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| Status |
Public on Sep 25, 2013 |
| Title |
Structural and functional characterization of the N-terminus of Schizosaccharomyces pombe Cwf10 |
| Organism |
Schizosaccharomyces pombe |
| Experiment type |
Expression profiling by high throughput sequencing
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| Summary |
The spliceosome is a dynamic macromolecular machine that catalyzes the removal of introns from pre-mRNA to make mature message. Schizosaccharomyces pombe Cwf10 (homolog Saccharomyces cerevisiae Snu114 and of Human U5-116K), an integral member of the U5 snRNP, is a GTPase that shares sequence homology with the eukaryotic translation elongation factor EF2. Cwf10 is required for pre-mRNA splicing; however, its mechanism(s) of action is still not understood. Cwf10/Snu114 family members contain a conserved N-terminal extension (NTE) that lacks homology with EF2 and has been predicted to be an intrinsically unfolded domain. Using S. pombe as a model system, we show that the NTE is not essential, but cells lacking this domain are defective in pre-mRNA splicing at all temperatures. Genetic interactions between cwf10-ΔNTE and other pre-mRNA splicing mutants are consistent with a role for the NTE in spliceosome activation. Characterization of Cwf10-NTE by various biophysical techniques shows the NTE contains both regions of structure and disorder in solution. The first twenty-three highly-conserved amino acids of the NTE are essential for its role in splicing, but are not sufficient to restore pre-mRNA splicing to wild-type levels in cwf10-∆NTE cells. When the NTE is overexpressed in the cwf10-ΔNTE background, it can complement the truncated Cwf10 protein in trans, and it also immunoprecipitates a complex similar in composition to the late-stage U5.U2/U6 spliceosome. These data show that the structurally flexible NTE is capable of making specific contacts within the spliceosome that may facilitate Cwf10’s overall role facilitating spliceosome rearrangements.
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| Overall design |
Interrogation of the S. pombe transcriptome using poly-A enriched RNA sequencing (Illumina HiSeq 2500) in wild type and cwf10-ΔNTE cultures. A total of 4 samples were analysed: two biological repeats of wild-type strain and two biological repeats of cwf10-ΔNTE
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| Contributor(s) |
Livesay SB, Collier SE, Bitton DA, Bähler J, Ohi MD |
| Citation(s) |
24014766 |
| |
| Submission date |
May 31, 2013 |
| Last update date |
May 15, 2019 |
| Contact name |
Melanie Ohi |
| E-mail(s) |
melanie.ohi@vanderbilt.edu
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| Organization name |
Vanderbilt University Medical School
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| Department |
Cell and Developmental Biology
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| Street address |
465 21st Avenue South
|
| City |
Nashville |
| State/province |
TN |
| ZIP/Postal code |
37232 |
| Country |
USA |
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| Platforms (1) |
| GPL17225 |
Illumina HiSeq 2500 (Schizosaccharomyces pombe) |
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| Samples (8)
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| Relations |
| BioProject |
PRJNA206185 |
| SRA |
SRP023504 |
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