronika A, additional, Briggs, Tracy A, additional, Hubbard, Simon J, additional, Hentges, Kathryn E, additional, Newman, William G, additional, and O’Keefe, Raymond T, additional

Published
2019
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50. A resource for functional profiling of noncoding RNA in the yeast Saccharomyces cerevisiae

Author

Parker, Steven, Fraczek, Marcin G., Wu, Jian, Shamsah, Sara, Manousaki, Alkisti, Dungrattanalert, Kobchai, de Almeida, Rogerio Alves, Estrada-Rivadeneyra, Diego, Omara, Walid, Delneri, Daniela, and O'Keefe, Raymond T.

Subjects
RNA, Untranslated, snRNA, Report, RNA, Fungal, Saccharomyces cerevisiae, yeast, Genome, Fungal, snoRNA, ncRNA, tRNA, Sequence Deletion
Abstract

Eukaryotic genomes are extensively transcribed, generating many different RNAs with no known function. We have constructed 1502 molecular barcoded ncRNA gene deletion strains encompassing 443 ncRNAs in the yeast Saccharomyces cerevisiae as tools for ncRNA functional analysis. This resource includes deletions of small nuclear RNAs (snRNAs), transfer RNAs (tRNAs), small nucleolar RNAs (snoRNAs), and other annotated ncRNAs as well as the more recently identified stable unannotated transcripts (SUTs) and cryptic unstable transcripts (CUTs) whose functions are largely unknown. Specifically, deletions have been constructed for ncRNAs found in the intergenic regions, not overlapping genes or their promoters (i.e., at least 200 bp minimum distance from the closest gene start codon). The deletion strains carry molecular barcodes designed to be complementary with the protein gene deletion collection enabling parallel analysis experiments. These strains will be useful for the numerous genomic and molecular techniques that utilize deletion strains, including genome-wide phenotypic screens under different growth conditions, pooled chemogenomic screens with drugs or chemicals, synthetic genetic array analysis to uncover novel genetic interactions, and synthetic dosage lethality screens to analyze gene dosage. Overall, we created a valuable resource for the RNA community and for future ncRNA research.

Published
2017

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