12 results on '"Christine M. Norman"'
Search Results
2. psiCLIP reveals dynamic RNA binding by DEAH-box helicases before and after exon ligation
- Author
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Lisa M., Strittmatter, Charlotte, Capitanchik, Andrew J., Newman, Martina, Hallegger, Christine M., Norman, Sebastian M., Fica, Chris, Oubridge, Nicholas M., Luscombe, Jernej, Ule, Kiyoshi, Nagai, Lisa M., Strittmatter, Charlotte, Capitanchik, Andrew J., Newman, Martina, Hallegger, Christine M., Norman, Sebastian M., Fica, Chris, Oubridge, Nicholas M., Luscombe, Jernej, Ule, and Kiyoshi, Nagai
- Abstract
RNA helicases remodel the spliceosome to enable pre-mRNA splicing, but their binding and mechanism of action remain poorly understood. To define helicase-RNA contacts in specific spliceosomal states, we develop purified spliceosome iCLIP (psiCLIP), which reveals dynamic helicase-RNA contacts during splicing catalysis. The helicase Prp16 binds along the entire available single-stranded RNA region between the branchpoint and 3'-splice site, while Prp22 binds diffusely downstream of the branchpoint before exon ligation, but then switches to more narrow binding in the downstream exon after exon ligation, arguing against a mechanism of processive translocation. Depletion of the exon-ligation factor Prp18 destabilizes Prp22 binding to the pre-mRNA, suggesting that proofreading by Prp22 may sense the stability of the spliceosome during exon ligation. Thus, psiCLIP complements structural studies by providing key insights into the binding and proofreading activity of spliceosomal RNA helicases., source:https://www.nature.com/articles/s41467-021-21745-9
- Published
- 2021
3. Post-catalytic spliceosome structure reveals mechanism of 3-splice site selection
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Wojciech P. Galej, Sebastian M. Fica, Kiyoshi Nagai, Andrew J. Newman, Max E. Wilkinson, and Christine M. Norman
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0303 health sciences ,Messenger RNA ,Spliceosome ,biology ,Chemistry ,Intron ,Cell biology ,03 medical and health sciences ,Exon ,0302 clinical medicine ,Docking (molecular) ,biology.protein ,RNase H ,Ligation ,030217 neurology & neurosurgery ,Small nuclear RNA ,030304 developmental biology - Abstract
Introns are removed from eukaryotic mRNA precursors by the spliceosome in two transesterification reactions – branching and exon ligation. Following branching, the 5'-exon remains paired to U5 snRNA loop 1, but the mechanism of 3'-splice site recognition during exon ligation has remained unclear. Here we present the 3.7Å cryo-EM structure of the yeast P complex spliceosome immediately after exon ligation. The 3'-splice site AG dinucleotide is recognised through non-Watson-Crick pairing with the 5'-splice site and the branch point adenosine. A conserved loop of Prp18 together with the α-finger and the RNaseH domain of Prp8 clamp the docked 3'-splice site and 3'-exon. The step 2 factors Prp18 and Slu7 and the C-terminal domain of Yju2 stabilise a conformation competent for 3'-splice site docking and exon ligation. The structure accounts for the strict conservation of the GU and AG dinucleotides of the introns and provides insight into the catalytic mechanism of exon ligation.
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- 2017
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4. Roles of the U5 snRNP in spliceosome dynamics and catalysis
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Ian A. Turner, Mark J. Churcher, Andrew J. Newman, and Christine M. Norman
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Genetics ,Spliceosome ,Exonic splicing enhancer ,Biology ,Ribonucleoproteins, Small Nuclear ,Heterogeneous ribonucleoprotein particle ,Models, Biological ,Biochemistry ,Catalysis ,Cell biology ,Kinetics ,Splicing factor ,RNA splicing ,Spliceosomes ,snRNP ,Ribonucleoprotein, U5 Small Nuclear ,Small nuclear ribonucleoprotein ,Ribonucleoprotein - Abstract
Most protein-coding genes in eukaryotes are interrupted by non-coding intervening sequences (introns), which must be precisely removed from primary gene transcripts (pre-mRNAs) before translation of the message into protein. Intron removal by pre-mRNA splicing occurs in the nucleus and is catalysed by complex ribonucleoprotein machines called spliceosomes. These molecular machines consist of several small nuclear RNA molecules and their associated proteins [together termed snRNP (small nuclear ribonucleoprotein) particles], plus multiple accessory factors. Of particular interest are the U2, U5 and U6 snRNPs, which play crucial roles in the catalytic steps of splicing. In the present review, we summarize our current understanding of the role played by the protein components of the U5 snRNP in pre-mRNA splicing, which include some of the largest and most highly conserved nuclear proteins.
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- 2004
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5. The Invariant U5 snRNA Loop 1 Sequence Is Dispensable for the First Catalytic Step of pre-mRNA Splicing in Yeast
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Andrew J. Newman, Raymond T. O'Keefe, and Christine M. Norman
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RNA Splicing ,Molecular Sequence Data ,Exonic splicing enhancer ,Prp24 ,Saccharomyces cerevisiae ,General Biochemistry, Genetics and Molecular Biology ,Structure-Activity Relationship ,Exon ,RNA, Small Nuclear ,RNA Precursors ,snRNP ,RNA, Messenger ,Sequence Deletion ,Sequence (medicine) ,Genetics ,Base Sequence ,biology ,Biochemistry, Genetics and Molecular Biology(all) ,Active site ,RNA, Fungal ,Exons ,Cell biology ,RNA splicing ,Mutagenesis, Site-Directed ,biology.protein ,Nucleic Acid Conformation ,Small nuclear RNA - Abstract
We have developed an in vitro reconstitution system to investigate the role of U5 snRNA in the two catalytic steps of pre-mRNA splicing. The invariant U5 loop 1 is known to interact with exon sequences at the 5′ splice site before the first catalytic step. Remarkably, analysis of U5 mutations in vitro reveals that the first transesterification occurs accurately in the absence of the U5 loop. Therefore this sequence is not an essential component of the spliceosomal active site for the first catalytic step. The second catalytic step, although strongly dependent on the presence of a U5 loop to tether the exon 1 splicing intermediate, is surprisingly tolerant of mutations in the invariant sequence.
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- 1996
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6. U5 snRNA interacts with exon sequences at 5′ and 3′ splice sites
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Andrew J. Newman and Christine M. Norman
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Models, Molecular ,Genetics ,Splice site mutation ,Base Sequence ,Models, Genetic ,RNA Splicing ,Genes, Fungal ,Molecular Sequence Data ,Intron ,Prp24 ,Exons ,Saccharomyces cerevisiae ,Biology ,General Biochemistry, Genetics and Molecular Biology ,Conserved sequence ,Mutagenesis, Insertional ,Exon ,RNA, Small Nuclear ,RNA splicing ,snRNP ,RNA Processing, Post-Transcriptional ,Small nuclear RNA - Abstract
U5 snRNA is an essential pre-mRNA splicing factor whose function remains enigmatic. Specific mutations in a conserved single-stranded loop sequence in yeast U5 snRNA can activate cleavage of G1 → A mutant pre-mRNAs at aberrant 5′ splice sites and facilitate processing of dead-end lariat intermediates to mRNA. Activation of aberrant 5′ cleavage sites involves base pairing between U5 snRNA and nucleotides upstream of the cleavage site. Processing of dead-end lariat intermediates to mRNA correlates with base pairing between U5 and the first two bases in exon 2. The loop sequence in U5 snRNA may therefore be intimately involved in the transesterification reactions at 5′ and 3′ splice sites. This pattern of interactions is strikingly reminiscent of exon recognition events in group II self-splicing introns and is consistent with the notion that U5 snRNA may be related to a specific functional domain from a group II-like self-splicing ancestral intron.
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- 1992
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7. Prp8p dissection reveals domain structure and protein interaction sites
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Andrew J. Newman, Jean D. Beggs, Richard J. Grainger, Kum-Loong Boon, and Christine M. Norman
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Genetics ,Binding Sites ,Saccharomyces cerevisiae Proteins ,Base Sequence ,Ribonucleoprotein, U4-U6 Small Nuclear ,Protein domain ,Molecular Sequence Data ,Nuclear Proteins ,Biology ,Protein Engineering ,Protein tertiary structure ,Recombinant Proteins ,Cell biology ,Protein Structure, Tertiary ,DDB1 ,Protein structure ,TAF4 ,GATAD2B ,Report ,HSPA2 ,Protein Interaction Mapping ,DNA Transposable Elements ,Molecular Biology ,Ribonucleoprotein, U5 Small Nuclear ,HSPA9 - Abstract
We describe a novel approach to characterize the functional domains of a protein in vivo. This involves the use of a custom-built Tn5-based transposon that causes the expression of a target gene as two contiguous polypeptides. When used as a genetic screen to dissect the budding yeast PRP8 gene, this showed that Prp8 protein could be dissected into three distinct pairs of functional polypeptides. Thus, four functional domains can be defined in the 2413-residue Prp8 protein, with boundaries in the regions of amino acids 394–443, 770, and 2170–2179. The central region of the protein was resistant to dissection by this approach, suggesting that it represents one large functional unit. The dissected constructs allowed investigation of factors that associate strongly with the N- or the C-terminal Prp8 protein fragments. Thus, the U5 snRNP protein Snu114p associates with Prp8p in the region 437–770, whereas fragmenting Prp8p at residue 2173 destabilizes its association with Aar2p.
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- 2006
8. Dissection of Prp8 protein defines multiple interactions with crucial RNA sequences in the catalytic core of the spliceosome
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Andrew J. Newman, Christine M. Norman, Ian A. Turner, and Mark J. Churcher
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Models, Molecular ,Spliceosome ,Saccharomyces cerevisiae Proteins ,Ribonucleoprotein, U4-U6 Small Nuclear ,RNA Splicing ,Computational biology ,Saccharomyces cerevisiae ,Biology ,Article ,Conserved sequence ,Minor spliceosome ,RNA, Small Nuclear ,Endopeptidases ,RNA Precursors ,Binding site ,Molecular Biology ,Conserved Sequence ,Ribonucleoprotein, U5 Small Nuclear ,Ribonucleoprotein ,Genetics ,Binding Sites ,Base Sequence ,Fungal genetics ,RNA ,RNA, Fungal ,Mutagenesis, Insertional ,RNA splicing ,Spliceosomes ,Nucleic Acid Conformation - Abstract
Current models of the core of the spliceosome include a network of RNA–RNA interactions involving the pre-mRNA and the U2, U5, and U6 snRNAs. The essential spliceosomal protein Prp8 interacts with U5 and U6 snRNAs and with specific pre-mRNA sequences that participate in catalysis. This close association with crucial RNA sequences, together with extensive genetic evidence, suggests that Prp8 could directly affect the function of the catalytic core, perhaps acting as a splicing cofactor. However, the sequence of Prp8 is almost entirely novel, and it offers few clues to the molecular basis of Prp8–RNA interactions. We have used an innovative transposon-based strategy to establish that catalytic core RNAs make multiple contacts in the central region of Prp8, underscoring the intimate relationship between this protein and the catalytic center of the spliceosome. Our analysis of RNA interactions identifies a discrete, highly conserved region of Prp8 as a prime candidate for the role of cofactor for the spliceosome’s RNA core.
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- 2006
9. Mutations in yeast U5 snRNA alter the specificity of 5' splice-site cleavage
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Christine M. Norman and Andrew J. Newman
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Base pair ,RNA Splicing ,Genes, Fungal ,Molecular Sequence Data ,Prp24 ,Saccharomyces cerevisiae ,Biology ,General Biochemistry, Genetics and Molecular Biology ,Substrate Specificity ,RNA, Small Nuclear ,RNA Precursors ,snRNP ,splice ,RNA Processing, Post-Transcriptional ,Genes, Suppressor ,Gene ,Genetics ,Base Composition ,Base Sequence ,Intron ,RNA, Fungal ,Introns ,RNA splicing ,Mutation ,Nucleic Acid Conformation ,Small nuclear RNA - Abstract
Recognition of 5' splice sites in pre-mRNA splicing is achieved in part by base pairing with U1 snRNA. We have used interactive suppression in the yeast Saccharomyces cerevisiae to look for other factors involved in 5' splice-site recognition. This approach identified an extragenic suppressor that activates a cryptic 5' splice site. The suppressor is a gene for U5 snRNA (snR7) with a single base mutation in a strictly conserved 9 base sequence. This suggests that U5 snRNA can play a part in determining the position of 5' splice-site cleavage. Consistent with this, we have been able to isolate other mutations in the 9 base element in U5 snRNA that specifically activate a second cryptic 5' splice site nearby.
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- 1991
10. Isolation and properties of cDNA clones encoding SRF, a transcription factor that binds to the c-fos serum response element
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Mike Runswick, Roy Pollock, Richard Treisman, and Christine M. Norman
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Serum Response Factor ,Base Sequence ,Transcription, Genetic ,Molecular Sequence Data ,Nuclear Proteins ,DNA ,Oncogenes ,Molecular cloning ,Biology ,Serum Response Element ,Molecular biology ,General Biochemistry, Genetics and Molecular Biology ,Gene Expression Regulation ,Transcription (biology) ,Complementary DNA ,Gene expression ,Serum response factor ,Humans ,Cloning, Molecular ,Nuclear protein ,Transcription factor ,HeLa Cells - Abstract
The serum response element (SRE) is a sequence required for transient transcriptional activation of genes in response to growth factors. We have isolated cDNA clones encoding serum response factor (SRF), a ubiquitous nuclear protein that binds to the SRE. The SRF gene is highly conserved through evolution, and in cultured cells its transcription is itself transiently increased following serum stimulation. A cDNA clone of SRF expressed in vitro generates protein that forms complexes indistinguishable from those formed with HeLa cell SRF, as judged by DNA binding specificity and the ability to promote SRE-dependent in vitro transcription. SRF binds DNA as a dimer, and the DNA binding/dimerization domain of the protein exhibits striking homology to two yeast regulatory proteins.
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- 1988
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11. Analysis of Serum Response Element Function In Vitro
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Richard Treisman and Christine M. Norman
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Serum Response Factor ,Transcription, Genetic ,Xenopus ,Molecular Sequence Data ,Biochemistry ,Proto-Oncogene Proteins ,Proto-Oncogenes ,Genetics ,Animals ,Humans ,Promoter Regions, Genetic ,Molecular Biology ,Base Sequence ,Chemistry ,Genes, Homeobox ,Nuclear Proteins ,Templates, Genetic ,Serum Response Element ,Actins ,In vitro ,Cell biology ,DNA-Binding Proteins ,Genes ,Protein Biosynthesis ,Proto-Oncogene Proteins c-fos ,Function (biology) ,Signal Transduction - Published
- 1988
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12. Postcatalytic spliceosome structure reveals mechanism of 3′–splice site selection
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Wojciech P. Galej, Kiyoshi Nagai, Christine M. Norman, Max E. Wilkinson, Andrew J. Newman, and Sebastian M. Fica
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0301 basic medicine ,Spliceosome ,Saccharomyces cerevisiae Proteins ,Base pair ,Protein Conformation ,RNA Splicing ,Saccharomyces cerevisiae ,Biology ,Article ,03 medical and health sciences ,Exon ,Protein structure ,Minor spliceosome ,Catalytic Domain ,RNA Precursors ,Base Pairing ,Messenger RNA ,Multidisciplinary ,Cryoelectron Microscopy ,Intron ,Exons ,Molecular biology ,Introns ,Cell biology ,030104 developmental biology ,RNA splicing ,Spliceosomes ,RNA Splice Sites - Abstract
Understanding splicing from the 3′ end The spliceosome removes introns from eukaryotic mRNA precursors and yields mature transcripts by joining exons. Despite decades of functional studies and recent progress in understanding the spliceosome structure, the mechanism by which the 3′ splice site (SS) is recognized by the spliceosome has remained unclear. Liu et al. and Wilkinson et al. report the high-resolution cryo-electron microscopy structures of the yeast postcatalytic spliceosome. The structures reveal that the 3′SS is recognized through non-Watson-Crick base pairing with the 5′SS and the branch point, stabilized by the intron region and protein factors. Science , this issue p. 1278 , p. 1283
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